Assessment of brain-derived extracellular vesicle enrichment for blood biomarker analysis in age-related neurodegenerative diseases: An international overview.

INTRODUCTION: Brain-derived extracellular vesicles (BEVs) in blood allows for minimally-invasive investigations of central nervous system (CNS) -specific markers of age-related neurodegenerative diseases (NDDs). Polymer-based EV- and immunoprecipitation (IP)-based BEV-enrichment protocols from blood have gained popularity. We systematically investigated protocol consistency across studies, and determined CNS-specificity of proteins associated with these protocols. METHODS: NDD articles investigating BEVs in blood using polymer-based and/or IP-based BEV enrichment protocols were systematically identified, and protocols compared. Proteins used for BEV-enrichment and/or post-enrichment were assessed for CNS- and brain-cell-type-specificity, extracellular domains (ECD+), and presence in EV-databases. RESULTS: A total of 82.1% of studies used polymer-based (ExoQuick) EV-enrichment, and 92.3% used L1CAM for IP-based BEV-enrichment. Centrifugation times differed across studies. A total of 26.8% of 82 proteins systematically identified were CNS-specific: 50% ECD+, 77.3% were listed in EV-databases. CONCLUSIONS: We identified protocol steps requiring standardization, and recommend additional CNS-specific proteins that can be used for BEV-enrichment or as BEV-biomarkers. HIGHLIGHTS: Across NDDs, we identified protocols commonly used for EV/BEV enrichment from blood. We identified protocol steps showing variability that require harmonization. We assessed CNS-specificity of proteins used for BEV-enrichment or found in BEV cargo. CNS-specific EV proteins with ECD+ or without were identified. We recommend evaluation of blood-BEV enrichment using these additional ECD+ proteins.

Rapid Activation of Neuroinflammation in Stroke: Plasma and Extracellular Vesicles Obtained on a Mobile Stroke Unit.

BACKGROUND: Neuroinflammation is ubiquitous in acute stroke and worsens outcome. However, the precise timing of the inflammatory response is unknown, hindering the design of acute anti-inflammatory therapeutic interventions. We sought to identify the onset of the neuroinflammatory cascade using a mobile stroke unit. METHODS: The study is a proof-of-concept, cohort investigation of ultra-early blood- and extracellular vesicle-derived markers of neuroinflammation and outcome in acute stroke. Blood was obtained, prehospital, on an mobile stroke unit. Outcomes were biomarker concentrations, modified Rankin Scale score, and National Institutes of Health Stroke Scale score. RESULTS: Forty-one adults were analyzed, including 15 patients treated on the mobile stroke unit between August 2021 and April 2022, and 26 healthy controls to establish biomarker reference levels. Median patient age was 74 (range, 36-97) years, 60% were female, and 80% White. Ten (67%) were diagnosed as stroke, with 8 (53%) confirmed and 2 likely transient ischemic attack or stroke averted by thrombolysis; 5 were stroke mimics. For strokes, median initial National Institutes of Health Stroke Scale score was 11 (range, 4-19) and 6 (75%) received tPA (tissue-type plasminogen activator). Blood was obtained a median of 58 (range, 36-133) minutes after symptom onset. Within 36 minutes after stroke, plasma IL-6 (interleukin-6), neurofilament light chain, UCH-L1 (ubiquitin C-terminal hydrolase L1), and GFAP (glial fibrillary acidic protein) were elevated by as much as 10 times normal. In EVs, MMP-9 (matrix metalloproteinase-9), CXCL4 (chemokine (C-X-C motif) ligand 4), CRP (C-reactive protein), IL-6, OPN (osteopontin), and PECAM1 (platelet and endothelial cell adhesion molecule 1) were elevated. Inflammatory markers increased rapidly in the first 2 hours and continued rising for 24 hours. CONCLUSIONS: The neuroinflammatory cascade was found to be activated within 36 to 133 minutes after stroke and progresses rapidly. This is earlier than observed previously in humans and suggests injury from neuroinflammation occurs faster than had been surmised. These findings could inform development of acute immunomodulatory stroke therapies and lead to new diagnostic tools and improved outcomes.

Plasma IgG aggregates as biomarkers for multiple sclerosis.

We recently reported that multiple sclerosis (MS) plasma contains IgG aggregates and induces complement-dependent neuronal cytotoxicity (Zhou et al., 2023). Using ELISA, we report herein that plasma IgG levels in the aggregates can be used as biomarkers for MS. We enriched the IgG aggregates from samples of two cohorts (190 MS and 160 controls) by collecting flow-through after plasma binding to Protein A followed by detection of IgG subclass. We show that there are significantly higher levels of IgG1, IgG3, and total IgG antibodies in MS IgG aggregates, with an AUC >90%; higher levels of IgG1 distinguish secondary progressive MS from relapsing-remitting MS (AUC = 91%). Significantly, we provided the biological rationale for MS plasma IgG biomarkers by demonstrating the strong correlation between IgG antibodies and IgG aggregate-induced neuronal cytotoxicity. These non-invasive, simple IgG-based blood ELISA assays can be adapted into clinical practice for diagnosing MS and SPMS and monitoring treatment responses.

Traumatic brain injury increases plasma astrocyte-derived exosome levels of neurotoxic complement proteins.

Possible involvement of complement (C) systems in the pathogenesis of traumatic brain injury (TBI) was investigated by quantifying Cproteins in plasma astrocyte-derived exosomes (ADEs) of subjects with sports-related TBI (sTBI) and TBI in military veterans (mtTBI) without cognitive impairment. All sTBI subjects (n = 24) had mild injuries, whereas eight of the mtTBI subjects had moderate, and 17 had mild injuries. Plasma levels of ADEs were decreased after acute sTBI and returned to normal within months. Cprotein levels in ADEs were from 12- to 35-fold higher than the corresponding levels in neuron-derived exosomes. CD81 exosome marker-normalized ADE levels of classical pathway C4b, alternative pathway factor D and Bb, lectin pathway mannose-binding lectin (MBL), and shared neurotoxic effectors C3b and C5b-9 terminal C complex were significantly higher and those of C regulatory proteins CR1 and CD59 were lower in the first week of acute sTBI (n = 12) than in controls (n = 12). Most C abnormalities were no longer detected in chronic sTBI at 3-12 months after acute sTBI, except for elevated levels of factor D, Bb, and MBL. In contrast, significant elevations of ADE levels of C4b, factor D, Bb, MBL, C3b and C5b-9 terminal C complex, and depressions of CR1 and CD59 relative to those of controls were observed after 1-4 years in early chronic mtTBI (n = 10) and persisted for decades except for normalization of Bb, MBL, and CD59 in late chronic mtTBI (n = 15). Complement inhibitors may be useful therapeutically in acute TBI and post-concussion syndrome.

RvE1 treatment prevents memory loss and neuroinflammation in the Ts65Dn mouse model of Down syndrome.

Inflammation can be resolved by pro-homeostatic lipids called specialized pro-resolving mediators (SPMs) upon activation of their receptors. Dysfunctional inflammatory resolution is now considered as a driver of chronic neuroinflammation and Alzheimer's disease (AD) pathogenesis. We have previously shown that SPM levels were reduced and also that SPM-binding receptors were increased in patients with AD compared to age-matched controls. Individuals with Down syndrome (DS) exhibit accelerated acquisition of AD neuropathology, dementia, and neuroinflammation at an earlier age than the general population. Beneficial effects of inducing resolution in DS have not been investigated previously. The effects of the SPM resolvin E1 (RvE1) in a DS mouse model (Ts65Dn) were investigated with regard to inflammation, neurodegeneration, and memory deficits. A moderate dose of RvE1 for 4 weeks in middle-aged Ts65Dn mice elicited a significant reduction in memory loss, along with reduced levels of serum pro-inflammatory cytokines, and reduced microglial activation in the hippocampus of Ts65Dn mice but had no effects in age-matched normosomic mice. There were no observable adverse side effects in Ts65Dn or in normosomic mice. These findings suggest that SPMs may represent a novel drug target for individuals with DS and others at risk of developing AD.

Inhibitory designer receptors aggravate memory loss in a mouse model of down syndrome.

The pontine nucleus locus coeruleus (LC) is the primary source of noradrenergic (NE) projections to the brain and is important for working memory, attention, and cognitive flexibility. Individuals with Down syndrome (DS) develop Alzheimer's disease (AD) with high penetrance and often exhibit working memory deficits coupled with degeneration of LC-NE neurons early in the progression of AD pathology. Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools that allow targeted manipulation of discrete neuronal populations in the brain without the confounds of off-target effects. We utilized male Ts65Dn mice (a mouse model for DS), and male normosomic (NS) controls to examine the effects of inhibitory DREADDs delivered via an AAV vector under translational control of the synthetic PRSx8, dopamine ß hydroxylase (DßH) promoter. This chemogenetic tool allowed LC inhibition upon administration of the inert DREADD ligand, clozapine-N-oxide (CNO). DREADD-mediated LC inhibition impaired performance in a novel object recognition task and reversal learning in a spatial task. DREADD-mediated LC inhibition gave rise to an elevation of α-adrenoreceptors both in NS and in Ts65Dn mice. Further, microglial markers showed that the inhibitory DREADD stimulation led to increased microglial activation in the hippocampus in Ts65Dn but not in NS mice. These findings strongly suggest that LC signaling is important for intact memory and learning in Ts65Dn mice and disruption of these neurons leads to increased inflammation and dysregulation of adrenergic receptors.

Altered levels of plasma neuron-derived exosomes and their cargo proteins characterize acute and chronic mild traumatic brain injury.

Neuron-derived exosomes (NDEs) were enriched by anti-L1CAM antibody immunoabsorption from plasmas of subjects ages 18-26 yr within 1 wk after a sports-related mild traumatic brain injury (acute mTBI) ( n = 18), 3 mo or longer after the last of 2-4 mTBIs (chronic mTBI) ( n = 14) and with no recent history of TBI (controls) ( n = 21). Plasma concentrations of NDEs, assessed by counts and levels of extracted exosome marker CD81, were significantly depressed by a mean of 45% in acute mTBI ( P < 0.0001), but not chronic mTBI, compared with controls. Mean CD81-normalized NDE levels of a range of functional brain proteins were significantly abnormal relative to those of controls in acute but not chronic mTBI, including ras-related small GTPase 10, 73% decrease; annexin VII, 8.8-fold increase; ubiquitin C-terminal hydrolase L1, 2.5-fold increase; AII spectrin fragments, 1.9-fold increase; claudin-5, 2.7-fold increase; sodium-potassium-chloride cotransporter-1, 2.8-fold increase; aquaporin 4, 8.9-fold increase (3.6-fold increase in chronic mTBI); and synaptogyrin-3, 3.1-fold increase (1.3-fold increase in chronic mTBI) (all acute mTBI proteins P < 0.0001). In chronic mTBI, there were elevated CD81-normalized NDE levels of usually pathologic ß-amyloid peptide 1-42 (1.6-fold, P < 0.0001), P-T181-tau (2.2-fold, P < 0.0001), P-S396-tau (1.6-fold, P < 0.01), IL-6 (16-fold, P < 0.0001), and prion cellular protein (PRPc) (5.1-fold, P < 0.0001) with lesser or greater (IL-6, PRPc) increases in acute mTBI. Increases in NDE levels of most neurofunctional proteins in acute, but not chronic, mTBI, and elevations of most NDE neuropathological proteins in chronic and acute mTBI delineated phase-specificity. Longitudinal studies of more mTBI subjects may identify biomarkers predictive of and etiologically involved in mTBI-induced neurodegeneration.-Goetzl, E. J., Elahi, F. M., Mustapic, M., Kapogiannis, D., Pryhoda, M., Gilmore, A., Gorgens, K. A., Davidson, B., Granholm, A.-C., Ledreux, A. Altered levels of plasma neuron-derived exosomes and their cargo proteins characterize acute and chronic mild traumatic brain injury.

Centre of pressure velocity shows impairments in NCAA Division I athletes six months post-concussion during standing balance.

Sport-related concussion return to play (RTP) decisions are largely based on the resolution of self-reported symptoms and neurocognitive function. Some evaluators also incorporate balance; however, an objective approach to balance that can detect effects beyond the acute condition is warranted. The purpose of this study is to examine linear measures of biomechanical balance up to 6 months post-concussion, and to present preliminary diagnostic thresholds useful for RTP. Each concussed athlete participated in instrumented standing balance tasks at 4 timepoints post-concussion. The measures from concussed athletes were compared to the sport-matched non-concussed athlete group at each timepoint. Centre of pressure (COP) mediolateral (ML) velocity in double-leg stance on a hard surface discriminated well between non-concussed and concussed athletes. COP anterior-posterior (AP) velocity in tandem stance on foam showed sensitivity to concussion. Sixty per cent of athletes at 6 months post-concussion did not recover to within the proposed COP ML velocity threshold in double-leg stance on a hard surface. Seventy-one per cent of athletes at 6 months post-concussion did not recover to within the COP AP velocity threshold in tandem stance on foam. This lack of recovery potentially indicates vestibular and motor control impairments long past the typical period of RTP.

Neuronal exosomes reveal Alzheimer's disease biomarkers in Down syndrome.

INTRODUCTION: Individuals with Down syndrome (DS) exhibit Alzheimer's disease (AD) neuropathology and dementia early in life. Blood biomarkers of AD neuropathology would be valuable, as non-AD intellectual disabilities of DS and AD dementia overlap clinically. We hypothesized that elevations of amyloid ß (Aß) peptides and phosphorylated-tau in neuronal exosomes may document preclinical AD. METHODS: AD neuropathogenic proteins Aß1-42, P-T181-tau, and P-S396-tau were quantified by enzyme-linked immunosorbent assays in extracts of neuronal exosomes purified from blood of individuals with DS and age-matched controls. RESULTS: Neuronal exosome levels of Aß1-42, P-T181-tau, and P-S396-tau were significantly elevated in individuals with DS compared with age-matched controls at all ages beginning in childhood. No significant gender differences were observed. DISCUSSION: These early increases in Aß1-42, P-T181-tau, and P-S396-tau in individuals with DS may provide a basis for early intervention as targeted treatments become available.

Extracellular vesicles from the CNS play pivotal roles in neuroprotection and neurodegeneration: lessons from in vitro experiments.

Intercellular communication between diverse cell types is crucial for the maintenance of the central nervous system, and exosomes have been shown to play an important role in this process. Exosomes are small extracellular vesicles (EVs) that are released by all cell types and carry cargoes that can elicit downstream effects in recipient cells. Exosomal communication in the central nervous system has been implicated in many neurodegenerative diseases, ranging from Alzheimer's disease to major depressive disorder. Though there remain many unknowns in the field of EV biology, in vitro experiments can provide many insights into their potential roles in health and disease. In this review, we discuss the findings of many in vitro EV experiments, with a focus on the potential roles in regulating cell viability, inflammation, oxidative stress, and neurite integrity in the central nervous system.

Assessment of brain-derived extracellular vesicle enrichment for blood biomarker analysis in age-related neurodegenerative diseases: An international overview.

INTRODUCTION: Brain-derived extracellular vesicles (BEVs) in blood allows for minimally- invasive investigations of CNS-specific markers of age-related neurodegenerative diseases (NDDs). Polymer-based EV- and immunoprecipitation (IP)-based BEV-enrichment protocols from blood have gained popularity. We systematically investigated protocol consistency across studies, and determined CNS-specificity of proteins associated with these protocols. METHODS: NDD articles investigating BEVs in blood using polymer-based and/or IP-based BEV enrichment protocols were systematically identified, and protocols compared. Proteins used for BEV-enrichment and/or post-enrichment were assessed for CNS- and brain-cell-type- specificity; extracellular domains (ECD+); and presence in EV-databases. RESULTS: 82.1% of studies used polymer-based (ExoQuick) EV-enrichment, and 92.3% used L1CAM for IP-based BEV-enrichment. Centrifugation times differed across studies. 26.8% of 82 proteins systematically identified were CNS-specific: 50% ECD+, 77.3% were listed in EV- databases. DISCUSSION: We identified protocol steps requiring standardization, and recommend additional CNS-specific proteins that can be used for BEV-enrichment or as BEV-biomarkers.

Recipient Reaction and Composition of Autologous Sural Nerve Tissue Grafts into the Human Brain.

Parkinson's disease (PD) is a severe neurological disease for which there is no effective treatment or cure, and therefore it remains an unmet need in medicine. We present data from four participants who received autologous transplantation of small pieces of sural nerve tissue into either the basal forebrain containing the nucleus basalis of Meynert (NBM) or the midbrain substantia nigra (SN). The grafts did not exhibit significant cell death or severe host-tissue reaction up to 55 months post-grafting and contained peripheral cells. Dopaminergic neurites showed active growth in the graft area and into the graft in the SN graft, and cholinergic neurites were abundant near the graft in the NBM. These results provide a histological basis for changes in clinical features after autologous peripheral nerve tissue grafting into the NBM or SN in PD.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins.

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins-okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)-were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose-response modeling and assay validation were designed after a round of "trial-and-error" process. For each toxin, the shape of the concentration-response curves and the IC(50) values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins.

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

BDNF mediates improvement in cognitive performance after computerized cognitive training in healthy older adults.

Introduction: The often-cited mechanism linking brain-derived neurotrophic factor (BDNF) to cognitive health has received limited experimental study. There is evidence that cognitive training, physical exercise, and mindfulness meditation may improve cognition. Here, we investigated whether improvements in cognition after these three types of structured interventions are facilitated by increases in BDNF. Methods: A total of 144 heathy older adults completed a 5-week intervention involving working memory/cognitive training, physical exercise, mindfulness meditation, or an active control condition. Serum BDNF levels and Digit Symbol Test (DST) performance were measured pre- and post-intervention. Results: Linear mixed models suggested that only the cognitive training group demonstrated augmentation of BDNF and DST performance relative to the control condition. Path analysis revealed that changes in BDNF mediate intervention-related improvement in task performance. Regression analyses showed that, across all intervention conditions, increased BDNF levels were associated with increased DST scores. Discussion: This study appears to be the first to suggest that BDNF helps mediate improvements in cognition after working memory training in healthy older adults. Highlights: Older adults were randomized to physical activity, mindfulness, cognitive training (computerized cognitive training (CCT), or control.CCT, but no other condition, led to increased serum brain-derived neurotrophic factor (BDNF) levels.CCT led to improvement on the untrained Digit Symbol Test (DST) of speed/working memory.Path analysis: increases in BDNF mediate intervention-related improvement on DST.Increases in BDNF associated with improved DST across all experimental groups.

Foldamers reveal and validate therapeutic targets associated with toxic α-synuclein self-assembly.

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. The pathological hallmark for PD involves the self-assembly of functional Alpha-Synuclein (αS) into non-functional amyloid structures. One of the potential therapeutic interventions against PD is the effective inhibition of αS aggregation. However, the bottleneck towards achieving this goal is the identification of αS domains/sequences that are essential for aggregation. Using a protein mimetic approach, we have identified αS sequences-based targets that are essential for aggregation and will have significant therapeutic implications. An extensive array of in vitro, ex vivo, and in vivo assays is utilized to validate αS sequences and their structural characteristics that are essential for aggregation and propagation of PD phenotypes. The study aids in developing significant mechanistic and therapeutic insights into various facets of αS aggregation, which will pave the way for effective treatments for PD.

Curiosity-Based Interventions Increase Everyday Functioning Score But Not Serum BDNF Levels in a Cohort of Healthy Older Adults.

An enriched environment is effective in stimulating learning and memory in animal models as well as in humans. Environmental enrichment increases brain-derived neurotrophic factor (BDNF) in aged rats and reduces levels of Alzheimer-related proteins in the blood, including amyloid-ß (Aß) peptides and misfolded toxic forms of tau. To address whether stimulation of curiosity, which is a form of enrichment, may provide a buffer against Alzheimer's disease (AD), we measured levels of biomarkers associated with AD at baseline and after a 6-week intervention in older adults (>65 years of age) randomized to one of three different intervention conditions. Specifically, in this pilot study, we tested the effectiveness of a traditional, structured learning environment compared to a self-motivated learning environment designed to stimulate curiosity. There were no significant differences from baseline to post-intervention in any of the groups for Aß42/Aß40 ratio or t-tau (total-tau) plasma levels. Serum BDNF levels decreased significantly in the control group. Interestingly, individuals who had the lowest serum BDNF levels at baseline experienced significantly higher increases in BDNF over the course of the 6-week intervention compared to individuals with higher serum BDNF levels at baseline. As expected, older individuals had lower MoCA scores. Years of education correlated negatively with Aß levels, suggesting a protective effect of education on levels of this toxic protein. ECog scores were negatively correlated with BDNF levels, suggesting that better performance on the ECog questionnaire was associated with higher BDNF levels. Collectively, these findings did not suggest that a 6-week cognitive training intervention focused on curiosity resulted in significant alterations in blood biomarkers but showed interesting correlations between cognitive scores and BDNF levels, further supporting the role of this trophic factor in brain health in older adults.

A multiplex chemiluminescent immunoassay for serological profiling of COVID-19-positive symptomatic and asymptomatic patients.

The COVID-19 pandemic affects more than 81 million people worldwide with over 1.7 million deaths. As the population returns to work, it is critical to develop tests that reliably detect SARS-CoV-2-specific antibodies. Here we present results from a multiplex serology test for assessing the antibody responses to COVID-19. In an initial large cohort, this test shows greater than 99% agreement with COVID-19 PCR test. In a second outpatient cohort consisting of adults and children in Colorado, the IgG responses are more robust in positive/symptomatic participants than in positive/asymptomatic participants, the IgM responses in symptomatic participants are transient and largely fall below the detection limit 30 days after symptom onset, and the levels of IgA against SARS-CoV-2 receptor binding domain are significantly increased in participants with moderate-to-severe symptoms compared to those with mild-to-moderate symptoms or asymptomatic individuals. Our results thus provide insight into serology profiling and the immune response to COVID-19.

Small Neuron-Derived Extracellular Vesicles from Individuals with Down Syndrome Propagate Tau Pathology in the Wildtype Mouse Brain.

Individuals with Down syndrome (DS) exhibit Alzheimer's disease (AD) pathology at a young age, including amyloid plaques and neurofibrillary tangles (NFTs). Tau pathology can spread via extracellular vesicles, such as exosomes. The cargo of neuron-derived small extracellular vesicles (NDEVs) from individuals with DS contains p-Tau at an early age. The goal of the study was to investigate whether NDEVs isolated from the blood of individuals with DS can spread Tau pathology in the brain of wildtype mice. We purified NDEVs from the plasma of patients with DS-AD and controls and injected small quantities using stereotaxic surgery into the dorsal hippocampus of adult wildtype mice. Seeding competent Tau conformers were amplified in vitro from DS-AD NDEVs but not NDEVs from controls. One month or 4 months post-injection, we examined Tau pathology in mouse brains. We found abundant p-Tau immunostaining in the hippocampus of the mice injected with DS-AD NDEVs compared to injections of age-matched control NDEVs. Double labeling with neuronal and glial markers showed that p-Tau staining was largely found in neurons and, to a lesser extent, in glial cells and that p-Tau immunostaining was spreading along the corpus callosum and the medio-lateral axis of the hippocampus. These studies demonstrate that NDEVs from DS-AD patients exhibit Tau seeding capacity and give rise to tangle-like intracellular inclusions.

Sample preparation and liquid chromatography-tandem mass spectrometry for the analysis of selected Pacific ciguatoxins in blood samples.

Consumption of ciguatoxin-contaminated seafood can lead to ciguatera poisoning (CP). The diagnosis of CP in humans is based on the clinical symptoms after eating the fish from tropical or subtropical areas because no confirmatory clinical tests are available. One of the challenges for ciguatoxin analysis is their extremely low but toxicologically relevant concentration in biological samples. We previously reported a method using acetonitrile to precipitate proteins and extract the ciguatoxins simultaneously in whole blood samples from animals for toxin quantification by N2A cell-based assay. However, a test method for unambiguous confirmation of exposure of marine animals or humans to ciguatoxins is still needed. In the present study, we adopted the acetonitrile extraction method and added sample clean-up in the sample preparation for the determination of Pacific ciguatoxins CTX1B (aka P-CTX-1), 52-epi-54-deoxyCTX1B (aka P-CTX-2), and CTX3C (aka P-CTX-3C) in blood plasma by LC-MS/MS. We investigated sample clean-up, LC mobile phases, LC solvent programming, and settings of the two mass spectrometers (4000 Q TRAP and AB SCIEX Triple Quad 5500) in order to improve the ability to detect the Pacific ciguatoxins at ppt level. Rat blood plasma was used for the method development. Average recoveries of the three toxins in the rat plasma samples ranged from 90% to 116% with relative standard deviations of less than 15%. The method detection limits were still not low enough for the determination of the Pacific ciguatoxins in individual blood samples from Hawaiian monk seals with the two LC-MS systems. The methods were applied to a pooled sample of blood plasma collected from Hawaiian monk seals for confirmation of toxin exposure. This study will benefit monitoring of Pacific ciguatoxins in marine mammals and potentially humans by LC-MS/MS.