Plasma H3Cit-DNA Discriminates Between Cancer and Inflammation in a Cohort of Patients with Unspecific Cancer Symptoms.

Cancer detection is challenging, especially in patients with unspecific cancer symptoms. Biomarkers could identify patients at high risk of cancer. Prior studies indicate that neutrophil extracellular traps (NETs) are associated with cancer, but also with autoimmune and infectious diseases. The objective of this prospective study was to investigate markers associated with NET formation (nucleosomal citrullinated histone 3 [H3Cit-DNA], cell free DNA [cfDNA] and neutrophil elastase [NE]), and c-reactive protein (CRP) in patients with unspecific cancer symptoms, such as fatigue, weight loss or radiological sign of malignancy without an apparent primary tumor, referred to the Diagnostic Center at Danderyd Hospital in Sweden. Blood samples were drawn on admission, before cancer diagnosis. Out of 475 patients, 160 (34%) were diagnosed with cancer, 56 (12%) with autoimmune disease, 32 (7%) with infectious disease, 71 (15%) with other diseases and 156 (33%) received no diagnosis. H3Cit-DNA, cfDNA, NE and CRP were significantly higher in patients with cancer compared to patients without cancer (p < 0.0001, p < 0.0001, p = 0.004, and p = 0.0002 respectively). H3Cit-DNA, but not cfDNA, NE or CRP, was significantly elevated in patients with cancer compared to patients with autoimmune disease (p = 0.0001). H3Cit-DNA, cfDNA, NE or CRP did not differ between cancer and infectious disease. In conclusion, H3Cit-DNA is elevated in patients diagnosed with cancer compared to non-cancer patients with the same symptomatology. Further studies should evaluate if H3Cit-DNA could aid in selecting patients that would benefit the most from a rapid cancer diagnostic work-up.

New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics.

Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC-MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact.

Unique pathways downstream of TLR-4 and TLR-7 activation: sex-dependent behavioural, cytokine, and metabolic consequences.

Introduction: Post-infection syndromes are characterised by fatigue, muscle pain, anhedonia, and cognitive impairment; mechanistic studies exploring these syndromes have focussed on pathways downstream of Toll-like receptor (TLR) 4 activation. Here, we investigated the mechanistic interplay between behaviour, metabolism, and inflammation downstream of TLR-7 activation compared to TLR-4 activation in male and female CD1 mice. Methods: Animals received either a TLR-4 (LPS; 0.83 mg/kg) or TLR-7 (R848, 5 mg/kg) agonist, or saline, and behaviour was analysed in an Open Field (OF) at 24 h (n = 20/group). Plasma, liver, and prefrontal cortex (PFC) were collected for gene expression analysis at 24 h and 1H-NMR metabolomics. Results: TLR-4 and TLR-7 activation decreased distance travelled and rearing in the OF, but activation of each receptor induced distinct cytokine responses and metabolome profiles. LPS increased IL-1ß expression and CXCL1 in the PFC, but TLR7 activation did not and strongly induced PFC CXCL10 expression. Thus, TLR7 induced sickness behaviour is independent of IL-1ß expression. In both cases, the behavioural response to TLR activation was sexually dimorphic: females were more resilient. However, dissociation was observed between the resilient female mice behaviour and the levels of gene cytokine expression, which was, in general, higher in the female mice. However, the metabolic shifts induced by immune activation were better correlated with the sex-dependent behavioural dimorphisms; increased levels of antioxidant potential in the female brain are intrinsic male/female metabolome differences. A common feature of both TLR4 and TLR7 activation was an increase in N-acetyl aspartate (NAA) in the PFC, which is likely be an allostatic response to the challenges as sickness behaviour is inversely correlated with NAA levels. Discussion: The results highlight how the cytokine profile induced by one PAMP cannot be extrapolated to another, but they do reveal how the manipulation of the conserved metabolome response might afford a more generic approach to the treatment of post-infection syndromes.

A multivariate blood metabolite algorithm stably predicts risk and resilience to major depressive disorder in the general population.

BACKGROUND: Socioeconomic pressures, sex, and physical health status strongly influence the development of major depressive disorder (MDD) and mask other contributing factors in small cohorts. Resilient individuals overcome adversity without the onset of psychological symptoms, but resilience, as for susceptibility, has a complex and multifaceted molecular basis. The scale and depth of the UK Biobank affords an opportunity to identify resilience biomarkers in rigorously matched, at-risk individuals. Here, we evaluated whether blood metabolites could prospectively classify and indicate a biological basis for susceptibility or resilience to MDD. METHODS: Using the UK Biobank, we employed random forests, a supervised, interpretable machine learning statistical method to determine the relative importance of sociodemographic, psychosocial, anthropometric, and physiological factors that govern the risk of prospective MDD onset (total n = 15,710). We then used propensity scores to rigorously match individuals with a history of MDD (n = 491) against a resilient subset of individuals without an MDD diagnosis (retrospectively or during follow-up; n = 491) using an array of key social, demographic, and disease-associated drivers of depression risk. 381 blood metabolites and clinical chemistry variables and 4 urine metabolites were integrated to generate a multivariate random forest-based algorithm using 10-fold cross-validation to predict prospective MDD risk and resilience. OUTCOMES: In unmatched individuals, a first case of MDD, with a median time-to-diagnosis of 72 years, can be predicted using random forest classification probabilities with an area under the receiver operator characteristic curve (ROC AUC) of 0.89. Prospective resilience/susceptibility to MDD was then predicted with a ROC AUC of 0.72 (x˜ = 3.2 years follow-up) and 0.68 (x˜ = 7.2 years follow-up). Increased pyruvate was identified as a key biomarker of resilience to MDD and was validated retrospectively in the TwinsUK cohort. INTERPRETATION: Blood metabolites prospectively associate with substantially reduced MDD risk. Therapeutic targeting of these metabolites may provide a framework for MDD risk stratification and reduction. FUNDING: New York Academy of Sciences' Interstellar Programme Award; Novo Fonden; Lincoln Kingsgate award; Clarendon Fund; Newton-Abraham studentship (University of Oxford). The funders had no role in the development of the present study.

Interleukin-3 coordinates glial-peripheral immune crosstalk to incite multiple sclerosis.

Glial cells and central nervous system (CNS)-infiltrating leukocytes contribute to multiple sclerosis (MS). However, the networks that govern crosstalk among these ontologically distinct populations remain unclear. Here, we show that, in mice and humans, CNS-resident astrocytes and infiltrating CD44hiCD4+ T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ). Astrocytic and T cell IL-3 elicited an immune migratory and chemotactic program by IL-3Rɑ+ myeloid cells that enhanced CNS immune cell infiltration, exacerbating MS and its preclinical model. Multiregional snRNA-seq of human CNS tissue revealed the appearance of IL3RA-expressing myeloid cells with chemotactic programming in MS plaques. IL3RA expression by plaque myeloid cells and IL-3 amount in the cerebrospinal fluid predicted myeloid and T cell abundance in the CNS and correlated with MS severity. Our findings establish IL-3:IL-3RA as a glial-peripheral immune network that prompts immune cell recruitment to the CNS and worsens MS.

Factors predicting the transition from acute to persistent pain in people with 'sciatica': the FORECAST longitudinal prognostic factor cohort study protocol.

INTRODUCTION: Sciatica is a common condition and is associated with higher levels of pain, disability, poorer quality of life, and increased use of health resources compared with low back pain alone. Although many patients recover, a third develop persistent sciatica symptoms. It remains unclear, why some patients develop persistent sciatica as none of the traditionally considered clinical parameters (eg, symptom severity, routine MRI) are consistent prognostic factors.The FORECAST study (factors predicting the transition from acute to persistent pain in people with 'sciatica') will take a different approach by exploring mechanism-based subgroups in patients with sciatica and investigate whether a mechanism-based approach can identify factors that predict pain persistence in patients with sciatica. METHODS AND ANALYSIS: We will perform a prospective longitudinal cohort study including 180 people with acute/subacute sciatica. N=168 healthy participants will provide normative data. A detailed set of variables will be assessed within 3 months after sciatica onset. This will include self-reported sensory and psychosocial profiles, quantitative sensory testing, blood inflammatory markers and advanced neuroimaging. We will determine outcome with the Sciatica Bothersomeness Index and a Numerical Pain Rating Scale for leg pain severity at 3 and 12 months.We will use principal component analysis followed by clustering methods to identify subgroups. Univariate associations and machine learning methods optimised for high dimensional small data sets will be used to identify the most powerful predictors and model selection/accuracy.The results will provide crucial information about the pathophysiological drivers of sciatica symptoms and may identify prognostic factors of pain persistence. ETHICS AND DISSEMINATION: The FORECAST study has received ethical approval (South Central Oxford C, 18/SC/0263). The dissemination strategy will be guided by our patient and public engagement activities and will include peer-reviewed publications, conference presentations, social media and podcasts. TRIAL REGISTRATION NUMBER: ISRCTN18170726; Pre-results.

HDL and LDL have distinct, opposing effects on LPS-induced brain inflammation.

Endotoxemia and sepsis induce neuroinflammation and increase the risk of neurodegenerative disorders although the mechanism by which peripheral infection leads to brain inflammation is not well understood. While circulating serum lipoproteins are known immunometabolites with the potential to modulate the acute phase response and cross the blood brain barrier, their contribution to neuroinflammation during systemic infection is unknown. The objective of this study was to elucidate the mechanisms by which lipoprotein subclasses modulate lipopolysaccharide (LPS)-induced neuroinflammation. Adult C57BL/6 mice were divided into 6 treatment groups, including a sterile saline vehicle control group (n = 9), an LPS group (n = 11), a premixed LPS + HDL group (n = 6), a premixed LPS + LDL group (n = 5), a HDL only group (n = 6) and an LDL only group (n = 3). In all cases injections were administered intraperitoneally. LPS was administered at 0.5 mg/kg, and lipoproteins were administered at 20 mg/kg. Behavioural testing and tissue collection was performed 6 h post-injection. The magnitude of peripheral and central inflammation was determined by qPCR of pro-inflammatory genes in fresh liver and brain. Metabolite profiles of liver, plasma and brain were determined by 1H NMR. Endotoxin concentration in the brain was measured by the Limulus Amoebocyte Lysate (LAL) assay. Co-administration of LPS + HDL exacerbated both peripheral and central inflammation, whilst LPS + LDL attenuated this inflammation. Metabolomic analysis identified several metabolites significantly associated with LPS-induced inflammation, which were partially rescued by LDL, but not HDL. Endotoxin was detected at significantly greater concentrations in the brains of animals that received LPS + HDL compared to LPS + saline, but not those that received LPS + LDL. These results suggest that HDL may promote neuroinflammation through direct shuttling of endotoxin to the brain. In contrast, LDL was shown to have anti-neuroinflammatory properties in this study. Our results indicate that lipoproteins may be useful targets in neuroinflammation and neurodegeneration associated with endotoxemia and sepsis.

Corrigendum: Determination of CSF GFAP, CCN5, and vWF levels enhances the diagnostic accuracy of clinically defined MS from non-MS patients with CSF oligoclonal bands.

[This corrects the article DOI: 10.3389/fimmu.2021.811351.].

In FUS[1-359]-tg mice O,S-dibenzoyl thiamine reduces muscle atrophy, decreases glycogen synthase kinase 3 beta, and normalizes the metabolome.

Mutations in the gene encoding the RNA/DNA-binding protein Fused in Sarcoma (FUS) have been detected in familial amyotrophic lateral sclerosis (ALS) patients. FUS has been found to be a critical component of the oxidative damage repair complex that might explain its role in neurodegeneration. Here, we examined what impact antioxidant treatment with thiamine (vitamine B1), or its more bioavailable derivative O,S-dibenzoylthiamine (DBT), would have on the hallmarks of pathology in the FUS[1-359]-transgenic mouse model of ALS. From 8-weeks old, in the pre-symptomatic phase of disease, animals received either thiamine, DBT (200 mg/kg/day), or vehicle for 6 weeks. We examined physiological, behavioral, molecular and histological outcomes, as well as the serum metabolome using nuclear magnetic resonance (NMR). The DBT-treated mice displayed improvements in physiological outcomes, motor function and muscle atrophy compared to vehicle, and the treatment normalized levels of brain glycogen synthase kinase-3ß (GSK-3ß), GSK-3ß mRNA and IL-1ß mRNA in the spinal cord. Analysis of the metabolome revealed an increase in the levels of choline and lactate in the vehicle-treated FUS mutants alone, which is also elevated in the cerebrospinal fluid of ALS patients, and reduced glucose and lipoprotein concentrations in the FUS[1-359]-tg mice, which were not the case in the DBT-treated mutants. The administration of thiamine had little impact on the outcome measures, but it did normalize circulating HDL levels. Thus, our study shows that DBT therapy in FUS mutants is more effective than thiamine and highlights how metabolomics may be used to evaluate therapy in this model.

Metabolomics detects clinically silent neuroinflammatory lesions earlier than neurofilament-light chain in a focal multiple sclerosis animal model.

BACKGROUND: Despite widespread searches, there are currently no validated biofluid markers for the detection of subclinical neuroinflammation in multiple sclerosis (MS). The dynamic nature of human metabolism in response to changes in homeostasis, as measured by metabolomics, may allow early identification of clinically silent neuroinflammation. Using the delayed-type hypersensitivity (DTH) MS rat model, we investigated the serum and cerebrospinal fluid (CSF) metabolomics profiles and neurofilament-light chain (NfL) levels, as a putative marker of neuroaxonal damage, arising from focal, clinically silent neuroinflammatory brain lesions and their discriminatory abilities to distinguish DTH animals from controls. METHODS: 1H nuclear magnetic resonance (NMR) spectroscopy metabolomics and NfL measurements were performed on serum and CSF at days 12, 28 and 60 after DTH lesion initiation. Supervised multivariate analyses were used to determine metabolomics differences between DTH animals and controls. Immunohistochemistry was used to assess the extent of neuroinflammation and tissue damage. RESULTS: Serum and CSF metabolomics perturbations were detectable in DTH animals (vs. controls) at all time points, with the greatest change occurring at the earliest time point (day 12) when the neuroinflammatory response was most intense (mean predictive accuracy [SD]-serum: 80.6 [10.7]%, p < 0.0001; CSF: 69.3 [13.5]%, p < 0.0001). The top discriminatory metabolites at day 12 (serum: allantoin, cytidine; CSF: glutamine, glucose) were all reduced in DTH animals compared to controls, and correlated with histological markers of neuroinflammation, particularly astrogliosis (Pearson coefficient, r-allantoin: r = - 0.562, p = 0.004; glutamine: r = - 0.528, p = 0.008). Serum and CSF NfL levels did not distinguish DTH animals from controls at day 12, rather, significant differences were observed at day 28 (mean [SEM]-serum: 38.5 [4.8] vs. 17.4 [2.6] pg/mL, p = 0.002; CSF: 1312.0 [379.1] vs. 475.8 [74.7] pg/mL, p = 0.027). Neither serum nor CSF NfL levels correlated with markers of neuroinflammation; serum NfL did, however, correlate strongly with axonal loss (r = 0.641, p = 0.001), but CSF NfL did not (p = 0.137). CONCLUSIONS: While NfL levels were elevated later in the pathogenesis of the DTH lesion, serum and CSF metabolomics were able to detect early, clinically silent neuroinflammation and are likely to present sensitive biomarkers for the assessment of subclinical disease activity in patients.

The serum metabolomic profile of a distinct, inflammatory subtype of acute psychosis.

A range of studies suggest that a proportion of psychosis may have an autoimmune basis, but this has not translated through into clinical practice-there is no biochemical test able to accurately identify psychosis resulting from an underlying inflammatory cause. Such a test would be an important step towards identifying who might require different treatments and have the potential to improve outcomes for patients. To identify novel subgroups within patients with acute psychosis we measured the serum nuclear magnetic resonance (NMR) metabolite profiles of 75 patients who had identified antibodies (anti-glycine receptor [GlyR], voltage-gated potassium channel [VGKC], Contactin-associated protein-like 2 [CASPR2], leucine-rich glioma inactivated 1 [LGI1], N-methyl-D-aspartate receptor [NMDAR] antibody) and 70 antibody negative patients matched for age, gender, and ethnicity. Clinical symptoms were assessed using the positive and negative syndrome scale (PANSS). Unsupervised principal component analysis identified two distinct biochemical signatures within the cohort. Orthogonal partial least squared discriminatory analysis revealed that the serum metabolomes of NMDAR, LGI1, and CASPR2 antibody psychosis patients were indistinct from the antibody negative control group while VGKC and GlyR antibody patients had significantly decreased lipoprotein fatty acids and increased amino acid concentrations. Furthermore, these patients had more severe presentation with higher PANSS scores than either the antibody negative controls or the NMDAR, LGI1, and CASPR2 antibody groups. These results suggest that a proportion of patients with acute psychosis have a distinct clinical and biochemical phenotype that may indicate an inflammatory subtype.

A novel serum metabolomic panel distinguishes IgG4-related sclerosing cholangitis from primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) and IgG4-related sclerosing cholangitis (IgG4-SC) are chronic fibro-inflammatory immune-mediated hepatobiliary conditions that are challenging to distinguish in a clinical setting. Accurate non-invasive biomarkers for discriminating PSC and IgG4-SC are important to ensure a correct diagnosis, prompt therapy and adequate cancer surveillance. METHODS: We performed nuclear magnetic resonance (NMR)-based metabolomic profiling using serum samples collected prospectively from patients with PSC (n = 100), IgG4-SC (n = 23) and healthy controls (HC; n = 16). RESULTS: Multivariate analysis of the serum metabolome discriminated PSC from IgG4-SC with greater accuracy (AUC 0.95 [95%CI 0.90-0.98]) than IgG4 titre (AUC 0.87 [95%CI 0.79-0.94]). When inflammatory bowel disease (IBD) was excluded as a comorbid condition (IgG4-SC n = 20, PSC n = 22), the diagnostic AUC increased to 1.0, suggesting that the metabolome differences identified are not a result of the increased prevalence of IBD in PSC relative to IgG4-SC patients. Serum lactate (p < .0001), glucose (p < .01) and glutamine (p < .01) metabolites were increased in IgG4-related disease (IgG4-RD) and IgG4-SC individuals compared to PSC, whereas mobile choline (p < .05), 3-hydroxybutyric acid (p < .01) and -CH3 lipoprotein resonances (p < .01) were decreased. CONCLUSIONS: Taken together, serum metabolomic profiling has the potential to be incorporated as a diagnostic criterion, independent of IgG4 titre, to improve the diagnosis of IgG4-RD and help distinguish IgG4-SC from PSC.

Modifying the maternal microbiota alters the gut-brain metabolome and prevents emotional dysfunction in the adult offspring of obese dams.

Maternal obesity disturbs brain-gut-microbiota interactions and induces negative affect in the offspring, but its impact on gut and brain metabolism in the offspring (F1) are unknown. Here, we tested whether perinatal intake of a multispecies probiotic could mitigate the abnormal emotional behavior in the juvenile and adult offspring of obese dams. Untargeted NMR-based metabolomic profiling and gene-expression analysis throughout the gut-brain axis were then used to investigate the biology underpinning behavioral changes in the dams and their offspring. Prolonged high-fat diet feeding reduced maternal gut short-chain fatty acid abundance, increased markers of peripheral inflammation, and decreased the abundance of neuroactive metabolites in maternal milk during nursing. Both juvenile (postnatal day [PND] 21) and adult (PND112) offspring of obese dams exhibited increased anxiety-like behavior, which were prevented by perinatal probiotic exposure. Maternal probiotic treatment increased gut butyrate and brain lactate in the juvenile and adult offspring and increased the expression of prefrontal cortex PFKFB3, a marker of glycolytic metabolism in astrocytes. PFKFB3 expression correlated with the increase in gut butyrate in the juvenile and adult offspring. Maternal obesity reduced synaptophysin expression in the adult offspring, while perinatal probiotic exposure increased expression of brain-derived neurotrophic factor. Finally, we showed that the resilience of juvenile and adult offspring to anxiety-like behavior was most prominently associated with increased brain lactate abundance, independent of maternal group. Taken together, we show that maternal probiotic supplementation exerts a long-lasting effect on offspring neuroplasticity and the offspring gut-liver-brain metabolome, increasing resilience to emotional dysfunction induced by maternal obesity.

Depressive symptoms in non-alcoholic fatty liver disease are identified by perturbed lipid and lipoprotein metabolism.

Non-alcoholic fatty liver disease (NAFLD) and depression are common disorders and have bidirectional contributing relationships to metabolic syndrome. We aimed to determine whether a fasting serum signature of recent, self-reported depressive symptoms could be identified in a heterogeneous NAFLD cohort using nuclear magnetic resonance (NMR)-based metabolomics integrated with clinical chemistry. Serum nuclear magnetic resonance (NMR) metabolite profiles and corresponding clinical chemistry were compared between patients with depressive symptoms in the last 12-months (n = 81) and patients without recent depressive symptoms (n = 137 controls) using multivariate statistics. Orthogonal partial least squares discriminant analysis (OPLS-DA) of the biochemical and metabolomic data identified NAFLD patients with recent depression with a cross-validated accuracy of 61.5%, independent of age, sex, medication, and other comorbidities. This led to the development of a diagnostic algorithm with AUC 0.83 for future testing in larger clinical cohorts. Serum triglycerides, VLDL cholesterol, and the inflammatory biomarker GlycA were key metabolites increased in patients with recent depressive symptoms, while serum glutamine level was reduced. Here, serum NMR metabolite analysis provides a link between disturbed lipid metabolism, inflammation, and active mental health issues in NAFLD, irrespective of disease severity.

Metabolomic Biomarkers in Blood Samples Identify Cancers in a Mixed Population of Patients with Nonspecific Symptoms.

PURPOSE: Early diagnosis of cancer is critical for improving patient outcomes, but cancers may be hard to diagnose if patients present with nonspecific signs and symptoms. We have previously shown that nuclear magnetic resonance (NMR) metabolomics analysis can detect cancer in animal models and distinguish between differing metastatic disease burdens. Here, we hypothesized that biomarkers within the blood metabolome could identify cancers within a mixed population of patients referred from primary care with nonspecific symptoms, the so-called "low-risk, but not no-risk" patient group, as well as distinguishing between those with and without metastatic disease. EXPERIMENTAL DESIGN: Patients (n = 304 comprising modeling, n = 192, and test, n = 92) were recruited from 2017 to 2018 from the Oxfordshire Suspected CANcer (SCAN) pathway, a multidisciplinary diagnostic center (MDC) referral pathway for patients with nonspecific signs and symptoms. Blood was collected and analyzed by NMR metabolomics. Orthogonal partial least squares discriminatory analysis (OPLS-DA) models separated patients, based upon diagnoses received from the MDC assessment, within 62 days of initial appointment. RESULTS: Area under the ROC curve for identifying patients with solid tumors in the independent test set was 0.83 [95% confidence interval (CI): 0.72-0.95]. Maximum sensitivity and specificity were 94% (95% CI: 73-99) and 82% (95% CI: 75-87), respectively. We could also identify patients with metastatic disease in the cohort of patients with cancer with sensitivity and specificity of 94% (95% CI: 72-99) and 88% (95% CI: 53-98), respectively. CONCLUSIONS: For a mixed group of patients referred from primary care with nonspecific signs and symptoms, NMR-based metabolomics can assist their diagnosis, and may differentiate both those with malignancies and those with and without metastatic disease. See related commentary by Van Tine and Lyssiotis, p. 1477.

Chronically elevated branched chain amino acid levels are pro-arrhythmic.

AIMS: Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. METHODS AND RESULTS: We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6-9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4-5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs-leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. CONCLUSIONS: Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.

Objective biomarkers for clinical relapse in multiple sclerosis: a metabolomics approach.

Accurate determination of relapses in multiple sclerosis is important for diagnosis, classification of clinical course and therapeutic decision making. The identification of biofluid markers for multiple sclerosis relapses would add to our current diagnostic armamentarium and increase our understanding of the biology underlying the clinical expression of inflammation in multiple sclerosis. However, there is presently no biofluid marker capable of objectively determining multiple sclerosis relapses although some, in particular neurofilament-light chain, have shown promise. In this study, we sought to determine if metabolic perturbations are present during multiple sclerosis relapses, and, if so, identify candidate metabolite biomarkers and evaluate their discriminatory abilities at both group and individual levels, in comparison with neurofilament-light chain. High-resolution global and targeted 1H nuclear magnetic resonance metabolomics as well as neurofilament-light chain measurements were performed on the serum in four groups of relapsing-remitting multiple sclerosis patients, stratified by time since relapse onset: (i) in relapse (R); (ii) last relapse (LR) ≥ 1 month (M) to < 6 M ago; (iii) LR ≥ 6 M to < 24 M ago; and (iv) LR ≥ 24 M ago. Two hundred and one relapsing-remitting multiple sclerosis patients were recruited: R (n = 38), LR 1-6 M (n = 28), LR 6-24 M (n = 34), LR ≥ 24 M (n = 101). Using supervised multivariate analysis, we found that the global metabolomics profile of R patients was significantly perturbed compared to LR ≥ 24 M patients. Identified discriminatory metabolites were then quantified using targeted metabolomics. Lysine and asparagine (higher in R), as well as, isoleucine and leucine (lower in R), were shortlisted as potential metabolite biomarkers. ANOVA of these metabolites revealed significant differences across the four patient groups, with a clear trend with time since relapse onset. Multivariable receiver operating characteristics analysis of these four metabolites in discriminating R versus LR ≥ 24 M showed an area under the curve of 0.758, while the area under the curve for serum neurofilament-light chain was 0.575. Within individual patients with paired relapse-remission samples, all four metabolites were significantly different in relapse versus remission, with the direction of change consistent with that observed at group level, while neurofilament-light chain was not discriminatory. The perturbations in the identified metabolites point towards energy deficiency and immune activation in multiple sclerosis relapses, and the measurement of these metabolites, either singly or in combination, are useful as biomarkers to differentiate relapse from remission at both group and individual levels.

Integrative biochemical, proteomics and metabolomics cerebrospinal fluid biomarkers predict clinical conversion to multiple sclerosis.

Eighty-five percent of multiple sclerosis cases begin with a discrete attack termed clinically isolated syndrome, but 37% of clinically isolated syndrome patients do not experience a relapse within 20 years of onset. Thus, the identification of biomarkers able to differentiate between individuals who are most likely to have a second clinical attack from those who remain in the clinically isolated syndrome stage is essential to apply a personalized medicine approach. We sought to identify biomarkers from biochemical, metabolic and proteomic screens that predict clinically defined conversion from clinically isolated syndrome to multiple sclerosis and generate a multi-omics-based algorithm with higher prognostic accuracy than any currently available test. An integrative multi-variate approach was applied to the analysis of cerebrospinal fluid samples taken from 54 individuals at the point of clinically isolated syndrome with 2-10 years of subsequent follow-up enabling stratification into clinical converters and non-converters. Leukocyte counts were significantly elevated at onset in the clinical converters and predict the occurrence of a second attack with 70% accuracy. Myo-inositol levels were significantly increased in clinical converters while glucose levels were decreased, predicting transition to multiple sclerosis with accuracies of 72% and 63%, respectively. Proteomics analysis identified 89 novel gene products related to conversion. The identified biochemical and protein biomarkers were combined to produce an algorithm with predictive accuracy of 83% for the transition to clinically defined multiple sclerosis, outperforming any individual biomarker in isolation including oligoclonal bands. The identified protein biomarkers are consistent with an exaggerated immune response, perturbed energy metabolism and multiple sclerosis pathology in the clinical converter group. The new biomarkers presented provide novel insight into the molecular pathways promoting disease while the multi-omics algorithm provides a means to more accurately predict whether an individual is likely to convert to clinically defined multiple sclerosis.

Foveal changes in aquaporin-4 antibody seropositive neuromyelitis optica spectrum disorder are independent of optic neuritis and not overtly progressive.

BACKGROUND AND PURPOSE: Foveal changes were reported in aquaporin-4 antibody (AQP4-Ab) seropositive neuromyelitis optica spectrum disorder (NMOSD) patients; however, it is unclear whether they are independent of optic neuritis (ON), stem from subclinical ON or crossover from ON in fellow eyes. Fovea morphometry and a statistical classification approach were used to investigate if foveal changes in NMOSD are independent of ON and progressive. METHODS: This was a retrospective longitudinal study of 27 AQP4-IgG + NMOSD patients (49 eyes; 15 ON eyes and 34 eyes without a history of ON [NON eyes]), follow-up median (first and third quartile) 2.32 (1.33-3.28), and 38 healthy controls (HCs) (76 eyes), follow-up median (first and third quartile) 1.95 (1.83-2.54). The peripapillary retinal nerve fibre layer thickness and the volume of combined ganglion cell and inner plexiform layer as measures of neuroaxonal damage from ON were determined by optical coherence tomography. Nineteen foveal morphometry parameters were extracted from macular optical coherence tomography volume scans. Data were analysed using orthogonal partial least squares discriminant analysis and linear mixed effects models. RESULTS: At baseline, foveal shape was significantly altered in ON eyes and NON eyes compared to HCs. Discriminatory analysis showed 81% accuracy distinguishing ON vs. HCs and 68% accuracy in NON vs. HCs. NON eyes were distinguished from HCs by foveal shape parameters indicating widening. Orthogonal partial least squares discriminant analysis discriminated ON vs. NON with 76% accuracy. In a follow-up of 2.4 (20.85) years, no significant time-dependent foveal changes were found. CONCLUSION: The parafoveal area is altered in AQP4-Ab seropositive NMOSD patients suggesting independent neuroaxonal damage from subclinical ON. Longer follow-ups are needed to confirm the stability of the parafoveal structure over time.

Determination of CSF GFAP, CCN5, and vWF Levels Enhances the Diagnostic Accuracy of Clinically Defined MS From Non-MS Patients With CSF Oligoclonal Bands.

Background: Inclusion of cerebrospinal fluid (CSF) oligoclonal IgG bands (OCGB) in the revised McDonald criteria increases the sensitivity of diagnosis when dissemination in time (DIT) cannot be proven. While OCGB negative patients are unlikely to develop clinically definite (CD) MS, OCGB positivity may lead to an erroneous diagnosis in conditions that present similarly, such as neuromyelitis optica spectrum disorders (NMOSD) or neurosarcoidosis. Objective: To identify specific, OCGB-complementary, biomarkers to improve diagnostic accuracy in OCGB positive patients. Methods: We analysed the CSF metabolome and proteome of CDMS (n=41) and confirmed non-MS patients (n=64) comprising a range of CNS conditions routinely encountered in neurology clinics. Results: OCGB discriminated between CDMS and non-MS with high sensitivity (85%), but low specificity (67%), as previously described. Machine learning methods revealed CCN5 levels provide greater accuracy, sensitivity, and specificity than OCGB (79%, +5%; 90%, +5%; and 72%, +5% respectively) while glial fibrillary acidic protein (GFAP) identified CDMS with 100% specificity (+33%). A multiomics approach improved accuracy further to 90% (+16%). Conclusion: The measurement of a few additional CSF biomarkers could be used to complement OCGB and improve the specificity of MS diagnosis when clinical and radiological evidence of DIT is absent.