Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells that detach from the original site and reach the bloodstream. The most aggressive CTCs survive various immune system attacks and initiate metastasis formation. Importantly, CTCs are not specifically targeted by the current immunotherapies due to the limited knowledge on specific targets. Proteomic profiling can be a powerful tool for understanding some of the immune evasion mechanisms used by cancer cells and particularly CTCs. These mechanisms are generally linked to the expression of specific surface proteins/peptides (i.e. the surfaceome). The study of the peptides that bind to class I molecules of the major histocompatibility complex (MHC-I) and of the various glycoproteins expressed on CTC surface may open a completely new avenue for the discovery of novel mechanisms of immune evasion. In this review, we discuss how immunopeptidomic and glycoproteomic studies of CTCs that interact with immune cells could help to better understand how metastasis-initiator CTCs escape the host immune response. We also describe how immunopeptidomic and glycoproteomic studies are carried out.

Increasing the sensitivity of Simoa via bead count reduction facilitates the quantification of pTau-181 in dried plasma spots.

Introduction: The exclusion of affected populations from Alzheimer's disease (AD) clinical research limits our understanding of disease heterogeneity and its impact on clinical care. While micro sampling with dried plasma spots (DPS) can promote inclusivity by enabling sample collection in remote areas, current techniques lack the sensitivity required for the quantification of phosphorylated tau at Thr181 (pTau-181) in DPS extracts. Methods: We developed an assay for pTau-181 with reduced bead count and improved bead read efficiency (BRE) using a prototype Simoa instrument. This novel assay's performance was evaluated against standard pTau-181 assays on two Simoa platforms, and DPS extracts were tested for pTau-181 quantification feasibility. Results: The novel assay quantifies pTau-181 at concentrations up to 16x lower than traditional pTau-181 assays on HD-X and SR-X platforms. DPS extracts tested with our low-bead assay were quantified considerably above the lower limit of quantification (LLOQ), indicating the suitability of this assay for future DPS extract measurements. Discussion: Implementing DPS sampling and pTau-181 quantification could increase participation from underrepresented groups in AD research. However, additional assay optimization and an in-depth study of preanalytical sample stability are essential for the transition to clinical applicability.

Inflammation biomarkers in the intracranial blood are associated with outcome in patients with ischemic stroke.

BACKGROUND: Performing endovascular treatment (EVT) in patients with acute ischemic stroke (AIS) allows a port of entry for intracranial biological sampling. OBJECTIVE: To test the hypothesis that specific immune players are molecular contributors to disease, outcome biomarkers, and potential targets for modifying AIS. METHODS: We examined 75 subjects presenting with large vessel occlusion of the anterior circulation and undergoing EVT. Intracranial blood samples were obtained by microcatheter aspiration, as positioned for stent deployment. Peripheral blood samples were collected from the femoral artery. Plasma samples were quality controlled by electrophoresis and analyzed using a Mesoscale multiplex for targeted inflammatory and vascular factors. RESULTS: We measured 37 protein biomarkers in our sample cohort. Through multivariate analysis, adjusted for age, intravenous thrombolysis, pretreatment National Institutes of Health Stroke Scale and Alberta Stroke Program Early CT scores, we found that post-clot blood levels of interleukin-6 (IL-6) were significantly correlated (adjusted P value <0.05) with disability assessed by the modified Rankin Scale (mRS) score at 90 days, with medium effect size. Chemokine (C-C) ligand 17 CCL17/TARC levels were inversely correlated with the mRS score. Examination of peripheral blood showed that these correlations did not reach statistical significance after correction. Intracranial biomarker IL-6 level was specifically associated with a lower likelihood of favorable outcome, defined as a mRS score of 0-2. CONCLUSIONS: Our findings show a signature of blood inflammatory factors at the cerebrovascular occlusion site. The correlations between these acute-stage biomarkers and mRS score outcome support an avenue for add-on and localized immune modulatory strategies in AIS.

Mass Spectrometry-Based Pipeline for Identifying RNA Modifications Involved in a Functional Process: Application to Cancer Cell Adaptation.

Cancer onset and progression are known to be regulated by genetic and epigenetic events, including RNA modifications (a.k.a. epitranscriptomics). So far, more than 150 chemical modifications have been described in all RNA subtypes, including messenger, ribosomal, and transfer RNAs. RNA modifications and their regulators are known to be implicated in all steps of post-transcriptional regulation. The dysregulation of this complex yet delicate balance can contribute to disease evolution, particularly in the context of carcinogenesis, where cells are subjected to various stresses. We sought to discover RNA modifications involved in cancer cell adaptation to inhospitable environments, a peculiar feature of cancer stem cells (CSCs). We were particularly interested in the RNA marks that help the adaptation of cancer cells to suspension culture, which is often used as a surrogate to evaluate the tumorigenic potential. For this purpose, we designed an experimental pipeline consisting of four steps: (1) cell culture in different growth conditions to favor CSC survival; (2) simultaneous RNA subtype (mRNA, rRNA, tRNA) enrichment and RNA hydrolysis; (3) the multiplex analysis of nucleosides by LC-MS/MS followed by statistical/bioinformatic analysis; and (4) the functional validation of identified RNA marks. This study demonstrates that the RNA modification landscape evolves along with the cancer cell phenotype under growth constraints. Remarkably, we discovered a short epitranscriptomic signature, conserved across colorectal cancer cell lines and associated with enrichment in CSCs. Functional tests confirmed the importance of selected marks in the process of adaptation to suspension culture, confirming the validity of our approach and opening up interesting prospects in the field.

Post-polio syndrome is not a dysimmune condition.

BACKGROUND: Poliomyelitis is a global disabling disease affecting 12-20 million of people. Post poliomyelitis syndrome (PPS) may affect up to 80% of polio survivors: increased muscle weakness, pain, fatigue, functional decline. It relies on aging of an impaired neuro-muscular system with ongoing denervation processes. A late involvement of humoral or cellular pro-inflammatory phenomena is also suspected. AIM: To assess the dysimmune hypothesis of PPS by comparing lymphocyte subpopulations and humoral immune factors between PPS patients and controls. DESIGN: Cross-sectional study. SETTING: Montpellier University Hospital. POPULATION: Forty-seven PPS and 27 healthy controls. METHODS: PPS patients and controls were compared on their lymphocyte subpopulations and humoral immune factors (IL-1ß, IL-6, IL-8, IL-17, IL-21, IL-22, IL-23, IFN-γ, TNF-α, GM-CSF, RANTES, MCP1, MIP-3a, IL-10, TGF-ß, IL4, IL13). Patients were further compared according to their dominant clinical symptoms. Sample size guaranteed a power >90% for all comparisons. RESULTS: PPS patients and controls were comparable in gender, age and corpulence. Most patients had lower limb motor sequelae (N.=45, 95.7%), a minority had upper limb motor impairment (N.=16, 34.0%). Forty-five were able to walk (94%), 35/45 with technical aids. The median of the two-minute walking test was 110 meters (interquartile range 55; 132). Eighteen (38%) required help in their daily life. Their quality of life was low (SF36). All described an increased muscular weakness, 40 (85%) a general fatigue, and 39 (83%) muscular or joint pain. Blood count, serum electrolytes, T and B lymphocyte subpopulations and cytokines were comparable between patients and controls, except for creatine phospho kinase that was significantly higher in PPS patients. None of these variables differed between the 20/47 patients whose late main symptoms were pain or fatigue, and other patients. CONCLUSIONS: Our results suggest that PPS is not a dysimmune disease. CLINICAL REHABILITATION IMPACT: Our results do not sustain immunotherapy for PPS. Our work suggest that PPS may be mostly linked to physiological age-related phenomena in a disabled neuromuscular condition. Thus, our results emphasize the role of prevention and elimination of aggravating factors to avoid late functional worsening, and the importance of rehabilitation programs that should be adapted to patients' specific conditions.

Use of dried blood spots for monitoring inflammatory and nutritional biomarkers in the elderly.

OBJECTIVES: Blood microsampling, particularly dried blood spots (DBSs), is an attractive minimally-invasive approach that is well suited for home sampling and predictive medicine associated with longitudinal follow-up of the elderly. However, in vitro diagnostic quantification of biomarkers from DBS poses a major challenge. Clinical mass spectrometry can reliably quantify blood proteins in various research projects. Our goal here was to use mass spectrometry of DBS in a real-world clinical setting and compared it to the standard immunoassay method. We also sought to correlate DBS mass spectrometry measurements with clinical indices. METHODS: A clinical trial of diagnostic equivalence was conducted to compare conventional venous samples quantified by immunoassay and DBSs quantified by mass spectrometry in an elderly population. We assayed three protein biomarkers of nutritional and inflammatory status: prealbumin (transthyretin), C-reactive protein, and transferrin. RESULTS: The analysis of DBSs showed satisfactory variability and low detection limits. Statistical analysis confirmed that the two methods give comparable results at clinical levels of accuracy. In conclusion, we demonstrated, in a real-life setting, that DBSs can be used to measure prealbumin, CRP and transferrin, which are commonly used markers of nutritional status and inflammation in the elderly. However, there was no correlation with patient frailty for these proteins. CONCLUSIONS: Early detection and regular monitoring of nutritional and inflammatory problems using DBS appear to be clinically feasible. This could help resolve major public health challenges in the elderly for whom frailty leads to serious risks of health complications.

A sensitive high-resolution mass spectrometry method for quantifying intact M-protein light chains in patients with multiple myeloma.

To determine the disease status and the response to treatment for patients with multiple myeloma, measuring serum M-protein levels is a widely used alternative to invasive punctures to count malignant plasma cells in the bone marrow. However, the quantification of this monoclonal antibody, which varies from patient to patient, poses significant analytical challenges. This paper describes a sensitive and specific mass spectrometry assay that addresses two objectives: to overcome the potential interference of biotherapeutics in the measurement of M-proteins, and to determine the depth of response to treatment by assessing minimal residual disease. After immunocapture of immunoglobulins and free light chains in serum, heavy and light chains were dissociated by chemical reduction and separated by liquid chromatography. M-proteins were analyzed by high-resolution mass spectrometry using a method combining a full MS scan for isotyping and identification and a targeted single ion monitoring scan for quantification. This method was able to discriminate M-protein from the therapeutic antibody in all patient samples analyzed and allowed quantification of M-protein with a LLOQ of 2.0 to 3.5 µg/ml in 5 out of 6 patients. This methodology appears to be promising for assessing minimal residual disease with sufficient sensitivity, specificity, and throughput.

Readthrough isoform of aquaporin-4 (AQP4) as a therapeutic target for Alzheimer's disease and other proteinopathies.

The glymphatic system is a crucial component in preserving brain homeostasis by facilitating waste clearance from the central nervous system (CNS). Aquaporin-4 (AQP4) water channels facilitate the continuous interchange between cerebrospinal fluid and brain interstitial fluid by convective flow movement. This flow is responsible for guiding proteins and metabolites away from the CNS. Proteinopathies are neurological conditions characterized by the accumulation of aggregated proteins or peptides in the brain. In Alzheimer's disease (AD), the deposition of amyloid-ß (Aß) peptides causes the formation of senile plaques. This accumulation has been hypothesized to be a result of the imbalance between Aß production and clearance. Recent studies have shown that an extended form of AQP4 increases Aß clearance from the brain. In this mini-review, we present a summary of these findings and explore the potential for future therapeutic strategies aiming to boost waste clearance in AD.

Determination of plasma concentration of Belimumab by LC-MS/MS: Method development, validation, and clinical application.

INTRODUCTION: Belimumab is a monoclonal antibody against B cell activating factor (BLyS). This monoclonal antibody (mAb) has been shown to be effective in reducing disease activity in patients with systemic lupus erythematosus (SLE). Belimumab is available in two forms as a lyophilized powder for intravenous (IV) use, or single-dose syringe for subcutaneous (SC) use. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of belimumab in human serum. MATERIAL AND METHODS: All analyses relied on nano-surface and molecular-orientation limited (nSMOL) proteolysis coupled with LC-MS/MS. Quantifications was performed in multiple reactions monitoring (MRM) mode, and electrospray ionization was conducted in positive mode. RESULTS: Belimumab was quantified with signature peptide QAPGQGLEWMGGIPFGTAK and normalized using P14R. The total run time per assay was 10 min. Linearity was measured from 5 to 800 µg/mL (r² > 0.995). Accuracy and precision based on three quality control levels range from 11.2 - 9.51 % and 1.24 - 13.12 % respectively. The carryover was less than 7 %. In all, 87 patient samples were processed (65, IV; 22, SC). Mean concentration of belimumab was significantly higher for SC (93.0 ± 74.0 µg/mL) than for IV (67.4 ± 38.9 µg/mL) administration. CONCLUSION: We have developed the first method of belimumab quantification combining LC-MS/MS and nSMOL proteolysis. It can be used for future clinical pharmacokinetic studies of belimumab and for investigating the relationship between belimumab concentration, efficacy, and toxicity in SLE patients.

Methods to Discover and Validate Biofluid-Based Biomarkers in Neurodegenerative Dementias.

Neurodegenerative dementias are progressive diseases that cause neuronal network breakdown in different brain regions often because of accumulation of misfolded proteins in the brain extracellular matrix, such as amyloids or inside neurons or other cell types of the brain. Several diagnostic protein biomarkers in body fluids are being used and implemented, such as for Alzheimer's disease. However, there is still a lack of biomarkers for co-pathologies and other causes of dementia. Such biofluid-based biomarkers enable precision medicine approaches for diagnosis and treatment, allow to learn more about underlying disease processes, and facilitate the development of patient inclusion and evaluation tools in clinical trials. When designing studies to discover novel biofluid-based biomarkers, choice of technology is an important starting point. But there are so many technologies to choose among. To address this, we here review the technologies that are currently available in research settings and, in some cases, in clinical laboratory practice. This presents a form of lexicon on each technology addressing its use in research and clinics, its strengths and limitations, and a future perspective.

Ultrasensitive digital immunoassays for SOD1 conformation in amyotrophic lateral sclerosis.

Aim: The aim of this study was to detect misfolded Cu/Zn SOD1 as a potential biomarker for amyotrophic lateral sclerosis (ALS). Materials & methods: Two ultrasensitive immunodetection assays were developed for the quantification of total and misfolded SOD1. Results: The detection of total and misfolded SOD1 was possible in human serum and cerebrospinal fluid. Total SOD1 was increased in cerebrospinal fluid from ALS patients. Misfolded SOD1 had low and variable expression in both control and ALS patient samples. Conclusion: These assays hold promise for improving our understanding of ALS and its detection, and could lead to more effective treatment options in the future. Further studies in larger cohorts are now required.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with protein misfolding, including Cu/Zn SOD1. In this study, we set up a method for detecting normal and pathological misfolded SOD1 in human serum and cerebrospinal fluid. SOD1 was increased in ALS and misfolded SOD1 had low and variable expression in both control and ALS. These assays holds promise for improving our understanding of ALS and its diagnosis.

Neurofilament-Light, a Promising Biomarker: Analytical, Metrological and Clinical Challenges.

Neurofilament-light chain (Nf-L) is a non-specific early-stage biomarker widely studied in the context of neurodegenerative diseases (NDD) and traumatic brain injuries (TBI), which can be measured in biofluids after axonal damage. Originally measured by enzyme-linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF), Nf-L can now be quantified in blood with the emergence of ultrasensitive assays. However, to ensure successful clinical implementation, reliable clinical thresholds and reference measurement procedures (RMP) should be developed. This includes establishing and distributing certified reference materials (CRM). As a result of the complexity of Nf-L and the number of circulating forms, a clear definition of what is measured when immunoassays are used is also critical to achieving standardization to ensure the long-term success of those assays. The use of powerful tools such as mass spectrometry for developing RMP and defining the measurand is ongoing. Here, we summarize the current methods in use for quantification of Nf-L in biofluid showing potential for clinical implementation. The progress and challenges in developing RMP and defining the measurand for Nf-L standardization of diagnostic tests are addressed. Finally, we discuss the impact of pathophysiological factors on Nf-L levels and the establishment of a clinical cut-off.

Lower systemic nitric oxide bioactivity, cerebral hypoperfusion and accelerated cognitive decline in formerly concussed retired rugby union players.

NEW FINDINGS: What is the central question of this study? What are the molecular, cerebrovascular and cognitive biomarkers of retired rugby union players with concussion history? What is the main finding and its importance? Retired rugby players compared with matched controls exhibited lower systemic nitric oxide bioavailability accompanied by lower middle cerebral artery velocity and mild cognitive impairment. Retired rugby players are more susceptible to accelerated cognitive decline. ABSTRACT: Following retirement from sport, the chronic consequences of prior-recurrent contact are evident and retired rugby union players may be especially prone to accelerated cognitive decline. The present study sought to integrate molecular, cerebrovascular and cognitive biomarkers in retired rugby players with concussion history. Twenty retired rugby players aged 64 ± 5 years with three (interquartile range (IQR), 3) concussions incurred over 22 (IQR, 6) years were compared to 21 sex-, age-, cardiorespiratory fitness- and education-matched controls with no prior concussion history. Concussion symptoms and severity were assessed using the Sport Concussion Assessment Tool. Plasma/serum nitric oxide (NO) metabolites (reductive ozone-based chemiluminescence), neuron specific enolase, glial fibrillary acidic protein and neurofilament light-chain (ELISA and single molecule array) were assessed. Middle cerebral artery blood velocity (MCAv, doppler ultrasound) and reactivity to hyper/hypocapnia ( CVR CO 2 hyper ${\mathrm{CVR}}_{{\mathrm{CO}}_{\mathrm{2}}{\mathrm{hyper}}}$ / CVR CO 2 hypo ${\mathrm{CVR}}_{{\mathrm{CO}}_{\mathrm{2}}{\mathrm{hypo}}}$ ) were assessed. Cognition was determined using the Grooved Pegboard Test and Montreal Cognitive Assessment. Players exhibited persistent neurological symptoms of concussion (U = 109(41) , P = 0.007), with increased severity compared to controls (U = 77(41) , P < 0.001). Lower total NO bioactivity (U = 135(41) , P = 0.049) and lower basal MCAv were apparent in players (F2,39  = 9.344, P = 0.004). This was accompanied by mild cognitive impairment (P = 0.020, 95% CI, -3.95 to -0.34), including impaired fine-motor coordination (U = 141(41) , P = 0.021). Retired rugby union players with history of multiple concussions may be characterised by impaired molecular, cerebral haemodynamic and cognitive function compared to non-concussed, non-contact controls.

Multiplexed LC-MS/MS quantification of salivary RNA modifications in periodontitis.

OBJECTIVE: To analyse the salivary epitranscriptomic profiles as periodontitis biomarkers using multiplexed mass spectrometry (MS). BACKGROUND: The field of epitranscriptomics, which relates to RNA chemical modifications, opens new perspectives in the discovery of diagnostic biomarkers, especially in periodontitis. Recently, the modified ribonucleoside N6-methyladenosine (m6A) was revealed as a crucial player in the etiopathogenesis of periodontitis. However, no epitranscriptomic biomarker has been identified in saliva to date. MATERIALS AND METHODS: Twenty-four saliva samples were collected from periodontitis patients (n = 16) and from control subjects (n = 8). Periodontitis patients were stratified according to stage and grade. Salivary nucleosides were directly extracted and, in parallel, salivary RNA was digested into its constituent nucleosides. Nucleoside samples were then quantified by multiplexed MS. RESULTS: Twenty-seven free nucleosides were detected and an overlapping set of 12 nucleotides were detected in digested RNA. Among the free nucleosides, cytidine and three other modified nucleosides (inosine, queuosine and m6Am) were significantly altered in periodontitis patients. In digested RNA, only uridine was significantly higher in periodontitis patients. Importantly there was no correlation between free salivary nucleoside levels and the levels of those same nucleotides in digested salivary RNA, except for cytidine, m5C and uridine. This statement implies that the two detection methods are complementary. CONCLUSION: The high specificity and sensitivity of MS allowed the detection and quantification of multiple nucleosides from RNA and free nucleosides in saliva. Some ribonucleosides appear to be promising biomarkers of periodontitis. Our analytic pipeline opens new perspectives for diagnostic periodontitis biomarkers.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

New Perspectives of Multiplex Mass Spectrometry Blood Protein Quantification on Microsamples in Biological Monitoring of Elderly Patients.

Blood microsampling combined with large panels of clinically relevant tests are of major interest for the development of home sampling and predictive medicine. The aim of the study was to demonstrate the practicality and medical utility of microsamples quantification using mass spectrometry (MS) in a clinical setting by comparing two types of microsamples for multiplex MS protein detection. In a clinical trial based on elderly population, we compared 2 µL of plasma to dried blood spot (DBS) with a clinical quantitative multiplex MS approach. The analysis of the microsamples allowed the quantification of 62 proteins with satisfactory analytical performances. A total of 48 proteins were significantly correlated between microsampling plasma and DBS (p < 0.0001). The quantification of 62 blood proteins allowed us to stratify patients according to their pathophysiological status. Apolipoproteins D and E were the best biomarker link to IADL (instrumental activities of daily living) score in microsampling plasma as well as in DBS. It is, thus, possible to detect multiple blood proteins from micro-samples in compliance with clinical requirements and this allows, for example, to monitor the nutritional or inflammatory status of patients. The implementation of this type of analysis opens new perspectives in the field of diagnosis, monitoring and risk assessment for personalized medicine approaches.

Serum neurofilament light chain cut-off definition for clinical diagnosis and prognosis of amyotrophic lateral sclerosis.

BACKGROUND: The neurofilament light chain (NfL) assay is gradually becoming an essential diagnostic tool for the diagnosis of many neurological diseases including amyotrophic lateral sclerosis (ALS). Different methods for the determination of this biomarker in serum have been developed in recent years. METHODS: We measured blood NfL in 429 patients referred to the tertiary ALS center of Montpellier, France using two different ultrasensitive methods (Ella™ and Simoa™) and we compared the clinical performances of these two approaches. We also converted NfL values into age and body mass index-adjusted Z-scores to assess cut-off values of this biomarker in this clinical context. RESULTS: We show comparable diagnostic and prognostic performance of Ella™ and Simoa™ technologies in ALS, with specificities and sensitivities exceeding 80% for both. We propose cut-off values for serum NfL in this clinical context, thus enabling the routine clinical use of this biomarker. CONCLUSION: The use of NfL in routine clinical practice will help predict survival and improve diagnostic accuracy by distinguishing ALS from other neurological diseases and motor neuron disease mimics.

Comparison of ultrasensitive and mass spectrometry quantification of blood-based amyloid biomarkers for Alzheimer's disease diagnosis in a memory clinic cohort.

BACKGROUND: Alzheimer's disease (AD) is a complex neurodegenerative disorder with ß-amyloid pathology as a key underlying process. The relevance of cerebrospinal fluid (CSF) and brain imaging biomarkers is validated in clinical practice for early diagnosis. Yet, their cost and perceived invasiveness are a limitation for large-scale implementation. Based on positive amyloid profiles, blood-based biomarkers should allow to detect people at risk for AD and to monitor patients under therapeutics strategies. Thanks to the recent development of innovative proteomic tools, the sensibility and specificity of blood biomarkers have been considerably improved. However, their diagnosis and prognosis relevance for daily clinical practice is still incomplete. METHODS: The Plasmaboost study included 184 participants from the Montpellier's hospital NeuroCognition Biobank with AD (n = 73), mild cognitive impairments (MCI) (n = 32), subjective cognitive impairments (SCI) (n = 12), other neurodegenerative diseases (NDD) (n = 31), and other neurological disorders (OND) (n = 36). Dosage of ß-amyloid biomarkers was performed on plasma samples using immunoprecipitation-mass spectrometry (IPMS) developed by Shimadzu (IPMS-Shim Aß42, Aß40, APP669-711) and Simoa Human Neurology 3-PLEX A assay (Aß42, Aß40, t-tau). Links between those biomarkers and demographical and clinical data and CSF AD biomarkers were investigated. Performances of the two technologies to discriminate clinically or biologically based (using the AT(N) framework) diagnosis of AD were compared using receiver operating characteristic (ROC) analyses. RESULTS: The amyloid IPMS-Shim composite biomarker (combining APP669-711/Aß42 and Aß40/Aß42 ratios) discriminated AD from SCI (AUC: 0.91), OND (0.89), and NDD (0.81). The IPMS-Shim Aß42/40 ratio also discriminated AD from MCI (0.78). IPMS-Shim biomarkers have similar relevance to discriminate between amyloid-positive and amyloid-negative individuals (0.73 and 0.76 respectively) and A-T-N-/A+T+N+ profiles (0.83 and 0.85). Performances of the Simoa 3-PLEX Aß42/40 ratio were more modest. Pilot longitudinal analysis on the progression of plasma biomarkers indicates that IPMS-Shim can detect the decrease in plasma Aß42 that is specific to AD patients. CONCLUSIONS: Our study confirms the potential usefulness of amyloid plasma biomarkers, especially the IPMS-Shim technology, as a screening tool for early AD patients.

Development of a candidate reference measurement procedure by ID-LC-MS/MS for total tau protein measurement in cerebrospinal fluid (CSF).

OBJECTIVES: In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF). METHODS: To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode. RESULTS: A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high). CONCLUSIONS: The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs.

Lifelong exposure to high-altitude hypoxia in humans is associated with improved redox homeostasis and structural-functional adaptations of the neurovascular unit.

High-altitude (HA) hypoxia may alter the structural-functional integrity of the neurovascular unit (NVU). Herein, we compared male lowlanders (n = 9) at sea level (SL) and after 14 days acclimatization to 4300 m (chronic HA) in Cerro de Pasco (CdP), Péru (HA), against sex-, age- and body mass index-matched healthy highlanders (n = 9) native to CdP (lifelong HA). Venous blood was assayed for serum proteins reflecting NVU integrity, in addition to free radicals and nitric oxide (NO). Regional cerebral blood flow (CBF) was examined in conjunction with cerebral substrate delivery, dynamic cerebral autoregulation (dCA), cerebrovascular reactivity to carbon dioxide (CVRCO2 ) and neurovascular coupling (NVC). Psychomotor tests were employed to examine cognitive function. Compared to lowlanders at SL, highlanders exhibited elevated basal plasma and red blood cell NO bioavailability, improved anterior and posterior dCA, elevated anterior CVRCO2 and preserved cerebral substrate delivery, NVC and cognition. In highlanders, S100B, neurofilament light-chain (NF-L) and T-tau were consistently lower and cognition comparable to lowlanders following chronic-HA. These findings highlight novel integrated adaptations towards regulation of the NVU in highlanders that may represent a neuroprotective phenotype underpinning successful adaptation to the lifelong stress of HA hypoxia. KEY POINTS: High-altitude (HA) hypoxia has the potential to alter the structural-functional integrity of the neurovascular unit (NVU) in humans. For the first time, we examined to what extent chronic and lifelong hypoxia impacts multimodal biomarkers reflecting NVU structure and function in lowlanders and native Andean highlanders. Despite lowlanders presenting with a reduction in systemic oxidative-nitrosative stress and maintained cerebral bioenergetics and cerebrovascular function during chronic hypoxia, there was evidence for increased axonal injury and cognitive impairment. Compared to lowlanders at sea level, highlanders exhibited elevated vascular NO bioavailability, improved dynamic regulatory capacity and cerebrovascular reactivity, comparable cerebral substrate delivery and neurovascular coupling, and maintained cognition. Unlike lowlanders following chronic HA, highlanders presented with lower concentrations of S100B, neurofilament light chain and total tau. These findings highlight novel integrated adaptations towards the regulation of the NVU in highlanders that may represent a neuroprotective phenotype underpinning successful adaptation to the lifelong stress of HA hypoxia.