Chemotherapy-induced cognitive impairment and its long-term development in patients with breast cancer: results from the observational CICARO study.

BACKGROUND: Chemotherapy-induced cognitive impairment (CICI) is a well-recognized side effect of breast cancer treatment. However, prospective long-term evaluations of CICI using standardized neuropsychological tests are scarce. PATIENTS AND METHODS: This prospective longitudinal cohort study investigated cognitive dysfunction and its impact on quality of life and everyday functioning in patients with breast cancer receiving first-line chemotherapy compared to patients with breast cancer without chemotherapy. Assessment occurred prior to chemotherapy, postchemotherapy (median 6 months), and 2-3 years later. We used standardized neuropsychological tests, questionnaires, and scales to assess patients' quality of life and functioning. Additionally, serum analysis for neurodegenerative markers and autoantibodies was conducted. RESULTS: We included n = 53 patients. Overall cognitive function declined statistically significantly (P = .046) postchemotherapy compared to control patients, mostly driven by a reduced figural memory (P = .011). Patients who received chemotherapy showed a greater reduction in quality of life (increased fatigue symptoms, P = .023; reduced Karnofsky index, P < .001); however, without a statistically significant effect on cognitive decline. The neurodegenerative markers Neurofilament light chain (NfL) and phosphorylated Neurofilament heavy chain (pNfH) increased statistically significantly (P < .001) postchemotherapy and pNfH correlated with overall cognitive function. After 2-3 years, both cognitive performance and quality of life were comparable between chemotherapy-treated and control patients. CONCLUSION: Our findings suggest that chemotherapy statistically significantly contributes to overall cognitive dysfunction in patients with breast cancer, which disappears after 2-3 years, indicating a recovery in both objectively measurable cognitive function and subjective quality of life. Future research should examine larger sample sizes and explore screening indicators, particularly pNfH.

Comprehensive analysis of the cerebrospinal fluid and serum metabolome in neurological diseases.

BACKGROUND: Comprehensive characterization of the metabolome in cerebrospinal fluid (CSF) and serum by Nuclear Magnetic Resonance (NMR) spectroscopy may identify biomarkers and contribute to the understanding of the pathophysiology of neurological diseases. METHODS: Metabolites were determined by NMR spectroscopy in stored CSF/serum samples of 20 patients with Parkinson's disease, 25 patients with other neuro-degenerative diseases, 22 patients with cerebral ischemia, 48 patients with multiple sclerosis, and 58 control patients with normal CSF findings. The data set was analysed using descriptive and multivariate statistics, as well as machine learning models. RESULTS: CSF glucose and lactic acid measured by NMR spectroscopy and routine clinical chemistry showed a strong correlation between both methods (glucose, R2 = 0.87, n = 173; lactic acid, R2 = 0.74, n = 173). NMR spectroscopy detected a total of 99 metabolites; 51 in both, CSF and serum, 16 in CSF only, and 32 in serum only. CSF concentrations of some metabolites increased with age and/or decreasing blood-brain-barrier function. Metabolite detection rates were overall similar among the different disease groups. However, in two-group comparisons, absolute metabolite levels in CSF and serum discriminated between multiple sclerosis and neurodegenerative diseases (area under the curve (AUC) = 0.96), multiple sclerosis and Parkinson's disease (AUC = 0.89), and Parkinson's disease and control patients (AUC = 0.91), as demonstrated by random forest statistical models. Orthogonal partial least square discriminant analysis using absolute metabolite levels in CSF and serum furthermore permitted separation of Parkinson's disease and neurodegenerative diseases. CSF propionic acid levels were about fourfold lower in Parkinson's disease as compared to neurodegenerative diseases. CONCLUSIONS: These findings outline the landscape of the CSF and serum metabolome in different categories of neurological diseases and identify age and blood-brain-barrier function as relevant co-factors for CSF levels of certain metabolites. Metabolome profiles as determined by NMR spectroscopy may potentially aid in differentiating groups of patients with different neurological diseases, including clinically meaningful differentiations, such as Parkinson's disease from other neurodegenerative diseases.

Ferroptosis inhibitor improves outcome after early and delayed treatment in mild spinal cord injury.

We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe2+) to ferric iron (Fe3+) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.

Targeting Transcutaneous Spinal Cord Stimulation Using a Supervised Machine Learning Approach Based on Mechanomyography.

Transcutaneous spinal cord stimulation (tSCS) provides a promising therapy option for individuals with injured spinal cords and multiple sclerosis patients with spasticity and gait deficits. Before the therapy, the examiner determines a suitable electrode position and stimulation current for a controlled application. For that, amplitude characteristics of posterior root muscle (PRM) responses in the electromyography (EMG) of the legs to double pulses are examined. This laborious procedure holds potential for simplification due to time-consuming skin preparation, sensor placement, and required expert knowledge. Here, we investigate mechanomyography (MMG) that employs accelerometers instead of EMGs to assess muscle activity. A supervised machine-learning classification approach was implemented to classify the acceleration data into no activity and muscular/reflex responses, considering the EMG responses as ground truth. The acceleration-based calibration procedure achieved a mean accuracy of up to 87% relative to the classical EMG approach as ground truth on a combined cohort of 11 healthy subjects and 11 patients. Based on this classification, the identified current amplitude for the tSCS therapy was in 85%, comparable to the EMG-based ground truth. In healthy subjects, where both therapy current and position have been identified, 91% of the outcome matched well with the EMG approach. We conclude that MMG has the potential to make the tuning of tSCS feasible in clinical practice and even in home use.

1,2-13C2-Glucose Tracing Approach to Assess Metabolic Alterations of Human Monocytes under Neuroinflammatory Conditions.

Neuroinflammation is one of the common features in most neurological diseases including multiple sclerosis (MScl) and neurodegenerative diseases such as Alzheimer's disease (AD). It is associated with local brain inflammation, microglial activation, and infiltration of peripheral immune cells into cerebrospinal fluid (CSF) and the central nervous system (CNS). It has been shown that the diversity of phenotypic changes in monocytes in CSF relates to neuroinflammation. It remains to be investigated whether these phenotypic changes are associated with functional or metabolic alteration, which may give a hint to their function or changes in cell states, e.g., cell activation. In this article, we investigate whether major metabolic pathways of blood monocytes alter after exposure to CSF of healthy individuals or patients with AD or MScl. Our findings show a significant alteration of the metabolism of monocytes treated with CSF from patients and healthy donors, including higher production of citric acid and glutamine, suggesting a more active glycolysis and tricarboxylic acid (TCA) cycle and reduced production of glycine and serine. These alterations suggest metabolic reprogramming of monocytes, possibly related to the change of compartment (from blood to CSF) and/or disease-related. Moreover, the levels of serine differ between AD and MScl, suggesting different phenotypic alterations between diseases.

Septin-3 autoimmunity in patients with paraneoplastic cerebellar ataxia.

BACKGROUND: Septins are cytoskeletal proteins with filament forming capabilities, which have multiple roles during cell division, cellular polarization, morphogenesis, and membrane trafficking. Autoantibodies against septin-5 are associated with non-paraneoplastic cerebellar ataxia, and autoantibodies against septin-7 with encephalopathy with prominent neuropsychiatric features. Here, we report on newly identified autoantibodies against septin-3 in patients with paraneoplastic cerebellar ataxia. We also propose a strategy for anti-septin autoantibody determination. METHODS: Sera from three patients producing similar immunofluorescence staining patterns on cerebellar and hippocampal sections were subjected to immunoprecipitation followed by mass spectrometry. The identified candidate antigens, all of which were septins, were expressed recombinantly in HEK293 cells either individually, as complexes, or combinations missing individual septins, for use in recombinant cell-based indirect immunofluorescence assays (RC-IIFA). Specificity for septin-3 was further confirmed by tissue IIFA neutralization experiments. Finally, tumor tissue sections were analyzed immunohistochemically for septin-3 expression. RESULTS: Immunoprecipitation with rat cerebellum lysate revealed septin-3, -5, -6, -7, and -11 as candidate target antigens. Sera of all three patients reacted with recombinant cells co-expressing septin-3/5/6/7/11, while none of 149 healthy control sera was similarly reactive. In RC-IIFAs the patient sera recognized only cells expressing septin-3, individually and in complexes. Incubation of patient sera with five different septin combinations, each missing one of the five septins, confirmed the autoantibodies' specificity for septin-3. The tissue IIFA reactivity of patient serum was abolished by pre-incubation with HEK293 cell lysates overexpressing the septin-3/5/6/7/11 complex or septin-3 alone, but not with HEK293 cell lysates overexpressing septin-5 as control. All three patients had cancers (2 × melanoma, 1 × small cell lung cancer), presented with progressive cerebellar syndromes, and responded poorly to immunotherapy. Expression of septin-3 was demonstrated in resected tumor tissue available from one patient. CONCLUSIONS: Septin-3 is a novel autoantibody target in patients with paraneoplastic cerebellar syndromes. Based on our findings, RC-IIFA with HEK293 cells expressing the septin-3/5/6/7/11 complex may serve as a screening tool to investigate anti-septin autoantibodies in serological samples with a characteristic staining pattern on neuronal tissue sections. Autoantibodies against individual septins can then be confirmed by RC-IIFA expressing single septins.

The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation.

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

Analysing cerebrospinal fluid with explainable deep learning: From diagnostics to insights.

AIM: Analysis of cerebrospinal fluid (CSF) is essential for diagnostic workup of patients with neurological diseases and includes differential cell typing. The current gold standard is based on microscopic examination by specialised technicians and neuropathologists, which is time-consuming, labour-intensive and subjective. METHODS: We, therefore, developed an image analysis approach based on expert annotations of 123,181 digitised CSF objects from 78 patients corresponding to 15 clinically relevant categories and trained a multiclass convolutional neural network (CNN). RESULTS: The CNN classified the 15 categories with high accuracy (mean AUC 97.3%). By using explainable artificial intelligence (XAI), we demonstrate that the CNN identified meaningful cellular substructures in CSF cells recapitulating human pattern recognition. Based on the evaluation of 511 cells selected from 12 different CSF samples, we validated the CNN by comparing it with seven board-certified neuropathologists blinded for clinical information. Inter-rater agreement between the CNN and the ground truth was non-inferior (Krippendorff's alpha 0.79) compared with the agreement of seven human raters and the ground truth (mean Krippendorff's alpha 0.72, range 0.56-0.81). The CNN assigned the correct diagnostic label (inflammatory, haemorrhagic or neoplastic) in 10 out of 11 clinical samples, compared with 7-11 out of 11 by human raters. CONCLUSIONS: Our approach provides the basis to overcome current limitations in automated cell classification for routine diagnostics and demonstrates how a visual explanation framework can connect machine decision-making with cell properties and thus provide a novel versatile and quantitative method for investigating CSF manifestations of various neurological diseases.

Humoral immune responses remain quantitatively impaired but improve qualitatively in anti-CD20-treated patients with multiple sclerosis after three or four COVID-19 vaccinations.

OBJECTIVE: To analyze anti-SARS-CoV-2-S1-IgG levels, avidity, Omicron BA.2 variant neutralizing capacity, and SARS-CoV-2-specific T cells in anti-CD20-treated patients with multiple sclerosis (aCD20pwMS) after two, three, or four COVID-19 vaccinations. RESULTS: Frequencies of aCD20pwMS with detectable SARS-CoV-2-S1-IgG increased moderately between two (31/61 (51%)), three (31/57 (54%)), and four (17/26 (65%)) vaccinations. However, among patients with detectable SARS-CoV-2-S1-IgG, frequencies of high avidity (6/31 (19%) vs 11/17 (65%)) and Omicron neutralizing antibodies (0/10 (0%) vs 6/10 (60%)) increased strongly between two and four vaccinations. SARS-CoV-2-specific T cells were detectable in >92% after two or more vaccinations. CONCLUSION: Additional vaccinations qualitatively improve SARS-CoV-2 antibody responses.

Microfibrillar-associated protein 4 as a potential marker of acute relapse in inflammatory demyelinating diseases of the central nervous system: Pathological and clinical aspects.

BACKGROUND: Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein not previously described in the human central nervous system (CNS). OBJECTIVES: We determined MFAP4 CNS expression and measured cerebrospinal fluid (CSF) and serum levels. METHODS: Tissue was sampled at autopsy from patients with acute multiple sclerosis (MS) (n = 3), progressive MS (n = 3), neuromyelitis optica spectrum disorder (NMOSD) (n = 2), and controls (n = 9), including 6 healthy controls (HC). MFAP4 levels were measured in 152 patients: 49 MS, 62 NMOSD, 22 myelin oligodendrocyte glycoprotein-associated disease (MOGAD), and 19 isolated optic neuritis (ION). RESULTS: MFAP4 localized to meninges and vascular/perivascular spaces, intense in the optic nerve. At sites of active inflammation, MFAP4 reactivity was reduced in NMOSD and acute MS and less in progressive MS. CSF MFAP4 levels were reduced during relapse and at the onset of diseases (mean U/mL: MS 14.3, MOGAD 9.7, and ION 14.6 relative to HC 17.9. (p = 0.013, p = 0.000, and p = 0.019, respectively). Patients with acute ON (n = 68) had reduced CSF MFAP4 (mean U/mL: 14.5, p = 0.006). CSF MFAP4 levels correlated negatively with relapse severity (rho = -0.41, p = 0.017). CONCLUSION: MFAP4 immunoreactivity was reduced at sites of active inflammation. CSF levels of MFAP4 were reduced following relapse and may reflect disease activity.

The spectrum of central nervous system involvement in Whipple's disease.

BACKGROUND AND PURPOSE: To assess the clinical spectrum of central nervous system (CNS) involvement as well as cerebrospinal fluid (CSF) and neuroimaging findings in patients with Whipple's disease (WD) and to analyze the association of neurological symptoms with CSF and imaging findings. METHODS: Neurological involvement was retrospectively analyzed in a series of 36 patients diagnosed with WD at a single center between 1992 and 2019. Findings of 81 comprehensive CSF examinations from 36 patients, including polymerase chain reaction (PCR) tests for Tropheryma whipplei (TW) in CSF from 35 patients, were systematically evaluated. The prevalence of ischemic stroke in patients with WD was compared to a matched control cohort. RESULTS: Neurological symptoms occurred in 23 of 36 (63.9%) patients, with cognitive, motor, and oculomotor dysfunction being most frequent. TW was detected by PCR in CSF of 13 of 22 (59.1%) patients with and four of 13 (30.8%, p = 0.0496) patients without neurological symptoms. Total CSF protein (p = 0.044) and lactate (p = 0.035) were moderately elevated in WD with neurologic symptoms compared with WD without. No intrathecal immunoglobulin synthesis was observed. Three of 36 (8.3%) patients had hydrocephalus due to aqueductal stenosis. Patients with WD had an unexpectedly high prevalence of ischemic stroke (10/36, 27.7%) compared to matched controls (10/360, 3.2%). CONCLUSIONS: Neurological involvement in patients with WD is common. Detection of TW DNA in CSF is only partly associated with neurological symptoms. Elevated CSF parameters suggest CNS parenchymal infection. Stroke is a hitherto underrecognized manifestation of WD. These findings suggest that mechanisms beyond CNS infection contribute to the spectrum of CNS involvement in WD.

Retinal ganglion cell loss is associated with future disability worsening in early relapsing-remitting multiple sclerosis.

BACKGROUND AND PURPOSE: Thinning of the retinal combined ganglion cell and inner plexiform layer (GCIP) as measured by optical coherence tomography (OCT) is a common finding in patients with multiple sclerosis. This study aimed to investigate whether a single retinal OCT analysis allows prediction of future disease activity after a first demyelinating event. METHODS: This observational cohort study included 201 patients with recently diagnosed clinically isolated syndrome or relapsing-remitting multiple sclerosis from two German tertiary referral centers. Individuals underwent neurological examination, magnetic resonance imaging, and OCT at baseline and at yearly follow-up visits. RESULTS: Patients were included at a median disease duration of 2.0 months. During a median follow-up of 59 (interquartile range = 43-71) months, 82% of patients had ongoing disease activity as demonstrated by failing the no evidence of disease activity 3 (NEDA-3) criteria, and 19% presented with confirmed disability worsening. A GCIP threshold of ≤77 µm at baseline identified patients with a high risk for NEDA-3 failure (hazard ratio [HR] = 1.7, 95% confidence interval [CI] = 1.1-2.8, p = 0.04), and GCIP measures of ≤69 µm predicted disability worsening (HR = 2.2, 95% CI = 1.2-4.3, p = 0.01). Higher rates of annualized GCIP loss increased the risk for disability worsening (HR = 2.5 per 1 µm/year increase of GCIP loss, p = 0.03). CONCLUSIONS: Ganglion cell thickness as measured by OCT after the initial manifestation of multiple sclerosis may allow early risk stratification as to future disease activity and progression.

Immunomodulatory Effects of New Phenanthrene Derivatives from Dendrobium crumenatum.

Three new phenanthrene derivatives (1, 2, 4), one new fluorenone (3), and four known compounds (5-8) were isolated from the ethyl acetate extract of Dendrobium crumenatum Sw. stems using column chromatography. The chemical structures were elucidated by analysis of spectroscopic data. The absolute configuration of 4 was determined by electronic circular dichroism calculation. We also evaluated the immunomodulatory effects of compounds isolated from D. crumenatum in human peripheral blood mononuclear cells from healthy individuals and those from patients with multiple sclerosis in vitro. Dendrocrumenol B (2) and dendrocrumenol D (4) showed strong immunomodulatory effects on both CD3+ T cells and CD14+ monocytes. Compounds 2 and 4 could reduce IL-2 and TNF production in T cells and monocytes that were treated with phorbol-12-myristate-13-acetate and ionomycin (PMA/Iono). Deep immune profiling using high-dimensional single-cell mass cytometry could confirm immunomodulatory effects of 4, quantified by the reduction of activated T cell population under PMA/Iono stimulation, in comparison to the stimulated T cells without treatment.

Serum tau protein elevation in migraine: a cross-sectional case-control study.

BACKGROUND: Migraine is a disorder associated with neuropeptide release, pain and inflammation. Tau protein has recently been linked to inflammatory diseases and can be influenced by neuropeptides such as CGRP, a key neurotransmitter in migraine. Here, we report serum concentrations of total-tau protein in migraine patients and healthy controls. METHODS: In this cross-sectional study, interictal blood samples from n = 92 patients with episodic migraine (EM), n = 93 patients with chronic migraine (CM), and n = 42 healthy matched controls (HC) were studied. We assessed serum total-tau protein (t-tau) and for comparison neurofilament light chain protein (NfL), glial fibrillary acidic protein (GFAP), and ubiquitin carboxy-terminal hydrolase L (UCH-L1) concentrations using the Neurology 4-plex kit, on a single molecule array HD-X Analyzer (Quanterix Corp Lexington, MA). Matched serum/cerebrospinal fluid (CSF) samples were used for post-hoc evaluations of a central nervous system (CNS) source of relevant findings. We applied non-parametric tests to compare groups and assess correlations. RESULTS: Serum t-tau concentrations were elevated in EM [0.320 (0.204 to 0.466) pg/mL] and CM [0.304 (0.158 to 0.406) pg/mL] patients compared to HC [0.200 (0.114 to 0.288) pg/mL] (p = 0.002 vs. EM; p = 0.025 vs. CM). EM with aura [0.291 (0.184 to 0.486 pg/mL); p = 0.013] and EM without aura [0.332 (0.234 to 0.449) pg/mL; p = 0.008] patients had higher t-tau levels than HC but did not differ between each other. Subgroup analysis of CM with/without preventive treatment revealed elevated t-tau levels compared to HC only in the non-prevention group [0.322 (0.181 to 0.463) pg/mL; p = 0.009]. T-tau was elevated in serum (p = 0.028) but not in cerebrospinal fluid (p = 0.760). In contrast to t-tau, all proteins associated with cell damage (NfL, GFAP, and UCH-L1), did not differ between groups. DISCUSSION: Migraine is associated with t-tau elevation in serum but not in the CSF. Our clinical study identifies t-tau as a new target for migraine research.

HLA-DRB1*15:01 is a co-receptor for Epstein-Barr virus, linking genetic and environmental risk factors for multiple sclerosis.

The strongest genetic and environmental risk factors for MS, an inflammatory CNS disease, are HLA-DRB1*15:01 and EBV. This work shows that HLA-DRB1*15:01 acts as a co-receptor for EBV infection of a B cell line, suggesting a mechanistic link between both risk factors for MS.

Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD.

BACKGROUND: In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We previously demonstrated that NMOSD neutrophils show functional deficiencies. Thus, we hypothesized that neutrophil accumulation in the CNS may be facilitated by impairments affecting mechanisms of neutrophil death. OBJECTIVE: To evaluate cell death in blood neutrophils from aquaporin-4 (AQP4)-IgG-seropositive NMOSD and MOGAD patients as well as matched healthy controls (HC) using in vitro assays. METHODS: Twenty-eight AQP4 + NMOSD and 19 MOGAD patients in stable disease phase as well as 45 age- and sex-matched HC were prospectively recruited. To induce cell death, isolated neutrophils were cultured with/without phorbol 12-myristate 13-acetate (PMA). Spontaneous and PMA-induced NETosis and apoptosis were analyzed using 7-AAD and annexin-V by flow cytometry. Caspase-3 was assessed by western blot. Myeloperoxidase-DNA complexes (MPO-DNA), MPO and elastase were evaluated by ELISA, and cell-free DNA (cfDNA) by a fluorescence-based assay. Reactive oxygen species (ROS) were evaluated by a dihydrorhodamine 123-based cytometric assay. Serum GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ and IL-10 were evaluated by multiplex assays, and neurofilament light chain (NfL) by single-molecule array assay. RESULTS: In response to PMA, neutrophils from AQP4 + NMOSD but not from MOGAD patients showed an increased survival, and subsequent reduced cell death (29.6% annexin V+ 7-AAD+) when compared to HC (44.7%, p = 0.0006). However, AQP4 + NMOSD also showed a mild increase in annexin V+ 7-AAD- early apoptotic neutrophils (24.5%) compared to HC (20.8%, p = 0.048). PMA-induced reduction of caspase-3 activation was more pronounced in HC (p = 0.020) than in AQP4 + NMOSD neutrophils (p = 0.052). No differences were observed in neutrophil-derived MPO-DNA or serum levels of MPO, elastase, IL-6, IL-8 and TNF-ɑ. IL-15 levels were increased in both groups of patients. In AQP4 + NMOSD, an increase in cfDNA, GM-CSF and IL-10 was found in serum. A positive correlation among cfDNA and NfL was found in AQP4 + NMOSD. CONCLUSIONS: AQP4 + NMOSD neutrophils showed an increased survival capacity in response to PMA when compared to matched HC neutrophils. Although the data indicate that the apoptotic but not the NETotic response is altered in these neutrophils, additional evaluations are required to validate this observation.

Cerebrospinal fluid findings in COVID-19: a multicenter study of 150 lumbar punctures in 127 patients.

BACKGROUND: Comprehensive data on the cerebrospinal fluid (CSF) profile in patients with COVID-19 and neurological involvement from large-scale multicenter studies are missing so far. OBJECTIVE: To analyze systematically the CSF profile in COVID-19. METHODS: Retrospective analysis of 150 lumbar punctures in 127 patients with PCR-proven COVID-19 and neurological symptoms seen at 17 European university centers RESULTS: The most frequent pathological finding was blood-CSF barrier (BCB) dysfunction (median QAlb 11.4 [6.72-50.8]), which was present in 58/116 (50%) samples from patients without pre-/coexisting CNS diseases (group I). QAlb remained elevated > 14d (47.6%) and even > 30d (55.6%) after neurological onset. CSF total protein was elevated in 54/118 (45.8%) samples (median 65.35 mg/dl [45.3-240.4]) and strongly correlated with QAlb. The CSF white cell count (WCC) was increased in 14/128 (11%) samples (mostly lympho-monocytic; median 10 cells/µl, > 100 in only 4). An albuminocytological dissociation (ACD) was found in 43/115 (37.4%) samples. CSF L-lactate was increased in 26/109 (24%; median 3.04 mmol/l [2.2-4]). CSF-IgG was elevated in 50/100 (50%), but was of peripheral origin, since QIgG was normal in almost all cases, as were QIgA and QIgM. In 58/103 samples (56%) pattern 4 oligoclonal bands (OCB) compatible with systemic inflammation were present, while CSF-restricted OCB were found in only 2/103 (1.9%). SARS-CoV-2-CSF-PCR was negative in 76/76 samples. Routine CSF findings were normal in 35%. Cytokine levels were frequently elevated in the CSF (often associated with BCB dysfunction) and serum, partly remaining positive at high levels for weeks/months (939 tests). Of note, a positive SARS-CoV-2-IgG-antibody index (AI) was found in 2/19 (10.5%) patients which was associated with unusually high WCC in both of them and a strongly increased interleukin-6 (IL-6) index in one (not tested in the other). Anti-neuronal/anti-glial autoantibodies were mostly absent in the CSF and serum (1509 tests). In samples from patients with pre-/coexisting CNS disorders (group II [N = 19]; including multiple sclerosis, JC-virus-associated immune reconstitution inflammatory syndrome, HSV/VZV encephalitis/meningitis, CNS lymphoma, anti-Yo syndrome, subarachnoid hemorrhage), CSF findings were mostly representative of the respective disease. CONCLUSIONS: The CSF profile in COVID-19 with neurological symptoms is mainly characterized by BCB disruption in the absence of intrathecal inflammation, compatible with cerebrospinal endotheliopathy. Persistent BCB dysfunction and elevated cytokine levels may contribute to both acute symptoms and 'long COVID'. Direct infection of the CNS with SARS-CoV-2, if occurring at all, seems to be rare. Broad differential diagnostic considerations are recommended to avoid misinterpretation of treatable coexisting neurological disorders as complications of COVID-19.

Multiple Sclerosis Is Rare in Epstein-Barr Virus-Seronegative Children with Central Nervous System Inflammatory Demyelination.

Although Epstein-Barr virus (EBV) is hypothesized to be a prerequisite for multiple sclerosis (MS), up to 15% of children with a diagnosis of MS were reported to be EBV-seronegative. When re-evaluating 25 EBV-seronegative children out of 189 pediatric patients with a diagnosis of clinically isolated syndrome/MS, we found anti-myelin oligodendrocyte glycoprotein (MOG) antibody in 11 of 25 (44%) EBV-seronegative but only 9 of 164 (5.5%, p < 0.001) EBV-seropositive patients. After critical review, MS remained a plausible diagnosis in only 4 of 14 EBV-seronegative/MOG antibody-negative patients. In children with an MS-like presentation, EBV seronegativity should alert clinicians to consider diagnoses other than MS, especially MOG-antibody disease. ANN NEUROL 2021;89:1234-1239.

Preserved T cell responses to SARS-CoV-2 in anti-CD20 treated multiple sclerosis.

BACKGROUND: Optimal management of anti-CD20-treated patients with multiple sclerosis (pwMS) is an important clinical task during the current severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. OBJECTIVES: To characterize humoral and cellular immune responses to SARS-CoV-2 vaccinations/infections in a longitudinal cohort of anti-CD20 treated (n = 175) and anti-CD20 therapy-naïve (n = 41) pwMS. METHODS: Anti-SARS-CoV-2 spike protein immunoglobulin G (IgG) and IgA, virus neutralizing capacity, IgG avidity and SARS-CoV-2-specific T cells were determined. RESULTS: Following two SARS-CoV-2 vaccinations, not only SARS-CoV-2 spike protein IgG and IgA, but also neutralizing capacity and avidity of SARS-CoV-2 IgG were lower in anti-CD20-treated (n = 51) than in anti-CD20 therapy-naïve pwMS (n = 14) and in healthy controls (HC, n = 19). However, in all anti-CD20-treated pwMS vaccinated twice (n = 26) or infected with SARS-CoV-2 (n = 2), in whom SARS-CoV-2-specific T cells were measured, SARS-CoV-2-specific T cells were detectable, at levels similar to those of twice-vaccinated anti-CD20 therapy-naïve pwMS (n = 7) and HC (n = 19). SARS-CoV-2-S1 IgG levels (r = 0.42, p = 0.002), antibody avidity (r = 0.7, p < 0.001), and neutralizing capacity (r = 0.44, p = 0.03) increased with time between anti-CD20 infusion and second vaccination. Based on detection of SARS-CoV-2 antibodies, SARS-CoV-2 infections occurred in 4 out of 175 (2.3%) anti-CD20-treated pwMS, all of whom recovered fully. CONCLUSIONS: These findings should inform treatment decisions and SARS-CoV-2 vaccination management in pwMS.

Impaired motion perception is associated with functional and structural visual pathway damage in multiple sclerosis and neuromyelitis optica spectrum disorders.

BACKGROUND: Decreased motion perception has been suggested as a marker for visual pathway demyelination in optic neuritis (ON) and/or multiple sclerosis (MS). OBJECTIVES: To examine the influence of neuro-axonal damage on motion perception in MS and neuromyelitis optica spectrum disorders (NMOSD). METHODS: We analysed motion perception with numbers-from-motion (NFM), visual acuity, (multifocal (mf)) VEP, optical coherence tomography in patients with MS (n = 38, confirmatory cohort n = 43), NMOSD (n = 13) and healthy controls (n = 33). RESULTS: NFM was lower compared with controls in MS (B = -12.37, p < 0.001) and NMOSD (B = -34.5, p < 0.001). NFM was lower in ON than in non-ON eyes (B = -30.95, p = 0.041) in NMOSD, but not MS. In MS and NMOSD, lower NFM was associated with worse visual acuity (B = -139.4, p < 0.001/B = -77.2, p < 0.001) and low contrast letter acuity (B = 0.99, p = 0.002/B = 1.6, p < 0.001), thinner peripapillary retinal nerve fibre layer (B = 1.0, p < 0.001/ B = 0.92, p = 0.016) and ganglion cell/inner plexiform layer (B = 64.8, p < 0.001/B = 79.5, p = 0.006), but not with VEP P100 latencies. In the confirmatory MS cohort, lower NFM was associated with thinner retinal nerve fibre layer (B = 1.351, p < 0.001) and increased mfVEP P100 latencies (B = -1.159, p < 0.001). CONCLUSIONS: Structural neuro-axonal visual pathway damage is an important driver of motion perception impairment in MS and NMOSD.