Human Cerebrospinal Fluid Samples Research Articles

Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. Challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

HGF secreted by hUC-MSCs mitigates neuronal apoptosis to repair the injured spinal cord via phosphorylation of Akt/FoxO3a pathway

Spinal cord injury (SCI) can cause severe and permanent neurological damage, and neuronal apoptosis could inhibit functional recovery of damaged spinal cord greatly. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have great potential to repair SCI because of a series of advantages, including inhibition of neuronal apoptosis and multiple differentiation. The former may play an important role. However, the detailed regulatory mechanism associated with the inhibition of neuronal apoptosis after hUC-MSCs administration has not been elucidated. In this study, proteomics analysis of precious human cerebrospinal fluid (CSF) samples collected from SCI subjects receiving hUC-MSCs delivery indicated that hepatocyte growth factor (HGF) is largely involved in SCI repair. Furthermore, overexpression of HGF derived from hUC-MSCs could decrease reactive oxygen species to prevent neuron apoptosis to the maximum, and thus lead to significant recovery of spinal cord dysfunction. Moreover, HGF could promote phosphorylation of Akt/FoxO3a pathway to decrease reactive oxygen species to reduce neuron apoptosis. For the first time, our research revealed that HGF secreted by hUC-MSCs inhibits neuron apoptosis by phosphorylation of Akt/FoxO3a to repair SCI. This study provides important clues associated with drug selection for the effective treatment of SCI in humans.

Changes of tRNA-Derived Fragments by Alzheimer's Disease in Cerebrospinal Fluid and Blood Serum.

Alzheimer's disease (AD) is the most common type of dementia, affecting individuals over 65. AD is also a multifactorial disease, with disease mechanisms incompletely characterized, and disease-modifying therapies are marginally effective. Biomarker signatures may shed light on the diagnosis, disease mechanisms, and the development of therapeutic targets. tRNA-derived RNA fragments (tRFs), a family of recently discovered small non-coding RNAs, have been found to be significantly enhanced in human AD hippocampus tissues. However, whether tRFs change in body fluids is unknown. To investigate whether tRFs in body fluids are impacted by AD. We first used T4 polynucleotide kinase-RNA-seq, a modified next-generation sequencing technique, to identify detectable tRFs in human cerebrospinal fluid and serum samples. The detectable tRFs were then compared in these fluids from control, AD, and mild cognitive impairment patients using tRF qRT-PCR. The stability of tRFs in serum was also investigated by checking the change in tRFs in response to protein digestion or exosome lysis. Among various tRFs, tRF5-ProAGG seemed to be impacted by AD in both cerebrospinal fluid and serum. AD-impacted serum tRF5-ProAGG showed a correlation with the AD stage. Putative targets of tRF5-ProAGG in the hippocampus were also predicted by a computational algorithm, with some targets being validated experimentally and one of them being in a negative correlation with tRF5-ProAGG even using a small size of samples. tRF5-ProAGG showed the potential as an AD biomarker and may play a role in disease progression.

Human Cerebrospinal Fluid Sample Preparation and Annotation for Integrated Lipidomics and Metabolomics Profiling Studies

Cerebrospinal fluid (CSF) is a metabolically diverse biofluid and a key specimen for exploring biochemical changes in neurodegenerative diseases. Detecting lipid species in CSF using mass spectrometry (MS)-based techniques remains challenging because lipids are highly complex in structure, and their concentrations span over a broad dynamic range. This work aimed to develop a robust lipidomics and metabolomics method based on commonly used two-phase extraction systems from human CSF samples. Prioritizing lipid detection, biphasic extraction methods, Folch, Bligh and Dyer (B&D), Matyash, and acidified Folch and B&D (aFolch and aB&D) were compared using 150 μL of human CSF samples for the simultaneous extraction of lipids and metabolites with a wide range of polarity. Multiple chromatographical separation approaches, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and gas chromatography (GC), were utilized to characterize human CSF metabolome. The aB&D method was found as the most reproducible technique (RSD < 15%) for lipid extraction. The aB&D and B&D yielded the highest peak intensities for targeted lipid internal standards and displayed superior extracting power for major endogenous lipid classes. A total of 674 unique metabolites with a wide polarity range were annotated in CSF using, combining RPLC-MS/MS lipidomics (n = 219), HILIC-MS/MS (n = 304), and GC-quadrupole time of flight (QTOF) MS (n = 151). Overall, our findings show that the aB&D extraction method provided suitable lipid coverage, reproducibility, and extraction efficiency for global lipidomics profiling of human CSF samples. In combination with RPLC-MS/MS lipidomics, complementary screening approaches enabled a comprehensive metabolite signature that can be employed in an array of clinical studies.Graphical abstract

B-083 Novel High Affinity Monoclonal Antibodies for Specific Detection of Neurofilament Light Protein in Serum and Cerebrospinal Fluid

Abstract Background Neurofilaments are very important protein components of axons, and they maintain the function of nervous system. Cerebrospinal fluid (CSF) and serum neurofilament levels elevate due to damages in nervous system making neurofilaments promising biomarkers for neural diseases and injuries, such as Alzheimer’s disease, traumatic brain injury and Multiple sclerosis. Based on their size, neurofilaments are divided in groups: Neurofilament light (NfL), neurofilament medium (NfM), neurofilament heavy (NfH), α-internexin, and peripherin. Of these, NfL is considered to be the most promising biomarker since it is the most abundant form of neurofilaments. Methods We have developed four mouse monoclonal antibodies against human NfL. These antibodies, designated as Anti-h NfL 12601, 12603, 12604 or 12605, were tested as pairs in sandwich fluorescence-based immunoassay (FIA) with NfL spiked serum and buffer to determine linear measuring ranges and the matrix effect. Furthermore, the ability of the antibodies to measure NfL in clinical samples was tested in sandwich FIA by testing 10 CSF samples in the range of 0 to 5 083 pg/mL. The antibodies were also compared against commercially available reference antibodies in FIA. Kinetic parameters were determined with bio-layer interferometry (BLI). Results In sandwich FIA, the widest linear measuring range of 10 to 40 960 pg/mL of recombinant human NfL spiked in normal serum was achieved with antibody pair 12603 and 12604. In contrast, the commercial reference antibody pair achieved a linear measuring range of 2560 to 40 960 pg/mL. Matrix effect caused by serum was negligible. With clinical samples, the best correlation with the reference method (NF-light CSF ELISA from Uman Diagnostics) with a coefficient of correlation of 0.986 was achieved with antibody pair 12604 and 12601. This pair also showed a good correlation with the commercial reference pair with a coefficient of correlation of 0.984. In kinetics measurements, none of four antibodies showed dissociation indicating very high affinity. The fastest association of 4.0 × 105 1/Ms was achieved with antibody 12605. Conclusion These results demonstrate that the newly developed mouse monoclonal NfL antibodies are promising tools for development of diagnostic immunoassays measuring NfL levels in human serum and CSF samples. Comparison against commercial reference antibodies indicates that higher NfL detection sensitivity and wider detection ranges can be achieved with antibody pairs developed in this study. Additionally, the kinetic measurement results forecast these antibodies to be suitable for fast reaction kinetic assay formats, such as lateral flow assays, which might become a key resolution in acute neural injury diagnostics.

Reliable HPLC-UV method for therapeutic levetiracetam monitoring in serum and cerebrospinal fluid of patients with epilepsy

The present study was to develop and validate a rapid analytical method of high-performance liquid chromatography with UV (HPLC/UV) detection for the determination of levetiracetam (LEV) concentration in human serum and cerebrospinal fluid (CSF) samples. The newly developed method was used to measure LEV concentration in the serum/CSF of 29 patients with different types of epilepsy; 52% (group I, n = 15) were treated with LEV in monotherapy, 48% (group II, n = 14) were co-administered with other AEDs. Serum and CSF levels of LEV were correlated with patients dosage, concomitant AEDs, therapeutic effect, and adverse drug reactions (ADRs). The calibration curves for serum and CSF were linear in the range 0.5-100 and 0.5-50 mg/L, respectively, with a coefficient correlation (R) value &gt; 0.998 in each case. The quantitation limit was 0.5 mg/L in serum and CSF. The validated method was found to be selective, precise, and accurate. Optimal Cssmin values according to International League Against Epilepsy (12-46 mg/L) were achieved in 40 and 50% of patients in group I and II, and did not correlate with treatment efficacy in 27 and 21% patients, respectively. The ratio of serum LEV to CSF concentration (Kp) after 2 h administration LEV ranged in group I from 0.31 to 0.92 and in group II from 0.18 to 0.65. These findings indicate that the developed simple and rapid HPLC/UV method may be useful for monitoring serum/CSF LEV concentrations in patients receiving standard doses.

Influence of apolipoprotein E (apoE) on ADAM10 secretase in cerebrospinal fluid of older adults with Alzheimer’s disease

AbstractBackgroundThe ε4 allele of APOE is the strongest genetic risk for sporadic late‐onset Alzheimer’s disease (AD), and is associated with an increase in the levels of β‐amyloid (Aβ) deposition. The main α‐secretase that inhibits the Aβ deposition is the ADAM10, which is altered in the blood and cerebrospinal fluid (CSF) of older adults with AD.MethodTo assess whether the levels of ADAM10 forms are altered in CSF of AD and non‐dementia control (NDC) individuals with different APOE genotype, we analysed a total of 13 samples from NDC (mean age = 66.3 ±9.8; T‐tau = 305 ±135 ng/L; Aβ42 = 780 ±253 ng/L) and 41 from AD (mean age 77.0 ±6.2; T‐tau = 845 ±356.1 ng/L; Aβ42 = 456 ± 66 ng/L), subdivided into NDC APOE ε3/ε3 (n = 8), NDC APOE ε3/ε4 (n = 5), AD APOE ε3/ε3 (n = 13), AD APOE ε3/ε4 (n = 14) and AD APOE ε4/ε4 (n = 14). ADAM10 was characterized in human CSF samples by western blotting.ResultAn antibody raised against to the ectodomain region of ADAM10, common to all species, recognized three bands, which might be the proADAM10 (immature form retaining the prodomain), ADAM10f (mature unprocessed full‐length form) and sADAM10 (truncated fragment). Regarding proADAM10, no differences were found among NDC with different APOE genotype. However, the AD APOE ε4/ε4 showed a reduction, which was significantly lower than AD APOE ε3/ε3 (p = 0.0011), NDC APOE ε3/ε3 (p = 0.0460) and NDC APOE ε3/ε3 (p = 0.0012), suggesting the impact of being homozygous for APOE ε4 allele on proADAM10 levels. In the ADAM10f and sADAM10, we observed similar findings. For both species of protein, AD groups carriers of APOE ε4 allele, that is, AD APOE ε3/ε4 and AD APOE ε4/ε4, presented significant lower levels of ADAM10f compared with both non‐carriers subgroups (AD APOE ε3/ε3 and NDC APOE ε3/ε3).ConclusionThe results indicated that being a carrier of the APOE ε4 allele and having developed AD are associated with lower levels of ADAM10f and sADAM10. This study contributes to a better understanding the impact of apoE on the disease.

Matrix disequilibrium in Alzheimer's disease and conditions that increase Alzheimer's disease risk.

Alzheimer's Disease (AD) and related dementias are a leading cause of death globally and are predicted to increase in prevalence. Despite this expected increase in the prevalence of AD, we have yet to elucidate the causality of the neurodegeneration observed in AD and we lack effective therapeutics to combat the progressive neuronal loss. Throughout the past 30 years, several non-mutually exclusive hypotheses have arisen to explain the causative pathologies in AD: amyloid cascade, hyper-phosphorylated tau accumulation, cholinergic loss, chronic neuroinflammation, oxidative stress, and mitochondrial and cerebrovascular dysfunction. Published studies in this field have also focused on changes in neuronal extracellular matrix (ECM), which is critical to synaptic formation, function, and stability. Two of the greatest non-modifiable risk factors for development of AD (aside from autosomal dominant familial AD gene mutations) are aging and APOE status, and two of the greatest modifiable risk factors for AD and related dementias are untreated major depressive disorder (MDD) and obesity. Indeed, the risk of developing AD doubles for every 5 years after ≥ 65, and the APOE4 allele increases AD risk with the greatest risk in homozygous APOE4 carriers. In this review, we will describe mechanisms by which excess ECM accumulation may contribute to AD pathology and discuss pathological ECM alterations that occur in AD as well as conditions that increase the AD risk. We will discuss the relationship of AD risk factors to chronic central nervous system and peripheral inflammation and detail ECM changes that may follow. In addition, we will discuss recent data our lab has obtained on ECM components and effectors in APOE4/4 and APOE3/3 expressing murine brain lysates, as well as human cerebrospinal fluid (CSF) samples from APOE3 and APOE4 expressing AD individuals. We will describe the principal molecules that function in ECM turnover as well as abnormalities in these molecular systems that have been observed in AD. Finally, we will communicate therapeutic interventions that have the potential to modulate ECM deposition and turnover in vivo.

Magnetic ionic liquid-based liquid-liquid microextraction followed by ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry for simultaneous determination of neurotransmitters in human cerebrospinal fluid and plasma

A green, efficient and easy sample pretreatment method of magnetic ionic liquid-based liquid-liquid microextraction (MIL-based LLME) combined with a sensitive, rapid and precise analytical method of ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-QqQ/MS2) was developed to simultaneously - determining of neurotransmitters (NTs) in biosamples. Two magnetic ionic liquids (MILs), [P6,6,6,14]3[GdCl6] and [P6,6,6,14]2[CoCl4] tested, and the latter was selected as the extraction solvent due to its advantages of visual recognition, paramagnetic behavior and higher extraction efficiency. Facile magnetic separation of MIL containing analytes from matrix was realized by applying external magnetic field without rather than centrifugation. Experimental parameters that would influence the extraction efficiency, including type and amount of MIL, extraction time, speed of the vortex process, salt concentration, and environmental pH, were optimized obtained. The proposed method was successfully applied to the simultaneous extraction and determination of 20 NTs in human cerebrospinal fluid and plasma samples. Excellent analytical performance indicates the broad potential of this method for clinical diagnosis and therapy of neurological diseases.

The neuronal pentraxin Nptx2 regulates complement activity and restrains microglia-mediated synapse loss in neurodegeneration.

Complement overactivation mediates microglial synapse elimination in neurological diseases such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), but how complement activity is regulated in the brain remains largely unknown. We identified that the secreted neuronal pentraxin Nptx2 binds complement C1q and thereby regulates its activity in the brain. Nptx2-deficient mice show increased complement activity, C1q-dependent microglial synapse engulfment, and loss of excitatory synapses. In a neuroinflammation culture model and in aged TauP301S mice, adeno-associated virus (AAV)-mediated neuronal overexpression of Nptx2 was sufficient to restrain complement activity and ameliorate microglia-mediated synapse loss. Analysis of human cerebrospinal fluid (CSF) samples from a genetic FTD cohort revealed reduced concentrations of Nptx2 and Nptx2-C1q protein complexes in symptomatic patients, which correlated with elevated C1q and activated C3. Together, these results show that Nptx2 regulates complement activity and microglial synapse elimination in the brain and that diminished Nptx2 concentrations might exacerbate complement-mediated neurodegeneration in patients with FTD.

CCR5 deficiency normalizes TIMP levels, working memory, and gamma oscillation power in APOE4 targeted replacement mice

The APOE4 allele increases the risk for Alzheimer's disease (AD) in a dose-dependent manner and is also associated with cognitive decline in non-demented elderly controls. In mice with targeted gene replacement (TR) of murine APOE with human APOE3 or APOE4, the latter show reduced neuronal dendritic complexity and impaired learning. APOE4 TR mice also show reduced gamma oscillation power, a neuronal population activity which is important to learning and memory. Published work has shown that brain extracellular matrix (ECM) can reduce neuroplasticity as well as gamma power, while attenuation of ECM can instead enhance this endpoint. In the present study we examine human cerebrospinal fluid (CSF) samples from APOE3 and APOE4 individuals and brain lysates from APOE3 and APOE4 TR mice for levels of ECM effectors that can increase matrix deposition and restrict neuroplasticity. We find that CCL5, a molecule linked to ECM deposition in liver and kidney, is increased in CSF samples from APOE4 individuals. Levels of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the activity of ECM-degrading enzymes, are also increased in APOE4 CSF as well as astrocyte supernatants brain lysates from APOE4 TR mice. Importantly, as compared to APOE4/wild-type heterozygotes, APOE4/CCR5 knockout heterozygotes show reduced TIMP levels and enhanced EEG gamma power. The latter also show improved learning and memory, suggesting that the CCR5/CCL5 axis could represent a therapeutic target for APOE4 individuals.

Automated and fast online method for simultaneously determining a broad spectrum of per- and polyfluoroalkyl substances in a small volume of cerebrospinal fluid.

Per- and polyfluoroalkyl substances (PFASs) are potentially neurotoxic compounds. Levels of PFASs in cerebrospinal fluid (CSF) could directly reflect their potential harm to the central nervous system. Because of the variety of PFASs and the rarity of CSF, there is an urgent need to establish a rapid online method to detect a broad spectrum of PFASs accurately and simultaneously by consuming a small amount of CSF. In this study, we developed a fast and automated method to analyze 52 PFASs in human CSF samples using online TurboFlow ultra-high-performance liquid chromatography-tandem mass spectrometry. Our method offered excellent matrix-matched standard curve linearity (correlation coefficient > 0.99), good limits of quantitation (MLOQs) (0.01 to 0.08 ng mL-1), satisfactory accuracy (recoveries of 74.6%-119.1%) and precision (relative standard deviations of 1.4%-13.2%), small sample amount consumption (50 μL), and fast analysis time (18 min per sample) without complex sample pretreatment procedures. These are advantageous for the high throughput screening of PFASs in environmental epidemiology studies. Repeated freeze-thaw experiments showed that it was better to perform the analytical process soon as possible after sample collection. The established method was used to analyze PFASs in 60 people. Short-chain PFASs, perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), and novel PFASs [sodium 2-(N-ethylperfluorooctane-1-sulfonamido)ethyl phosphate (SAmPAP), perfluoroethylcyclohexanesulfonate (PFECHS), and perfluoro-3, 7-dimethyloctanoic acid (P37DMOA)] were reported in CSF for the first time. PFBA and PFPeA were detected in all samples with mean concentrations of 0.24 and 0.22 ng mL-1, respectively. We also calculated the blood-brain barrier transmission efficiency of PFASs (RPFAS), and the mean RPFBA value was above 1, which indicated that PFBA might transfer from serum to CSF.

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.

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).

Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay

Background:The use of biomarkers has become a major component of clinical trial design. In Huntington’s disease (HD), quantifying the amount of huntingtin protein (HTT) in patient cerebrospinal fluid (CSF) has served as a pharmacodynamic readout for HTT-lowering therapeutic approaches and is a potential disease progression biomarker. To date, an ultrasensitive immunoassay to quantify mutant HTT protein (mHTT) has been used, but additional assays are needed to measure other forms of HTT protein.Objective:We aimed to develop an ultrasensitive immunoassay to quantify HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild type HTT combined) in control and HD participant CSF samples.Methods:An ultrasensitive, bead-based, single molecule counting (SMC) immunoassay platform was used for the detection of HTT protein in human CSF samples.Results:A novel ultrasensitive SMC immunoassay was developed to quantify HTT protein in a polyglutamine length-independent manner and shown to measure HTT in both control and HD participant CSF samples. We validate the selectivity and specificity of the readout using biochemical and molecular biology tools, and we undertook a preliminary analytical qualification of this assay to enable its clinical use. We also used this novel assay, along with the previously described mHTT assay, to analyze CSF from control and HD participants. The results of this preliminary set suggests that correlation is present between mHTT and the polyglutamine length-independent HTT levels in human CSF.Conclusion:We have developed a novel ultrasensitive immunoassay that is able to quantify HTT protein in a polyglutamine length-independent manner in control and HD participant CSF.

Boost-DiLeu: Enhanced Isobaric N,N-Dimethyl Leucine Tagging Strategy for a Comprehensive Quantitative Glycoproteomic Analysis.

Intact glycopeptide analysis has been of great interest because it can elucidate glycosylation site information and glycan structural composition at the same time. However, mass spectrometry (MS)-based glycoproteomic analysis is hindered by the low abundance and poor ionization efficiency of glycopeptides. Relatively large amounts of starting materials are needed for the enrichment, which makes the identification and quantification of intact glycopeptides from samples with limited quantity more challenging. To overcome these limitations, we developed an improved isobaric labeling strategy with an additional boosting channel to enhance N,N-dimethyl leucine (DiLeu) tagging-based quantitative glycoproteomic analysis, termed as Boost-DiLeu. With the integration of a one-tube sample processing workflow and high-pH fractionation, 3514 quantifiable N-glycopeptides were identified from 30 μg HeLa cell tryptic digests with reliable quantification performance. Furthermore, this strategy was applied to human cerebrospinal fluid (CSF) samples to differentiate N-glycosylation profiles between Alzheimer's disease (AD) patients and non-AD donors. The results revealed processes and pathways affected by dysregulated N-glycosylation in AD, including platelet degranulation, cell adhesion, and extracellular matrix, which highlighted the involvement of N-glycosylation aberrations in AD pathogenesis. Moreover, weighted gene coexpression network analysis (WGCNA) showed nine modules of glycopeptides, two of which were associated with the AD phenotype. Our results demonstrated the feasibility of using this strategy for in-depth glycoproteomic analysis of size-limited clinical samples. Taken together, we developed and optimized a strategy for the enhanced comprehensive quantitative intact glycopeptide analysis with DiLeu labeling, showing significant promise for identifying novel therapeutic targets or biomarkers in biological systems with a limited sample quantity.

Simultaneous determination of LY3214996, abemaciclib, and M2 and M20 metabolites in human plasma, cerebrospinal fluid, and brain tumor by LC-MS/MS

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of total and unbound concentrations of LY3214996, an extracellular signal-regulated kinase inhibitor; abemaciclib, a cyclin-dependent kinase 4/6 inhibitor; and abemaciclib active metabolites, M2 and M20, in human plasma, brain tumor, and cerebrospinal fluid samples. The method was validated over a concentration range of 0.2–500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex™ F5 column. Detection was performed on a Sciex QTRAP 6500+ mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. The intra- and inter-batch accuracy as well as the precision of the method for all matrices was within ±20% and ≤20% at the lower limit of quantification, and within ±15% and ≤15% for other quality control levels for all analytes. The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis. The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib.

Quantification of endocannabinoids in human cerebrospinal fluid using a novel micro-flow liquid chromatography-mass spectrometry method

The endocannabinoid system (ECS) is implicated in various brain disorders. Changes in the composition of the cerebrospinal fluid (CSF) may be associated with ECS-related pathologies. Endocannabinoids (eCBs) and their analogues are present at low concentrations in human CSF, which hampered the investigation of the ECS in this body fluid. In this study, we developed a highly sensitive and selective micro-flow liquid chromatography-tandem mass spectrometry (micro-LC-MS/MS) method for the analysis of eCBs and eCB analogues in human CSF. The developed method allowed for the quantitative analysis of 16 eCBs and their analogues in human CSF. Micro-LC-MS/MS analyses were performed at a flow-rate of 4 μL min−1 with a 0.3-mm inner diameter column. A minor modification of a novel spray needle was carried out to improve the robustness of our method. By using an injection volume of 3 μL, our method reached limits of detection in the range from 0.6 to 1293.4 pM and limits of quantification in range from 2.0 to 4311.3 pM while intra- and interday precisions were below 13.7%. The developed workflow was successfully used for the determination of eCBs in 288 human CSF samples. It is anticipated that the proposed approach will contribute to a deeper understanding of the role of ECS in various brain disorders.

First Indications of Omsk Haemorrhagic Fever Virus beyond Russia.

Omsk haemorrhagic fever virus (OHFV) is the agent leading to Omsk haemorrhagic fever (OHF), a viral disease currently only known in Western Siberia in Russia. The symptoms include fever, headache, nausea, muscle pain, cough and haemorrhages. The transmission cycle of OHFV is complex. Tick bites or contact with infected small mammals are the main source of infection. The Republic of Kazakhstan is adjacent to the endemic areas of OHFV in Russia and febrile diseases with haemorrhages occur throughout the country—often with unclear aetiology. In this study, we examined human cerebrospinal fluid samples of patients with suspected meningitis or meningoencephalitis with unknown origins for the presence of OHFV RNA. Further, reservoir hosts such as rodents and ticks from four Kazakhstan regions were screened for OHFV RNA to clarify if this virus could be the causative agent for many undiagnosed cases of febrile diseases in humans in Kazakhstan. Out of 130 cerebrospinal fluid samples, two patients (1.53%) originating from Almaty city were positive for OHFV RNA. Screening of tick samples revealed positive pools from different areas in the Akmola region. Of the caught rodents, 1.1% out of 621 were positive for OHFV at four trapping areas from the West Kazakhstan region. In this paper, we present a broad investigation of the spread of OHFV in Kazakhstan in human cerebrospinal fluid samples, rodents and ticks. Our study shows for the first time that OHFV can not only be found in the area of Western Siberia in Russia, but can also be detected up to 1.600 km away in the Almaty region in patients and natural foci.