Human Cerebrospinal Fluid Research Articles

Quantitation of osimertinib, alectinib and lorlatinib in human cerebrospinal fluid by UPLC-MS/MS

Overall survival in metastatic lung cancer has been dramatically improved with the use of small molecule kinase inhibitors (SMKIs). Quantification of SMKI in cerebrospinal fluid (CSF) can be used to assess penetration of these drugs into the central nervous system. This paper describes an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantification of the SMKIs alectinib, lorlatinib and osimertinib in human CSF. Alectinib-d8 and dasatinib-d8 were used as internal standards. Aliquots with 25 µL CSF/30% albumin (9:1,v/v) were mixed with 100 µL internal standard solution consisting of 1 ng/mL dasatinib-d8 and alectinib-d8 in acetonitrile. The analytes were separated by an Acquity UPLC® HSS T3 column (2.1 ×150 mm, 1.8 µm), using gradient elution (ammonium formate pH 4.5, acetonitrile) with a flow rate of 0.400 mL/min. All calibration curves were linear for the concentration range from 2.50 to 250 ng/mL. Within-run and between-run precision varied from 0.72% to 11.7%, with accuracy ranging from 95.3% to 113.2%. For all compounds, a high degree of non-specific binding to the vacutainer was observed. This issue could be countered easily by a combination of pre-coating with BSA solution (30%) in phosphate buffer pH 4.2, and immediate sample mixture with BSA solution after collection. To test the clinical applicability, CSF was collected in seven unique patients using alectinib (n = 1), lorlatinib (n = 2), and osimertinib (n = 4). Measured CSF trough concentrations ranged between 3.37 and 116 ng/mL.

Pd nanoparticles decorated ultrathin 2D metal-organic framework nanosheets with enhanced peroxidase-mimic activity and colorimetric assay of glucose.

In addition to size, shape and morphology, enzyme-mimetic property could be efficiently regulated by controlling composition, forming complexes or hybrids, and surface modification. Herein, Pd nanoparticles with an average diameter of 2.52 nm were decorated on ultrathin 2D copper(ii)-porphyrin derived metal-organic framework (MOF) nanosheets by a simple reduction method for catalytic activity regulation. In comparison with other nanozymes, the as-synthesized Pd modified 2D MOF hybrid nanosheets (Pd@Cu-TCPP(Fe)) presented excellent peroxidase-mimic activity, exhibiting an even superior catalytic ability towards H2O2 with a Michaelis-Menten constant as low as 2.33 mM. Based on a cascade reaction between glucose oxidase and Pd@Cu-TCPP(Fe), a colorimetric method for the detection of glucose was established and validated with a wide linear range (0.2-8.0 mM), good recovery (89.5-94.2%) and nice reproducibility (3.65%). All these features guaranteed its excellent ability for glucose determination in human cerebrospinal fluids. This study could offer a valuable reference for constructing novel optical biosensors.

Identification and quantification of (poly)phenol and methylxanthine metabolites in human cerebrospinal fluid: evidence of their ability to cross the BBB.

Increasing evidence suggests that dietary (poly)phenols and methylxanthines have neuroprotective effects; however, little is known about whether they can cross the blood-brain barrier (BBB) and exert direct effects on the brain. We investigated the presence of (poly)phenol and methylxanthine metabolites in plasma and cerebrospinal fluid (CSF) from 90 individuals at risk of dementia using liquid chromatography-mass spectrometry and predicted their mechanism of transport across the BBB using in silico modelling techniques. A total of 123 and 127 metabolites were detected in CSF and plasma, respectively. In silico analysis suggests that 5 of the 20 metabolites quantified in CSF can cross the BBB by passive diffusion, while at least 9 metabolites require the aid of cell transporters to cross the BBB. Our results showed that (poly)phenols and methylxanthines are bioavailable, can cross the BBB via passive diffusion or transport carriers, and can reach brain tissues to exert neuroprotective effects.

Exploitation of multi-walled carbon nanotubes/Cu(ii)-metal organic framework based glassy carbon electrode for the determination of orphenadrine citrate.

Metal organic frameworks (MOFs), with structural tunability, high metal content and large surface area have recently attracted the attention of researchers in the field of electrochemistry. In this work, an unprecedented use of multi-walled carbon nanotubes (MWCNTs)/copper-based metal-organic framework (Cu-BTC MOF) composite as an ion-to-electron transducer in a potentiometric sensor is proposed for the determination of orphenadrine citrate. A comparative study was conducted between three proposed glassy carbon electrodes, Cu-MOF, (MWCNTs) and MWCNTs/Cu-MOF composite based sensors, where Cu-MOF, MWCNTs and their composite were utilized as the ion-to-electron transducers. The sensors were developed for accurate and precise determination of orphenadrine citrate in pharmaceutical dosage form, spiked real human plasma and artificial cerebrospinal fluid (ACSF). The sensors employed β-cyclodextrin as a recognition element with the aid of potassium tetrakis(4-chlorophenyl)borate (KTpCIPB) as a lipophilic ion exchanger. The sensors that were assessed based on the guidelines recommended by IUPAC and demonstrated a linear response within the concentration range of 10-7 M to 10-3 M, 10-6 M to 10-2 M and 10-8 M to 10-2 M for Cu-MOF, MWCNTs and MWCNTs/Cu-MOF composite based sensors, respectively. MWCNTs/Cu-MOF composite based sensor showed superior performance over other sensors regarding lower limit of detection (LOD), wider linearity range and faster response. The sensors demonstrated their potential as effective options for the analysis of orphenadrine citrate in quality control laboratories and in different healthcare activities.

Development and Validation of an LC-MS/MS Method for the Quantitative Determination of Contezolid in Human Plasma and Cerebrospinal Fluid

To develop and verify a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining contezolid in plasma and cerebrospinal fluid (CSF). Protein precipitation was performed on samples using linezolid as the internal standard. We used an Agilent EclipsePlus C18 column operating at 0.4 mL/min in conjunction with acetonitrile and water mobile phases for the LC-MS/MS analysis. Using the precursor-product ion pairs 409.15→269.14 (contezolid) and 338.14→195.1 (linezolid), multiple reaction monitoring was used to quantify the compounds. Plasma linearity range was 50.0 to 5000 ng/mL, and CSF was 20.0 to 1000 ng/mL (r2 = 0.999). The inter-batch and intra-batch precisions were ≤2.57% and ≤5.79%, respectively. Plasma recovered 92.94%, and CSF recovered 97.83%. Plasma, CSF, hemolytic plasma, and hyperlipidemic plasma all showed a coefficient of variation ≤ 7.44%. The stability and dilution integrity of this method were also acceptable. The study also demonstrated that artificial CSF can be used as a matrix for the preparation of standard curve samples. A simple and accurate method was developed and validated for the determination of contezolid concentrations in human plasma and CSF, which may be useful for monitoring the therapeutic effect of central nervous system medications.

Global transcriptional responses of pneumococcus to human blood components and cerebrospinal fluid.

Streptococcus pneumoniae (pneumococcus) is a leading cause of severe invasive infectious diseases such as sepsis and meningitis. Understanding how pneumococcus adapts and survive in the human bloodstream environment and cerebrospinal fluid (CSF) is important for development of future treatment strategies. This study investigates the global transcriptional response of pneumococcus to human blood components and CSF acquired from discarded and anonymized patient samples. Extensive transcriptional changes to human blood components were observed during early stages of interaction. Plasma-specific responses were primarily related to metabolic components and include strong downregulation of fatty acid biosynthesis genes, and upregulation of nucleotide biosynthesis genes. No transcriptional responses specific to the active plasma proteins (e.g., complement proteins) were observed during early stages of interaction as demonstrated by a differential expression analysis between plasma and heat-inactivated plasma. The red blood cell (RBC)-specific response was far more complex, and included activation of the competence system, differential expression of several two-component systems, phosphotransferase systems and transition metal transporter genes. Interestingly, most of the changes observed for CSF were also observed for plasma. One of the few CSF-specific responses, not observed for plasma, was a strong downregulation of the iron acquisition system piuBCDA. Intriguingly, this transcriptomic analysis also uncovers significant differential expression of more than 20 small non-coding RNAs, most of them in response to RBCs, including small RNAs from uncharacterized type I toxin-antitoxin systems. In summary, this transcriptomic study identifies key pneumococcal metabolic pathways and regulatory genes involved with adaptation to human blood and CSF. Future studies should uncover the potential involvement of these factors with virulence in-vivo.

Hevin/Sparcl1 drives pathological pain through spinal cord astrocyte and NMDA receptor signaling.

High endothelial venule protein/SPARC-like 1 (hevin/Sparcl1) is an astrocyte-secreted protein that regulates synapse formation in the brain. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared with WT mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than WT mice from neuropathic pain after nerve injury. Intrathecal injection of WT hevin was sufficient to induce persistent mechanical allodynia in naive mice. In hevin-null mice with nerve injury, adeno-associated-virus-mediated (AAV-mediated) re-expression of hevin in glial fibrillary acidic protein-expressing (GFAP-expressing) spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin-potentiated N-Methyl-D-aspartic acid (NMDA) currents are mediated by GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing Ab against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin that was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal Ab may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.

ULTRASENSITIVE NEUROFILAMENT LIGHT CHAIN (NfL) IMMUNOASSAY FOR THE SMCxPRO™ ASSAY PLATFORM

AbstractBackgroundThe recent advent of ultrasensitive assay technologies has enabled the measurement of low‐abundance blood‐based biomarkers of neurodegenerative conditions, thereby addressing the need for invasive nature of cerebrospinal fluid (CSF) collection, allowing for large scale sample acquisition and screening in research, and empowering studies into the transition between health and disease. Utilizing the SMCxPRO™ ultrasensitive immunoassay platform, we have developed an assay for the detection of neurofilament light chain (NfL) in human serum, plasma and CSF samples across healthy and diseased states. NfL is a valuable neurodegenerative biomarker for accessing progression and treatment of Alzheimer’s Disease (AD), Multiple Sclerosis (MS) and Huntington’s Disease, and is under investigation as a potential biomarker for several other neurological disorders.MethodRigorous assay development procedures were undertaken in an effort to produce an NfL immunoassay capable of accurately and reproducibly detecting NfL in human serum, plasma, and CSF samples.ResultA robust kit for consistent and reliable NfL measurement at sub‐pg/mL concentrations has been developed and characterized using Single Molecule Counting (SMC™) ultrasensitive immunoassay technology. The developed assay demonstrates excellent performance characteristics for spike/recovery values and dilutional linearity (90‐110% for all three matrices). NfL was detected in all sample types tested. Increased NfL sample levels in AD and MS samples as compared to normal serum, plasma and CSF samples was also observed.ConclusionOur SMC™ NfL immunoassay kit provides a valuable tool for research in the areas of neurodegenerative disease and neurological injury.

Detection of Alzheimer’s by Machine Learning-assisted Vibrational Spectroscopy in Human Cerebrospinal Fluid

Nowadays, the diagnosis of Alzheimer’s disease is a complex process that involves several clinical tests. Cerebrospinal fluid contains common Alzheimer-related biomarkers that include amyloid beta 1-42 (Aβ1-42) and tau proteins. In this work, we propose vibrational spectroscopy techniques supported by machine learning for the detection of biomarkers in cerebrospinal fluid that are related with Alzheimer’s by prediction models. Vibrational spectroscopy provides the entire biochemical composition of the body fluid, and thus, small but typical physiological changes related with the pathology can be ascertained. Within a machine learning framework, Raman and FTIR spectra were analyzed, which were taken from samples of healthy volunteers in comparison with samples from patients clinically diagnosed with Alzheimer’s. We find that a logistic regression model can discriminate between healthy control and Alzheimer’s patients with a precision of 98%, when the input for the model combines data from both vibrational spectroscopy methods. Our approach shows high discriminative capabilities and constitutes a proof of concept for an alternative and accurate tool for the diagnosis of Alzheimer’s disease.

Gut microbe‐modulated metabolites are longitudinally associated with higher neurodegeneration biomarkers in cerebrospinal fluid (CSF)

AbstractBackgroundGut bacterial metabolites in circulation and postmortem brain tissue differ in abundance between cognitively unimpaired (CU) and Alzheimer’s disease (AD) individuals. Furthermore, in mice chronic injection of bacterial components exacerbates AD pathology. In CSF, we found the microbially‐produced trimethylamine‐N‐oxide was linked to AD and neurodegeneration biomarkers. However, the broader extent to which microbe‐modulated metabolites relate to AD and neurodegeneration is unknown. We tested the hypothesis that in CSF of non‐demented adults, elevated metabolites would longitudinally associate with higher biomarkers.MethodCSF was collected biannually in the Wisconsin Alzheimer’s Disease Research Center and Wisconsin Registry for Alzheimer’s Prevention studies. Biomarkers of pTau181, Aβ42, tTau, axonal (NfL) and synaptic (neurogranin, α‐synuclein) degeneration were quantified using Elecsys assays (NeuroToolKit robust prototype, Roche Diagnostics). Metabolite relative abundances were quantified via untargeted metabolomics analysis (ultrahigh performance liquid chromatography‐tandem mass spectrometry, Metabolon). A priori literature and database review identified 76 detected microbe‐modulated metabolites. 200 CU participants with two or more analyzed CSF samples were included in statistical analysis (Table 1). For each metabolite‐biomarker pair, multilevel linear growth modeling tested person‐level mean metabolite effects on biomarker slope and intercept, and time‐varying metabolite effects on biomarker intercept (Figure 1). Covariates included age, sex, and APOE risk score; Benjamini‐Hochberg corrections adjusted for Type I error inflation.ResultSeven metabolites were positively associated with biomarker slope or intercept (Table 2). Mean gluconate, glycerate, and oxalate levels were correlated with increased tTau slope, and N‐acetylaspartate, N‐acetylglutamate and cholesterol were correlated with higher a‐synuclein or neurogranin intercepts. Phenol sulfate was linked to tTau and α‐synuclein trajectories. To our knowledge, this is the first study detecting phenol sulfate in human CSF. No metabolites significantly associated with NfL or pTau181/Aβ42.ConclusionIn CSF, participants with higher mean levels of bacteria‐utilized metabolites exhibited accelerated neurodegeneration (tTau). Gluconate is metabolized by Bifidobacterium and Lactobacillus, two genera reduced in AD. Furthermore, the microbially‐produced phenol sulfate predicted greater tTau development and synaptic degeneration. Uremic toxins related to phenol sulfate dysregulate endothelial barriers in the central nervous system and vascular tissues. Additional studies should evaluate microbial metabolites as mediators of gut microbiome‐neurodegeneration relationships, and should determine if they represent modifiable pathways.

Identifying risk genes and therapeutics for Alzheimer’s disease through integration multi‐omics and drug‐perturbation signatures

AbstractBackgroundAlzheimer’s disease (AD) is a severe and irreversible neurodegenerative disease with no effective treatments currently availableMethodTo better understand AD mechanisms and identify therapeutics, we collected AD GWAS loci from the largest meta‐analysis and incorporated multiple lines of genomic evidence, including human brain chromatin interaction map in microglia, brain cell‐type specific expression and gene‐gene interaction network, to infer high‐confidence risk genes (HRGs) at each GWAS locus. We then used the HRGs and network propagation to construct a genetic perturbation signature of AD, and queried a database of drug‐induced transcriptomic signatures (LINCS 1000) to evaluate compounds that are able to reverse the genetic perturbation signature.ResultThis resulted in 72 HRGs, which were further validated in multiple independent datasets of human microglia expression quantitative trait loci (eQTLs) and splicing quantitative trait loci (sQTLs), protein quantitative trait loci (pQTLs) in human brain, cerebrospinal fluid (CSF) and plasma, and genes of protein targets in brain and CSF associated with asymptomatic AD. Pathway analysis implicates the involvement of endocytosis and amyloid pathways, and highlights cellular compartments such as gamma‐secretase complex, NF‐kappaB complex, neurofibrillary tangle and endosome. Query of the genetic perturbation signature of AD against LINCS 1000 revealed several drugs that can reverse the genetic perturbation signature.ConclusionIn conclusion, by incorporating multiple lines of evidence and resources, our study offers new insights into the pathological processes underlying AD and potential therapeutic drugs.

A single-cell analysis framework allows for characterization of CSF leukocytes and their tissue of origin in multiple sclerosis.

Peripheral central nervous system (CNS)-infiltrating lymphocytes are a hallmark of relapsing-remitting multiple sclerosis. Tissue-resident memory T cells (TRM) not only populate the healthy CNS parenchyma but also are suspected to contribute to multiple sclerosis pathology. Because cerebrospinal fluid (CSF), unlike CNS parenchyma, is accessible for diagnostics, we evaluated whether human CSF, apart from infiltrating cells, also contains TRM cells and CNS-resident myeloid cells draining from the parenchyma or border tissues. Using deep generative models, we integrated 41 CSF and 14 CNS parenchyma single-cell RNA sequencing (scRNAseq) samples from eight independent studies, encompassing 120,629 cells. By comparing CSF immune cells collected during multiple sclerosis relapse with cells collected during therapeutic very late antigen-4 blockade, we could identify immune subsets with tissue provenance across multiple lineages, including CNS border-associated macrophages, CD8 and CD4 TRM cells, and tissue-resident natural killer cells. All lymphocytic CNS-resident cells shared expression of CXCR6 but showed differential ITGAE expression (encoding CD103). A common signature defined CD4 and CD8 TRM cells by expression of ZFP36L2, DUSP1, and ID2. We further developed a user interface-driven application based on this analysis framework for atlas-level cell identity transfer onto new CSF scRNAseq data. Together, these results define CNS-resident immune cells involved in multiple sclerosis pathology that can be detected and monitored in CSF. Targeting these cell populations might be promising to modulate immunopathology in progressive multiple sclerosis and other neuroinflammatory diseases.

Conserved YKL-40 changes in mice and humans after postoperative delirium

Delirium is a common postoperative neurologic complication among older adults. Despite its prevalence (14%–50%) and likely association with inflammation, the exact mechanisms that underpin postoperative delirium are unclear. This project aimed to characterize systemic and central nervous system (CNS) inflammatory changes following surgery in mice and humans. Matched plasma and cerebrospinal fluid (CSF) samples from the “Investigating Neuroinflammation Underlying Postoperative Brain Connectivity Changes, Postoperative Cognitive Dysfunction, Delirium in Older Adults” (INTUIT; NCT03273335) study were compared to murine endpoints. Delirium-like behavior was evaluated in aged mice using the 5-Choice Serial Reaction Time Test (5-CSRTT). Using a well established orthopedic surgical model in the FosTRAP reporter mouse we detected neuronal changes in the prefrontal cortex, an area implicated in attention, but notably not in the hippocampus. In aged mice, plasma interleukin-6 (IL-6), chitinase-3-like protein 1 (YKL-40), and neurofilament light chain (NfL) levels increased after orthopedic surgery, but hippocampal YKL-40 expression was decreased. Given the growing evidence for a YKL-40 role in delirium and other neurodegenerative conditions, we assayed human plasma and CSF samples. Plasma YKL-40 levels were similarly increased after surgery, with a trend toward a greater postoperative plasma YKL-40 increase in patients with delirium. However, YKL-40 levels in CSF decreased following surgery, which paralleled the findings in the mouse brain. Finally, we confirmed changes in the blood-brain barrier (BBB) as early as 9 h after surgery in mice, which warrants more detailed and acute evaluations of BBB integrity following surgery in humans. Together, these results provide a nuanced understanding of neuroimmune interactions underlying postoperative delirium in mice and humans, and highlight translational biomarkers to test potential cellular targets and mechanisms.

DDEL-07. DETERMINATION OF TOTAL AND UNBOUND LEVELS OF A POTENT INHIBITOR OF POLY ADP-RIBOSE POLYMERASE, NIRAPARIB, IN HUMAN PLASMA, CEREBROSPINAL FLUID AND BRAIN TUMOR BY A VALIDATED LC-MS/MS ASSAY

Abstract BACKGROUND Niraparib is an orally available inhibitor of poly ADP-ribose polymerase with high selectivity for PARP1 and PARP2. It is currently approved in the United States for the treatment of ovarian, fallopian tube, and primary peritoneal cancer. Herein we present the development and validation of an LC-MS/MS method for determination of total and unbound levels of niraparib in human plasma, cerebrospinal fluid (CSF) and glioblastoma tissue. METHODS Niraparib was extracted from human plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Unbound fractions of niraparib in plasma and brain tissues were determined by equilibrium dialysis. Validation was performed using Sciex ExionLC UHPLC system coupled with Sciex QTRAP 6500+ MS/MS detector using positive electrospray ionization mode. The method was validated according to FDA guidelines and CAP/CLIA regulations. RESULTS The method was validated for 1-10,000 nmol/L concentration range using plasma as matrix for all biospecimens. The chromatographic separation was performed on Phenomenex Kinetex™ PS C18 column with total run time of 3.5 min with gradient elution. Maximum coefficient of variation for intra- and inter-day precision was 10.6% and the accuracy was within 92.8-118.5% in all matrices. The analyte is stable in plasma and brain homogenate for at least 6 hours at room temperature (RT) and at least 32 days at -20°C. Stability of stock and working solutions was demonstrated for at least 21 hours (RT) and 41 days (4°C). The fraction unbound of niraparib in human plasma and brain were determined to be 0.15 and 0.05, respectively. CONCLUSIONS A sensitive and rapid bioanalytical method is developed and validated to quantify total and unbound niraparib levels in human plasma, CSF and glioblastoma tissue. This method is currently employed to assess plasma pharmacokinetics and CNS penetration of niraparib in newly-diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513).

Upconversion NaYF4:Yb/Er-TiO2-Ti3C2 Heterostructure-Based Near-Infrared Light-Driven Photoelectrochemical Biosensor for Highly Sensitive and Selective d-Serine Detection.

A near-infrared (NIR) light-driven NaYF4:Yb/Er-TiO2-Ti3C2 (NYF-TiO2-Ti3C2) heterostructure-based photoelectrochemical (PEC) biosensing platform was constructed for highly sensitive d-serine (d-ser) detection. Accurate d-ser detection depends on the model biocatalyst, d-amino acid oxidase (DAAO), which converts d-ser into hydroxypyruvate and an equimolar concentration of hydrogen peroxide (H2O2) via an enzymatic reaction. The TiO2-Ti3C2 semiconductor and NaYF4:Yb/Er optical transducer formed a Schottky junction that provided an irreversible channel for electron transfer. Infrared light was converted into absorbable multiemission light, thereby effectively increasing light absorption. Simultaneously, the generated H2O2 rapidly scavenged photogenerated holes to separate electron-hole pairs, which amplified the photocurrent signal. Under optimal conditions, the NIR light-driven PEC biosensor exhibited an excellent PEC performance for d-ser detection, with a wide linear range of 2-1650 μmol L-1 and detection limit as low as 0.286 μmol L-1. Importantly, high detection reproducibility and accuracy were achieved using this strategy for analyzing human serum and rat cerebrospinal fluid (CSF) specimens. The admirable applicability of the NYF-TiO2-Ti3C2-based PEC biosensor for detecting d-ser may lead to further opportunities for detecting other disease-related biomarkers.

Wasteosomes (corpora amylacea) of human brain can be phagocytosed and digested by macrophages

BackgroundCorpora amylacea of human brain, recently renamed as wasteosomes, are granular structures that appear during aging and also accumulate in specific areas of the brain in neurodegenerative conditions. Acting as waste containers, wasteosomes are formed by polyglucosan aggregates that entrap and isolate toxic and waste substances of different origins. They are expelled from the brain to the cerebrospinal fluid (CSF), and can be phagocytosed by macrophages. In the present study, we analyze the phagocytosis of wasteosomes and the mechanisms involved in this process. Accordingly, we purified wasteosomes from post-mortem extracted human CSF and incubated them with THP-1 macrophages. Immunofluorescence staining and time-lapse recording techniques were performed to evaluate the phagocytosis. We also immunostained human hippocampal sections to study possible interactions between wasteosomes and macrophages at central nervous system interfaces.ResultsWe observed that the wasteosomes obtained from post-mortem extracted CSF are opsonized by MBL and the C3b complement protein. Moreover, we observed that CD206 and CD35 receptors may be involved in the phagocytosis of these wasteosomes by THP-1 macrophages. Once phagocytosed, wasteosomes become degraded and some of the resulting fractions can be exposed on the surface of macrophages and interchanged between different macrophages. However, brain tissue studies show that, in physiological conditions, CD206 but not CD35 receptors may be involved in the phagocytosis of wasteosomes.ConclusionsThe present study indicates that macrophages have the machinery required to process and degrade wasteosomes, and that macrophages can interact in different ways with wasteosomes. In physiological conditions, the main mechanism involve CD206 receptors and M2 macrophages, which trigger the phagocytosis of wasteosomes without inducing inflammatory responses, thus avoiding tissue damage. However, altered wasteosomes like those obtained from post-mortem extracted CSF, which may exhibit waste elements, become opsonized by MBL and C3b, and so CD35 receptors constitute another possible mechanism of phagocytosis, leading in this case to inflammatory responses.

Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer.

Targeted mass spectrometric analysis is widely employed across various omics fields. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and small molecules. Selected reaction monitoring and multiple reaction monitoring (MRM) have been a method of choice for targeted structural studies of biomolecules. However, innovations in instrument designs have led to the development of parallel reaction monitoring (PRM). PRM detects all product ions simultaneously rather than optimizing/preselecting the target glycan transitions, simplifying the analytical workflow. By reducing background interference, increasing selectivity/specificity, and improving data quality, PRM allows reliable quantification of target glycans in complex matrices. PRM can also improve sensitivity for detecting low-abundance target glycans and reduce low-level limit of quantification values with an improved S/N ratio. PRM's advantages are attributed to the development of sensitive and highly selective mass analyzers, orbitrap, and time of flight. In this study, we developed a sensitive PRM method for the quantitative analysis of permethylated N-glycans, an important class of disease biomarkers, using a quadrupole-orbitrap hybrid mass spectrometer. Pooled human cerebrospinal fluid was used for the study as a source of permethylated N-glycans. The method illustrates the fragmentation of N-glycans at different collision energies as well as the optimization of collision energy. The method also detects low-abundance N-glycans more efficiently than MRM. This study is the first attempt to develop a sensitive PRM-based method to analyze permethylated N-glycans.

Validated UPLC-MS/MS method for the analysis of vitamin B6 pyridoxal 5́-phosphate, pyridoxal, pyridoxine, pyridoxamine, and pyridoxic acid in human cerebrospinal fluid

Vitamin B6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories.Until today, vitamin B6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory.

UHPLC-ESI-MS/MS assay for quantification of endocannabinoids in cerebrospinal fluid using surrogate calibrant and surrogate matrix approaches

Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90–3802 pM for 1-AG, 90–724 pM for 2-AG, 12–5226 pM for LEA, 33–13,942 for OEA, 34–23,850 pM for 2-AGE, 72–30,190 for PEA and 10–4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of − 14.7 to + 12.3% at LLOQ and − 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and <15% for the remaining levels) as well. It was finally used to quantify endocannabinoids in human cerebrospinal fluid obtained from 118 donors. Accurate quantification of endogenous compounds in biological samples was achieved by using two different principal approaches (surrogate matrix for AEA, 2-AG, OEA, 2-AGE, LEA and PEA, and surrogate calibrant for AEA only) and they were evaluated by use of the Passing-Bablok regression. Concentrations (median) of CSF samples of patients suffering from CNS infection and controls were found to be around 160 pM for 1- and 2-AG, 86 pM for AEA, 62 for 2-AGE, 58 for LEA, 93 pM for PEA, and 83 pM for OEA.

Recommendation: Treatment of clinical long COVID encephalopathies with nasal administered mesenchymal stromal cell extracellular vesicles

We propose therapy with extracellular vesicles (EVs) for dominant central nervous system aspects of chronic Long COVID Syndromes (LCS). These clinical conditions have a delayed onset of 1–3 months following the cessation of active SARS-CoV-2 virus infections that cause an acute disease called COVID-19. The therapy of LCS will be achieved by direct access to the central nervous system (CNS) by nasal administration of small EVs derived from Mesenchymal Stromal Cells (MSC). When administered nasally, they target CNS microglia and endothelia involved in LCS encephalopathy, as indicated by experimental animal models and human autopsy and spinal fluid studies. Underlying this approach is the discovery that MSC-sEV treatment for healing neuro injury targets, microglia, and macrophages that then likely release secondary trophic EVs that affect the local capillary endothelial cells to restore vascular integrity. It is postulated that the pathways of endothelial and neural pathologies in acute SARS-CoV-2 virus infections may carry over to produce underlying vascular and neurological defects mediating LCS that are susceptible to this proposed nasal therapy with MSC-sEVs.