Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with ...conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases.
Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma ...membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.
Background and aims
Patients infected with SARS-CoV-2 range from asymptomatic, to mild, moderate or severe disease evolution including fatal outcome. Thus, early predictors of clinical outcome are ...highly needed. We investigated markers of neural tissue damage as a possible early sign of multisystem involvement to assess their clinical prognostic value on survival or transfer to intensive care unit (ICU).
Methods
We collected blood from 104 patients infected with SARS-CoV-2 the day of admission to the emergency room and measured blood neurofilament light chair (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and total tau protein levels.
Results
We found that NfL, GFAP, and tau were significantly increased in patients with fatal outcome, while NfL and UCH-L1 in those needing ICU transfer. ROC and Kaplan–Meier curves indicated that total tau levels at admission accurately predict mortality.
Conclusions
Blood neural markers may provide additional prognostic value to conventional biomarkers used to predict COVID-19 outcome.
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.
Background Oxytocin (OT) has been suggested as a treatment to improve social behavior in autistic patients. Accordingly, the OT ( Oxt −/− ) and the OT receptor null mice ( Oxtr −/− ) display ...autistic-like deficits in social behavior, increased aggression, and reduced ultrasonic vocalization. Methods Oxtr −/− mice were characterized for general health, sociability, social novelty, cognitive flexibility, aggression, and seizure susceptibility. Because vasopressin (AVP) and OT cooperate in controlling social behavior, learning, and aggression, they were tested for possible rescue of the impaired behaviors. Primary hyppocampal cultures from Oxtr +/+ and Oxtr −/− mouse embryos were established to investigate the balance between gamma-aminobutyric acid (GABA) and glutamate synapses and the expression levels of OT and AVP (V1a) receptors were determined by autoradiography. Results Oxtr−/− mice display two additional, highly relevant, phenotypic characteristics: 1) a resistance to change in a learned pattern of behavior, comparable to restricted interests and repetitive behavior in autism, and 2) an increased susceptibility to seizures, a frequent and clinically relevant symptom of autism. We also show that intracerebral administration of both OT and AVP lowers aggression and fully reverts social and learning defects by acting on V1a receptors and that seizure susceptibility is antagonized by peripherally administered OT. Finally, we detect a decreased ratio of GABA–ergic versus total presynapses in hippocampal neurons of Oxtr −/− mice. Conclusions Autistic-like symptoms are rescued on administration of AVP and OT to young Oxtr −/− adult animals. The Oxtr −/− mouse is thus instrumental to investigate the neurochemical and synaptic abnormalities underlying autistic-like disturbances and to test new strategies of pharmacologic intervention.
Patients with bipolar disorder (BD) show higher immuno-inflammatory setpoints, with in vivo alterations in white matter (WM) microstructure and post-mortem infiltration of T cells in the brain. ...Cytotoxic CD8
T cells can enter and damage the brain in inflammatory disorders, but little is known in BD. Our study aimed to investigate the relationship between cytotoxic T cells and WM alterations in BD. In a sample of 83 inpatients with BD in an active phase of illness (68 depressive, 15 manic), we performed flow cytometry immunophenotyping to investigate frequencies, activation status, and expression of cytotoxic markers in CD8
and tested for their association with diffusion tensor imaging (DTI) measures of WM microstructure. Frequencies of naïve and activated CD8
cell populations expressing Perforin, or both Perforin and Granzyme, negatively associated with WM microstructure. CD8
Naïve cells negative for Granzyme and Perforin positively associates with indexes of WM integrity, while the frequency of CD8
memory cells negatively associates with index of WM microstructure, irrespective of toxins expression. The resulting associations involve measures representative of orientational coherence and myelination of the fibers (FA and RD), suggesting disrupted oligodendrocyte-mediated myelination. These findings seems to support the hypothesis that immunosenescence (less naïve, more memory T cells) can detrimentally influence WM microstructure in BD and that peripheral CD8
T cells may participate in inducing an immune-related WM damage in BD mediated by killer proteins.
The approval of immune checkpoint inhibitors (ICIs) by the Food and Drug Administration (FDA) led to an improvement in the treatment of several types of cancer. The main targets of these drugs are ...cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein-1/programmed death-ligand 1 pathway (PD-1/PD-L1), which are important inhibitory molecules for the immune system. Besides being generally safer than common chemotherapy, the use of ICIs has been associated with several immune-related adverse effects (irAEs). Although rare, neurological adverse effects are reported within the irAEs in clinical trials, particularly in patients treated with anti-PD-1 antibodies or a combination of both anti-CTLA-4 and PD-1 drugs. The observations obtained from clinical trials suggest that the PD-1 axis may play a remarkable role in the regulation of neuroinflammation. Moreover, numerous studies in preclinical models have demonstrated the involvement of PD-1 in several neurological disorders. However, a comprehensive understanding of these cellular mechanisms remains elusive. Our review aims to summarize the most recent evidence concerning the regulation of neuroinflammation through PD-1/PD-L signaling, focusing on cell populations that are involved in this pathway.
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.
Microvesicles (MVs) are membrane particles of 200-500 nm released by all cell types constitutively. MVs of myeloid origin are found increased in the cerebrospinal fluid (CSF) of patients suffering ...from neuroinflammatory disorders, although the factors triggering their production have never been defined. Here, we report that both pro- and anti-inflammatory cytokines, specifically interferon-γ and interleukin-4, are equally able to stimulate the production of MVs from microglia cells and monocytes. Additionally, we found this process to be independent from the best characterized molecular pathway so far described for membrane shedding, which is centered on the purinergic receptor P2X7, whose activation by high concentrations of extracellular ATP (exATP) results in membrane blebbing operated by the secreted enzyme acid sphingomyelinase (ASMase). Moreover, a potent inhibitor of ASMase, injected in a mouse model of multiple sclerosis, failed to reduce the number of MVs in their CSF. This suggests that cytokines, rather than exATP, may exert a long-term control of MV production in the context of chronic inflammation, where both pro- and anti-inflammatory factors play coordinated roles.
Specific proinflammatory and anti-inflammatory molecules could represent useful cerebrospinal fluid (CSF) biomarkers to predict the clinical course of multiple sclerosis (MS). The proinflammatory ...molecule interleukin (IL)-6 has been investigated in the pathophysiology of MS, and has been associated in previous, smaller studies to increased disability and disease activity. CSF IL-6 detectability in MS 2 This is a provisional file, not the final typeset article Here, we wanted to further address IL-6 as a possible CSF biomarker of MS by investigating its detectability in a large cohort of 534 MS patients and in 100 individuals with other non-inflammatory neurological diseases. In these newly diagnosed patients, we also explored correlations between IL-6 detectability, MS phenotypes and disease characteristics.We found that IL-6 was more frequently detectable in the CSF of MS patients compared with their control counterparts, as significant differences emerged between patients with clinically isolated syndrome, relapsing remitting, secondary progressive and primary progressive MS, compared to noninflammatory controls. IL-6 was equally present in the CSF of all MS phenotypes. In MS patients, IL-6 detectability was found to signal clinically and/or radiologically defined disease activity, among all other clinical characteristics.Our results add further evidence that CSF proinflammatory cytokines could be useful for the identification of those MS patients who are prone to increased prospective disease activity. In particular, IL-6 could represent an interesting prognostic biomarker of MS, as also demonstrated in other diseases where CSF IL-6 was found to identify patients with worse disease severity.
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