Low-dose interleukin 2 antidepressant potentiation in unipolar and bipolar depression: Safety, efficacy, and immunological biomarkers.

Immune-inflammatory mechanisms are promising targets for antidepressant pharmacology. Immune cell abnormalities have been reported in mood disorders showing a partial T cell defect. Following this line of reasoning we defined an antidepressant potentiation treatment with add-on low-dose interleukin 2 (IL-2). IL-2 is a T-cell growth factor which has proven anti-inflammatory efficacy in autoimmune conditions, increasing thymic production of naïve CD4 + T cells, and possibly correcting the partial T cell defect observed in mood disorders. We performed a single-center, randomised, double-blind, placebo-controlled phase II trial evaluating the safety, clinical efficacy and biological responses of low-dose IL-2 in depressed patients with major depressive (MDD) or bipolar disorder (BD). 36 consecutively recruited inpatients at the Mood Disorder Unit were randomised in a 2:1 ratio to receive either aldesleukin (12 MDD and 12 BD) or placebo (6 MDD and 6 BD). Active treatment significantly potentiated antidepressant response to ongoing SSRI/SNRI treatment in both diagnostic groups, and expanded the population of T regulatory, T helper 2, and percentage of Naive CD4+/CD8 + immune cells. Changes in cell frequences were rapidly induced in the first five days of treatment, and predicted the later improvement of depression severity. No serious adverse effect was observed. This is the first randomised control trial (RCT) evidence supporting the hypothesis that treatment to strengthen the T cell system could be a successful way to correct the immuno-inflammatory abnormalities associated with mood disorders, and potentiate antidepressant response.

A machine learning approach on whole blood immunomarkers to identify an inflammation-associated psychosis onset subgroup.

Psychosis onset is a transdiagnostic event that leads to a range of psychiatric disorders, which are currently diagnosed through clinical observation. The integration of multimodal biological data could reveal different subtypes of psychosis onset to target for the personalization of care. In this study, we tested the existence of subgroups of patients affected by first-episode psychosis (FEP) with a possible immunopathogenic basis. To do this, we designed a data-driven unsupervised machine learning model to cluster a sample of 127 FEP patients and 117 healthy controls (HC), based on the peripheral blood expression levels of 12 psychosis-related immune gene transcripts. To validate the model, we applied a resampling strategy based on the half-splitting of the total sample with random allocation of the cases. Further, we performed a post-hoc univariate analysis to verify the clinical, cognitive, and structural brain correlates of the subgroups identified. The model identified and validated two distinct clusters: 1) a FEP cluster characterized by the high expression of inflammatory and immune-activating genes (IL1B, CCR7, IL12A and CXCR3); 2) a cluster consisting of an equal number of FEP and HC subjects, which did not show a relative over or under expression of any immune marker (balanced subgroup). None of the subgroups was related to specific symptoms dimensions or longitudinal diagnosis of affective vs non-affective psychosis. FEP patients included in the balanced immune subgroup showed a thinning of the left supramarginal and superiorfrontal cortex (FDR-adjusted p-values < 0.05). Our results demonstrated the existence of a FEP patients' subgroup identified by a multivariate pattern of immunomarkers involved in inflammatory activation. This evidence may pave the way to sample stratification in clinical studies aiming to develop diagnostic tools and therapies targeting specific immunopathogenic pathways of psychosis.

Inflammatory Markers Predict Blood Neurofilament Light Chain Levels in Acute COVID-19 Patients.

Acute coronavirus disease 2019 (COVID-19) is paralleled by a rise in the peripheral levels of neurofilament light chain (NfL), suggesting early nervous system damage. In a cohort of 103 COVID-19 patients, we studied the relationship between the NfL and peripheral inflammatory markers. We found that the NfL levels are significantly predicted by a panel of circulating cytokines/chemokines, including CRP, IL-4, IL-8, IL-9, Eotaxin, and MIP-1ß, which are highly up-regulated during COVID-19 and are associated with clinical outcomes. Our findings show that peripheral cytokines influence the plasma levels of the NfL, suggesting a potential role of the NfL as a marker of neuronal damage associated with COVID-19 inflammation.

Interaction between miR-142-3p and BDNF Val/Met Polymorphism Regulates Multiple Sclerosis Severity.

MiR-142-3p has recently emerged as key factor in tailoring personalized treatments for multiple sclerosis (MS), a chronic autoimmune demyelinating disease of the central nervous system (CNS) with heterogeneous pathophysiology and an unpredictable course. With its involvement in a detrimental regulatory axis with interleukin-1beta (IL1ß), miR-142-3p orchestrates excitotoxic synaptic alterations that significantly impact both MS progression and therapeutic outcomes. In this study, we investigated for the first time the influence of individual genetic variability on the miR-142-3p excitotoxic effect in MS. We specifically focused on the single-nucleotide polymorphism Val66Met (rs6265) of the brain-derived neurotrophic factor (BDNF) gene, known for its crucial role in CNS functioning. We assessed the levels of miR-142-3p and IL1ß in cerebrospinal fluid (CSF) obtained from a cohort of 114 patients with MS upon diagnosis. By stratifying patients according to their genetic background, statistical correlations with clinical parameters were performed. Notably, in Met-carrier patients, we observed a decoupling of miR-142-3p levels from IL1ß levels in the CSF, as well as from of disease severity (Expanded Disability Status Score, EDSS; Multiple Sclerosis Severity Score, MSSS; Age-Related Multiple Sclerosis Severity Score, ARMSS) and progression (Progression Index, PI). Our discovery of the interference between BDNF Val66Met polymorphism and the synaptotoxic IL1ß-miR-142-3p axis, therefore hampering miR-142-3p action on MS course, provides valuable insights for further development of personalized medicine in the field.

An IL-5 Single-Nucleotide Polymorphism Influences Neuroinflammation and Prospective Disease Activity in Multiple Sclerosis.

(1) Multiple sclerosis (MS) is identified by a complex interaction between central inflammation and neurodegeneration. Genetic individual variability could play a significative role in clinical presentation. The interleukin-5 (IL-5) rs2069812 single-nucleotide polymorphism (SNP) seems to define the clinical course of Th2 autoimmune diseases, while its role in MS has never been investigated. (2) In a group of 230 patients diagnosed with relapsing-remitting MS (RR-MS) or progressive MS (P-MS) and controls (IC), rs2069812 polymorphism, cerebrospinal fluid (CSF) levels of inflammatory mediators, and clinical and demographic characteristics were determined. In RR-MS patients, No Evidence of Disease Activity (NEDA-3) at three years of follow-up was detected. (3) We identified higher levels of proinflammatory cytokines, particularly IL-2 (median [IQR], RR-MS = 0.2 [0-0.7]; P-MS = 0.1 [0-1.6]; IC = 0.1 [0.0-0.1]; p < 0.005), IL-6 (RR-MS = 0.9 [0.3-2.3]; P-MS = 0.8 [0.1-2.7]; IC = 0.1 [0.0-0.5]; p < 0.005), IL-12 (RR-MS = 0.5 [0-1.1]; P-MS = 0.5 [0-1.1]; IC = 0.0 [0.0-0.3]; p < 0.005), and GM-CSF (RR-MS = 15.6 [4.8-26.4]; P-MS = 14 [3.3-29.7]; IC = 8.9 [4.7-11.7]; p < 0.005) in MS patients compared with IC. Conversely, anti-inflammatory cytokines, specifically IL-5 (RR-MS = 0.65 [0-2.4]; P-MS = 0.1 [0-0.8]; IC = 1.7 [0.6-2.8]; p < 0.005) and IL-1ra (RR-MS = 14.7 [4.9-26.4]; P-MS = 13.1 [4.7-22.2]; IC = 27.8 [17.7-37.6]; p < 0.005) were higher in controls. According to rs2069812, in MS patients, the T-allele was associated with higher concentrations of proinflammatory mediators (IL-2, CT/TT = 0.2 [0.0-2.0]; CC = 0.1 [0.0-0.4], p = 0.015; IL-6, CT/TT = 1.2 [0.4-3.2] vs. CC = 0.7 [0.1-1.7], p = 0.007; IL-15, CT/TT = 0.1 [0.0-9.5] vs. CC = 0.0 [0.0-0.1], p = 0.019; and GM-CSF, CT/TT = 0.1 [0.0-0.6] vs. CC = 0.05 [0.0-0.1], p < 0.001), and CC was associated with anti-inflammatory mediators (IL-5, CT/TT = 0.03 [0.0-1.9] vs. CC = 1.28 [0.0-2.7], p = 0.001; IL-1ra, CT/TT = 12.1 [4.1-25.9] vs. CC = 18.1 [12.1-26.9], p = 0.006). We found the same differences in RR-MS patients (IL-2, T-allele median [IQR] = 0.3 [0.0-2.0] vs. C-allele, median [IQR] = 0.04 [0.0-0.3]; p = 0.005; IL-6, T-allele, median [IQR] = 1.3 [0.4-3.3] vs. C-allele, median [IQR] = 0.6 [0.03-1.5]; p = 0.001; IL-15, T-allele, median [IQR] = 0.1 [0.0-9.5] vs. C-allele, median [IQR] = 0.0 [0.0-0.1]; p = 0.008; GM-CSF, T-allele, median [IQR] = 0.1 [0.0-97.9] vs. C-allele, median [IQR] = 0.0 [0.0-0.001]; p < 0.001; IL-5, T-allele, median [IQR] = 0.02 [0.0-2.2] vs. C-allele, median [IQR] = 1.5 [0.0-2.9]; p = 0.016; and IL-1ra, T-allele, median [IQR] = 12.1 [4.3-26.4] vs. C-allele, median [IQR] = 18.5 [12.7-28.3]; p = 0.006) but not in P-MS, except for IL-5 (T-allele, median [IQR] = 0.1 [0-0.23] vs. C-allele, median [IQR] = 0.6 [0.0-2.5]; p = 0.022). Finally, we identified an association between CC in RR-MS patients and NEDA-3 after three years of follow-up (p = 0.007). (4) We describe, for the first time, the role of an SNP of the IL-5 gene in regulating central neuroinflammation and influencing clinical course in MS patients.

Polarized cells display asymmetric release of extracellular vesicles.

Extracellular vesicles (EVs), a broad term for the lipid microparticles known as microvesicles and exosomes, are discharged by cells into their surrounding space. Microvesicles are discharged upon outward plasma membrane budding, while exosomes are secreted after multivesicular body (MVB) fusion with the plasma membrane. The majority of information regarding EV biology comes from studies performed in non-polarized cells. Here we characterize EV release in polarized cells. We found a substantial asymmetry in the number and composition of EVs produced and released from the apical membrane of epithelial cells as compared to the basolateral membrane. We showed that the quantitative difference is related to the polarized distribution of two phosphoinositide species between the two cell surfaces and that the peculiar biochemical composition of resultant EVs reflects their site of origin. In particular, apical and basolateral exosomes may derive from distinct classes of MVBs originating from and fusing with the same plasma membrane. We identify VAMP8/Endobrevin as a regulator of the basolateral release of exosomes, whereas the mechanism responsible for apical EV release requires further study.

Cerebrospinal fluid, brain, and spinal cord levels of L-aspartate signal excitatory neurotransmission abnormalities in multiple sclerosis patients and experimental autoimmune encephalomyelitis mouse model.

The neuroinflammatory process characterizing multiple sclerosis (MS) is associated with changes in excitatory synaptic transmission and altered central concentrations of the primary excitatory amino acid, L-glutamate (L-Glu). Recent findings report that cerebrospinal fluid (CSF) levels of L-Glu positively correlate with pro-inflammatory cytokines in MS patients. However, to date, there is no evidence about the relationship between the other primary excitatory amino acid, L-aspartate (L-Asp), its derivative D-enantiomer, D-aspartate, and the levels of pro-inflammatory and anti-inflammatory cytokines in the CSF of MS. In the present study, we measured by HPLC the levels of these amino acids in the cortex, hippocampus, cerebellum, and spinal cord of mice affected by experimental autoimmune encephalomyelitis (EAE). Interestingly, in support of glutamatergic neurotransmission abnormalities in neuroinflammatory conditions, we showed reduced L-Asp levels in the cortex and spinal cord of EAE mice and increased D-aspartate/total aspartate ratio within the cerebellum and spinal cord of these animals. Additionally, we found significantly decreased CSF levels of L-Asp in both relapsing-remitting (n = 157) MS (RR-MS) and secondary progressive/primary progressive (n = 22) (SP/PP-MS) patients, compared to control subjects with other neurological diseases (n = 40). Importantly, in RR-MS patients, L-Asp levels were correlated with the CSF concentrations of the inflammatory biomarkers G-CSF, IL-1ra, MIP-1ß, and Eotaxin, indicating that the central content of this excitatory amino acid, as previously reported for L-Glu, reflects a neuroinflammatory environment in MS. In keeping with this, we revealed that CSF L-Asp levels were positively correlated with those of L-Glu, highlighting the convergent variation of these two excitatory amino acids under inflammatory synaptopathy occurring in MS.

iAstrocytes do not restrain T cell proliferation in vitro.

The cross-talk between T cells and astrocytes occurring under physiological and, even more, neuroinflammatory conditions may profoundly impact the generation of adaptive immune responses in the nervous tissue. In this study, we used a standardized in vitro co-culture assay to investigate the immunomodulatory properties of astrocytes differing for age, sex, and species. Mouse neonatal astrocytes enhanced T cell vitality but suppressed T lymphocyte proliferation in response to mitogenic stimuli or myelin antigens, regardless of the Th1, Th2 or Th17 T cell phenotype. Studies comparing glia cells from adult and neonatal animals showed that adult astrocytes were more efficient in inhibiting T lymphocyte activation than neonatal astrocytes, regardless of their sex. Differently from primary cultures, mouse and human astrocytes derived from reprogrammed fibroblasts did not interfere with T cell proliferation. Overall, we describe a standardized astrocyte-T cell interaction in vitro assay and demonstrate that primary astrocytes and iAstrocytes may differ in modulating T cell function.

Genetic regulation of IL-8 influences disease presentation of multiple sclerosis.

BACKGROUND: Individual genetic variability may influence the course of multiple sclerosis (MS). The interleukin (IL)-8C>T rs2227306 single nucleotide polymorphism (SNP) regulates IL-8 activity in other clinical conditions; however, its role in MS has never been investigated. OBJECTIVES: To explore the association between IL-8 SNP rs2227306, cerebrospinal fluid (CSF) IL-8 concentrations, clinical, and radiological characteristics in a group of newly diagnosed MS patients. METHODS: In 141 relapsing-remitting (RR)-MS patients, rs2227306 polymorphism, CSF levels of IL-8, clinical, and demographical characteristics were determined. In 50 patients, structural magnetic resonance imaging (MRI) measures were also assessed. RESULTS: An association between CSF IL-8 and Expanded Disability Status Scale (EDSS) at diagnosis was found in our set of patients (r = 0.207, p = 0.014). CSF IL-8 concentrations were significantly higher in patients carrying the T variant of rs2227306 (p = 0.004). In the same group, a positive correlation emerged between IL-8 and EDSS (r = 0.273, p = 0.019). Finally, a negative correlation between CSF levels of IL-8 and cortical thickness emerged in rs2227306T carriers (r = -0.498, p = 0.005). CONCLUSION: We describe for the first time a role of SNP rs2227306 of IL-8 gene in regulating the expression and the activity of this inflammatory cytokine in MS.

Distinct intrathecal inflammatory signatures following relapse and anti-COVID-19 mRNA vaccination in multiple sclerosis.

BACKGROUND: The role of vaccine-mediated inflammation in exacerbating multiple sclerosis (MS) is a matter of debate. OBJECTIVE: In this cross-sectional study, we compared the cerebrospinal fluid (CSF) inflammation associated with MS relapses or anti-COVID-19 mRNA vaccinations in relapsing-remitting multiple sclerosis (RRMS). METHODS: We dosed CSF cytokines in 97 unvaccinated RRMS patients with clinical relapse within the last 100 days. In addition, we enrolled 29 stable RRMS and 24 control patients receiving COVID-19 vaccine within the last 100 days. RESULTS: In RRMS patients, a negative association was found between relapse distance and the CSF concentrations of the pro-inflammatory cytokines interleukin (IL)-2 (beta = -0.265, p = 0.016), IL-6 (beta = -0.284, p = 0.01), and IL-17 (beta = -0.224, p = 0.044). Conversely, vaccine distance positively correlated with a different set of cytokines including IL-12 (beta = 0.576, p = 0.002), IL-13 (beta = 0.432, p = 0.027), and IL-1ra (beta = 0.387, p = 0.05). These associations were significant also considering other clinical characteristics. No significant associations emerged between vaccine distance and CSF molecules in the control group. CONCLUSION: Vaccine for COVID-19 induces a central inflammatory response in RRMS patients that is qualitatively different from that associated with disease relapse.

Preventive exercise attenuates IL-2-driven mood disorders in multiple sclerosis.

BACKGROUND: Elevated levels of specific proinflammatory molecules in the cerebrospinal fluid (CSF) have been associated with disability progression, enhanced neurodegeneration and higher incidence of mood disorders in people with multiple sclerosis (MS). Studies in animal models of MS suggest that preventive exercise may play an immunomodulatory activity, with beneficial effects on both motor deficits and behavioral alterations. Here we explored the impact of lifestyle physical activity on clinical presentation and associated central inflammation in a large group of newly diagnosed patients with MS. Furthermore, we addressed the causal link between exercise-mediated immunomodulation and mood symptoms in the animal setting. METHODS: A cross-sectional study was conducted on 235 relapsing-remitting MS patients at the time of the diagnosis. Patients were divided into 3 groups ("sedentary", "lifestyle physical activity" and "exercise") according to the level of physical activity in the six months preceding the evaluation. Patients underwent clinical, neuropsychological and psychiatric evaluation, magnetic resonance imaging and lumbar puncture for diagnostic purposes. The CSF levels of proinflammatory and anti-inflammatory cytokines were analyzed and compared with a group of 80 individuals with non-inflammatory and non-degenerative diseases. Behavioral and electrophysiological studies were carried out in control mice receiving intracerebral injection of IL-2 or vehicle. Behavior was also assessed in mice with experimental autoimmune encephalomyelitis (EAE), animal model of MS, reared in standard (sedentary group) or running wheel-equipped (exercise group) cages. RESULTS: In exercising MS patients, depression and anxiety were reduced compared to sedentary patients. The CSF levels of the interleukin-2 and 6 (IL-2, IL-6) were increased in MS patients compared with control individuals. In MS subjects exercise was associated with normalized CSF levels of IL-2. In EAE mice exercise started before disease onset reduced both behavioral alterations and striatal IL-2 expression. Notably, a causal role of IL-2 in mood disorders was shown. IL-2 administration in control healthy mice induced anxious- and depressive-like behaviors and impaired type-1 cannabinoid (CB1) receptor-mediated neurotransmission at GABAergic synapses, mimicking EAE-induced synaptic dysfunction. CONCLUSIONS: Our results indicate an immunomodulatory effect of exercise in MS patients, associated with reduced CSF expression of IL-2, which might result in reduced mood disorders. These data suggest that exercise in the early stages may act as a disease-modifying therapy in MS although further longitudinal studies are needed to clarify this issue.

MiR-142-3p regulates synaptopathy-driven disease progression in multiple sclerosis.

AIM: We recently proposed miR-142-3p as a molecular player in inflammatory synaptopathy, a new pathogenic hallmark of multiple sclerosis (MS) and of its mouse model experimental autoimmune encephalomyelitis (EAE), that leads to neuronal loss independently of demyelination. MiR-142-3p seems to be unique among potential biomarker candidates in MS, since it is an inflammatory miRNA playing a dual role in the immune and central nervous systems. Here, we aimed to verify the impact of miR-142-3p circulating in the cerebrospinal fluid (CSF) of MS patients on clinical parameters, neuronal excitability and its potential interaction with disease modifying therapies (DMTs). METHODS AND RESULTS: In a cohort of 151 MS patients, we found positive correlations between CSF miR-142-3p levels and clinical progression, IL-1ß signalling as well as synaptic excitability measured by transcranial magnetic stimulation. Furthermore, therapy response of patients with 'low miR-142-3p' to dimethyl fumarate (DMF), an established disease-modifying treatment (DMT), was superior to that of patients with 'high miR-142-3p' levels. Accordingly, the EAE clinical course of heterozygous miR-142 mice was ameliorated by peripheral DMF treatment with a greater impact relative to their wild type littermates. In addition, a central protective effect of this drug was observed following intracerebroventricular and ex vivo acute treatments of EAE wild type mice, showing a rescue of miR-142-3p-dependent glutamatergic alterations. By means of electrophysiology, molecular and biochemical analysis, we suggest miR-142-3p as a molecular target of DMF. CONCLUSION: MiR-142-3p is a novel and potential negative prognostic CSF marker of MS and a promising tool for identifying personalised therapies.

Spinal Fluid Myeloid Microvesicles Predict Disease Course in Multiple Sclerosis.

OBJECTIVE: In vivo measures of myeloid activity are promising biomarkers in multiple sclerosis. We previously demonstrated that cerebrospinal fluid (CSF) myeloid microvesicles are markers of microglial/macrophage activity and neuroinflammation in multiple sclerosis. Here, we aimed at investigating the diagnostic and prognostic value of myeloid microvesicles in a clinical setting. METHODS: Six hundred one patients discharged with a diagnosis of neuroinflammatory, neurodegenerative, or no neurological disease were enrolled. Myeloid microvesicles were measured with flow cytometry as isolectin B4-positive events in fresh CSF. Clinical, demographical, and magnetic resonance imaging (MRI) data were collected at diagnosis (all patients) and during follow-up (n = 176). RESULTS: CSF myeloid microvesicles were elevated in neuroinflammatory patients compared to the neurodegenerative and control groups. In multiple sclerosis, microvesicles were higher in patients with MRI disease activity and their concentration increased along with the number of enhancing lesions (p < 0.0001, Jonckheere-Terpstra test). CSF myeloid microvesicles were also higher in patients with higher disease activity in the month and year preceding diagnosis. Microvesicles excellently discriminated between the relapsing-remitting and control groups (receiver operator characteristic curve, area under the curve = 0.939, p < 0.0001) and between radiologically isolated syndrome and unspecific brain lesions (0.942, p < 0.0001). Furthermore, microvesicles were independent predictors of prognosis for both the relapsing-remitting and progressive groups. Microvesicles independently predicted future disease activity in relapsing-remitting patients (hazard ratio [HR] = 1.967, 95% confidence interval [CI] = 1.147-3.372), correcting for prognostic factors of standard clinical use. In the progressive group, microvesicles were independent predictors of disability accrual (HR = 10.767, 95% CI = 1.335-86.812). INTERPRETATION: Our results confirm that CSF myeloid microvesicles are a clinically meaningful biomarker of neuroinflammation and microglial/macrophage activity in vivo. These findings may support a possible use in clinical practice during diagnostic workup and prognostic assessment. ANN NEUROL 2021;90:253-265.

Anti-SARS-CoV-2 T-stem cell memory persists in ocrelizumab-treated MS patients.

BACKGROUND: Development of long-lasting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) T-cell responses in persons with multiple sclerosis (pwMS) treated with ocrelizumab is questioned. OBJECTIVE: Investigate antiviral T-cell responses after infection with SARS-CoV-2 in ocrelizumab-treated pwMS. Control groups included ocrelizumab-treated pwMS without SARS-CoV-2 infection, and non-MS individuals with and without SARS-CoV-2 infection. METHODS: Peripheral blood mononuclear cells were stimulated with SARS-CoV-2 peptide pools and T-cell reactivity was assessed by ELISPOT for interferon (IFN)-γ detection, and by multiparametric fluorescence-activated cell sorting (FACS) analyses for assessment and characterization of T-cell activation. RESULTS: ELISPOT assay against the spike and the N protein of SARS-CoV-2 displayed specific T-cell reactivity in 28/29 (96%) pwMS treated with ocrelizumab and infected by SARS-CoV-2, similar to infected persons without MS. This reactivity was present 1 year after infection and independent from the time of ocrelizumab infusion. FACS analysis following stimulation with SARS-CoV-2 peptide pools showed the presence of activation-induced markers (AIMs) in both CD4+ and CD8+ T-cell subsets in 96% and 92% of these individuals, respectively. Within naïve AIM+ CD4+ and CD8+ T-cells, we detected T memory stem cells, suggesting the acquisition of long-term memory. CONCLUSIONS: B-cell depletion using ocrelizumab does not impair the development of long-lasting anti-SARS-CoV-2 T-cell responses.

Subclinical anterior optic pathway involvement in early multiple sclerosis and clinically isolated syndromes.

Optical coherence tomography (OCT) is gaining increasing relevance in the assessment of patients with multiple sclerosis. Converging evidence point to the view that neuro-retinal changes, in eyes without acute optic neuritis, reflect inflammatory and neurodegenerative processes taking place throughout the CNS. The present study aims at exploring the usefulness of OCT as a marker of inflammation and disease burden in the earliest phases of the disease. Thus, a cohort of 150 consecutive patients underwent clinical, neurophysiological and brain MRI assessment as well as lumbar puncture as part of their diagnostic workup for a neurological episode suggestive of inflammatory CNS disorder; among those 32 patients had another previous misdiagnosed episode. For the present study, patients also received a visual pathway assessment (OCT, visual evoked potentials, visual acuity), measurement of CSF inflammatory markers (17 cytokines-chemokines, extracellular vesicles of myeloid origin), and dosage of plasma neurofilaments. Subclinical optic nerve involvement is frequently found in clinically isolated syndromes by visual evoked potentials (19.2%). OCT reveals ganglion cell layer asymmetries in 6.8% of patients; retinal fibre layer asymmetries, despite being more frequent (17.8%), display poor specificity. The presence of subclinical involvement is associated with a greater disease burden. Second, ganglion cell layer thinning reflects the severity of disease involvement even beyond the anterior optic pathway. In fact, the ganglion cell layer in eyes without evidence of subclinical optic involvement is correlated with Expanded Disability Status Scale, low contrast visual acuity, disease duration, brain lesion load, presence of gadolinium enhancing lesions, abnormalities along motor and somatosensory evoked potentials, and frequency of CSF-specific oligoclonal bands. Third, the inner nuclear layer thickens in a post-acute (1.1-3.7 months) phase after a relapse, and this phenomenon is counteracted by steroid treatment. Likewise, a longitudinal analysis on 65 patients shows that this swelling is transient and returns to normal values after 1 year follow-up. Notwithstanding, the clinical, MRI, serological and CSF markers of disease activity considered in the study are strictly associated with one another, but none of them are associated with the inner nuclear layer. Our findings challenge the current hypothesis that the inner nuclear layer is an acute phase marker of inflammatory activity. The present study suggests that instrumental evidence of subclinical optic nerve involvement is associated with a greater disease burden in clinically isolated syndrome. Neuro-retinal changes are present since the earliest phases of the disease and yield important information regarding the neurodegenerative and inflammatory processes occurring in the CNS.

Cerebrospinal fluid levels of L-glutamate signal central inflammatory neurodegeneration in multiple sclerosis.

Excessive extracellular concentrations of L-glutamate (L-Glu) can be neurotoxic and contribute to neurodegenerative processes in multiple sclerosis (MS). The association between cerebrospinal fluid (CSF) L-Glu levels, clinical features, and inflammatory biomarkers in patients with MS remains unclear. In 179 MS patients (relapsing remitting, RR, N = 157; secondary progressive/primary progressive, SP/PP, N = 22), CSF levels of L-Glu at diagnosis were determined and compared with those obtained in a group of 40 patients with non-inflammatory/non-degenerative disorders. Disability at the time of diagnosis, and after 1 year follow-up, was assessed using the Expanded Disability Status Scale (EDSS). CSF concentrations of lactate and of a large set of pro-inflammatory and anti-inflammatory molecules were explored. CSF levels of L-Glu were slightly reduced in MS patients compared to controls. In RR-MS patients, L-Glu levels correlated with EDSS after 1 year follow-up. Moreover, in MS patients, significant correlations were found between L-Glu and both CSF levels of lactate and the inflammatory molecules interleukin (IL)-2, IL-6, and IL-1 receptor antagonist. Altered expression of L-Glu is associated with disability progression, oxidative stress, and inflammation. These findings identify CSF L-Glu as a candidate neurochemical marker of inflammatory neurodegeneration in MS.

MYD88 L265P mutation and interleukin-10 detection in cerebrospinal fluid are highly specific discriminating markers in patients with primary central nervous system lymphoma: results from a prospective study.

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut-MYD88) and interleukin-10 (IL-10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra-CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut-MYD88 was assessed by TaqMan-based polymerase chain reaction. IL-6 and IL-10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme-linked immunosorbent assay. Mut-MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue-CSF samples. IL-10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL-6 transcripts was negligible. In CSF samples, mut-MYD88 and high IL-10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL-6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL-10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut-MYD88 and IL-10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

CSF extracellular vesicles and risk of disease activity after a first demyelinating event.

BACKGROUND: Extracellular vesicles (EVs), a recently described mechanism of cell communication, are released from activated microglial cells and macrophages and are a candidate biomarker in diseases characterized by chronic inflammatory process such as multiple sclerosis (MS). METHODS: We explored cerebrospinal fluid extracellular vesicle (CSF EV) of myeloid origin (MEVs), cytokine and chemokine levels in patients with clinically isolated syndrome (CIS). RESULTS: We found that CSF MEVs were significantly higher in CIS patients than in controls and were inversely correlated to CSF CCL2 levels. MEVs level were significantly associated with an shorter time to evidence of disease activity (hazard ratio: 1.01, 95% confidence interval: 1.00-1.02, p < 0.01) independently from other known prognostic markers. CONCLUSION: After a first demyelinating event, CSF EVs may improve risk stratification of these patients and allow more targeted intervention strategies.

Serum neurofilaments increase at progressive multifocal leukoencephalopathy onset in natalizumab-treated multiple sclerosis patients.

This study analyzed serum neurofilament light chains (NfL) in 2 European cohorts of 312 multiple sclerosis (MS) patients to investigate whether NfL are biomarkers of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. The cohort comprised 25 PML, 136 natalizumab-treated, and 151 untreated MS patients. Patients subsequently developing PML had similar NfL to other natalizumab-treated MS patients. At PML onset, NfL were 10-fold higher than in the pre-PML condition and in natalizumab-treated or untreated MS patients, and NfL continued to increase until onset of immune reconstitution inflammatory syndrome. The results suggest that in natalizumab-treated patients, NfL may represent an early and accessible marker of PML. Ann Neurol 2019;85:606-610.

Obesity worsens central inflammation and disability in multiple sclerosis.

BACKGROUND: Previous studies evidenced a link between metabolic dysregulation, inflammation, and neurodegeneration in multiple sclerosis (MS). OBJECTIVES: To explore whether increased adipocyte mass expressed as body mass index (BMI) and increased serum lipids influence cerebrospinal fluid (CSF) inflammation and disease severity. METHODS: In this cross-sectional study, 140 consecutive relapsing-remitting (RR)-MS patients underwent clinical assessment, BMI evaluation, magnetic resonance imaging scan, and blood and CSF collection before any specific drug treatment. The CSF levels of the following cytokines, adipocytokines, and inflammatory factors were measured: interleukin (IL)-6, IL-13, granulocyte macrophage colony-stimulating factor, leptin, ghrelin, osteoprotegerin, osteopontin, plasminogen activator inhibitor-1, resistin, and Annexin A1. Serum levels of triglycerides, total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were assessed. RESULTS: A positive correlation emerged between BMI and Expanded Disability Status Scale score. Obese RR-MS patients showed higher clinical disability, increased CSF levels of the proinflammatory molecules IL-6 and leptin, and reduced concentrations of the anti-inflammatory cytokine IL-13. Moreover, both the serum levels of triglycerides and TC/HDL-C ratio showed a positive correlation with IL-6 CSF concentrations. CONCLUSION: Obesity and altered lipid profile are associated with exacerbated central inflammation and higher clinical disability in RR-MS at the time of diagnosis. Increased adipocytokines and lipids can mediate the negative impact of high adiposity on RR-MS course.