Acute Hyperglycemia Induced by Hyperglycemic Clamp Affects Plasma Amyloid-ß in Type 2 Diabetes.

Background: Individuals with type 2 diabetes (T2D) have an increased risk of cognitive symptoms and Alzheimer's disease (AD). Mis-metabolism with aggregation of amyloid-ß peptides (Aß) play a key role in AD pathophysiology. Therefore, human studies on Aß metabolism and T2D are warranted. Objective: The objective of this study was to examine whether acute hyperglycemia affects plasma Aß 1-40 and Aß 1-42 concentrations in individuals with T2D and matched controls. Methods: Ten participants with T2D and 11 controls (median age, 69 years; range, 66-72 years) underwent hyperglycemic clamp and placebo clamp (saline infusion) in a randomized order, each lasting 4 hours. Aß 1-40, Aß 1-42, and insulin-degrading enzyme (IDE) plasma concentrations were measured in blood samples taken at 0 and 4 hours of each clamp. Linear mixed-effect regression models were used to evaluate the 4-hour changes in Aß 1-40 and Aß 1-42 concentrations, adjusting for body mass index, estimated glomerular filtration rate, and 4-hour change in insulin concentration. Results: At baseline, Aß 1-40 and Aß 1-42 concentrations did not differ between the two groups. During the hyperglycemic clamp, Aß decreased in the control group, compared to the placebo clamp (Aß 1-40: p = 0.034, Aß 1-42: p = 0.020), IDE increased (p = 0.016) during the hyperglycemic clamp, whereas no significant changes in either Aß or IDE was noted in the T2D group. Conclusions: Clamp-induced hyperglycemia was associated with increased IDE levels and enhanced Aß 40 and Aß 42 clearance in controls, but not in individuals with T2D. We hypothesize that insulin-degrading enzyme was inhibited during hyperglycemic conditions in people with T2D.

Multivariate GWAS of Alzheimer's disease CSF biomarker profiles implies GRIN2D in synaptic functioning.

BACKGROUND: Genome-wide association studies (GWAS) of Alzheimer's disease (AD) have identified several risk loci, but many remain unknown. Cerebrospinal fluid (CSF) biomarkers may aid in gene discovery and we previously demonstrated that six CSF biomarkers (ß-amyloid, total/phosphorylated tau, NfL, YKL-40, and neurogranin) cluster into five principal components (PC), each representing statistically independent biological processes. Here, we aimed to (1) identify common genetic variants associated with these CSF profiles, (2) assess the role of associated variants in AD pathophysiology, and (3) explore potential sex differences. METHODS: We performed GWAS for each of the five biomarker PCs in two multi-center studies (EMIF-AD and ADNI). In total, 973 participants (n = 205 controls, n = 546 mild cognitive impairment, n = 222 AD) were analyzed for 7,433,949 common SNPs and 19,511 protein-coding genes. Structural equation models tested whether biomarker PCs mediate genetic risk effects on AD, and stratified and interaction models probed for sex-specific effects. RESULTS: Five loci showed genome-wide significant association with CSF profiles, two were novel (rs145791381 [inflammation] and GRIN2D [synaptic functioning]) and three were previously described (APOE, TMEM106B, and CHI3L1). Follow-up analyses of the two novel signals in independent datasets only supported the GRIN2D locus, which contains several functionally interesting candidate genes. Mediation tests indicated that variants in APOE are associated with AD status via processes related to amyloid and tau pathology, while markers in TMEM106B and CHI3L1 are associated with AD only via neuronal injury/inflammation. Additionally, seven loci showed sex-specific associations with AD biomarkers. CONCLUSIONS: These results suggest that pathway and sex-specific analyses can improve our understanding of AD genetics and may contribute to precision medicine.

Harmonization and standardization of biofluid-based biomarker measurements for AT(N) classification in Alzheimer's disease.

Fluid biomarkers are currently measured in cerebrospinal fluid and blood for Alzheimer's disease diagnosis and are promising targets for drug development and for patients' follow-up in clinical trials. These biomarkers have been grouped in an unbiased research framework, the amyloid (Aß), tau, and neurodegeneration (AT[N]) biomarker system to aid patients' early diagnosis and stratification. Metrological approaches relying on mass spectrometry have been used for the development of reference materials and reference measurement procedures. Despite their excellent performances as clinical tools, fluid biomarkers often present an important between-laboratory variation. Standardization efforts were carried out on the biomarkers currently included in the AT(N) classification system, involving the collaboration of national metrology institutes, clinicians, researchers, and in vitro diagnostic providers. This article provides an overview of current activities towards standardization. These reference methods and reference materials may be used for recalibration of immunoassays and the establishment of standardized cutoff values allowing a better stratification of Alzheimer's disease patients. Highlights: The AT(N) biomarker system allows stratifying AD patients on the basis of biomarker profiles.Fluid biomarker measurements often present an important between-laboratory variation preventing the establishment of standardized cutoff values.Overview on the standardization initiatives involving the fluid biomarkers currently included in the AT(N) framework.

α-Synuclein seed amplification assay as a diagnostic tool for parkinsonian disorders.

INTRODUCTION: Synucleinopathies such as Parkinson's disease (PD) and multiple system atrophy (MSA) can be challenging to diagnose due to the symptom overlap with, for example, atypical parkinsonisms like progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Seed amplification assays (SAA), developed for the detection of α-synuclein (αSyn) aggregates in CSF, have been successful when used as a biomarker evaluation for synucleinopathies. In this study, we investigated the potential of this assay to not only detect αSyn seeds in CSF, but also discriminate between movement disorders. METHODS: The αSyn-SAA was tested in a Scandinavian cohort composed of 129 CSF samples from patients with PD (n = 55), MSA (n = 27), CBD (n = 7), and PSP (n = 16), as well as healthy controls (HC, n = 24). RESULTS: The αSyn seed amplification assay (αSyn-SAA) was able to correctly identify all PD samples as positive (sensitivity of 100%) while also discriminating the PD group from HC (70.8% specificity, p < 0.0001) and tauopathies [CBD (71% specificity) and PSP (75% specificity), p < 0.0001)]. The αSyn-SAA was also able to identify almost all MSA samples as positive for αSyn aggregation (sensitivity of 92.6%). In general, this assay is able to discriminate between the synucleinopathies and tauopathies analyzed herein (p < 0.0001) despite the overlapping symptoms in these diseases. CONCLUSION: These findings suggest the αSyn-SAA is a useful diagnostic tool for differentiating between different parkinsonian disorders, although further optimization may be needed.

Prevalence and Clinical Implications of a ß-Amyloid-Negative, Tau-Positive Cerebrospinal Fluid Biomarker Profile in Alzheimer Disease.

Importance: Knowledge is lacking on the prevalence and prognosis of individuals with a ß-amyloid-negative, tau-positive (A-T+) cerebrospinal fluid (CSF) biomarker profile. Objective: To estimate the prevalence of a CSF A-T+ biomarker profile and investigate its clinical implications. Design, Setting, and Participants: This was a retrospective cohort study of the cross-sectional multicenter University of Gothenburg (UGOT) cohort (November 2019-January 2021), the longitudinal multicenter Alzheimer Disease Neuroimaging Initiative (ADNI) cohort (individuals with mild cognitive impairment [MCI] and no cognitive impairment; September 2005-May 2022), and 2 Wisconsin cohorts, Wisconsin Alzheimer Disease Research Center and Wisconsin Registry for Alzheimer Prevention (WISC; individuals without cognitive impairment; February 2007-November 2020). This was a multicenter study, with data collected from referral centers in clinical routine (UGOT) and research settings (ADNI and WISC). Eligible individuals had 1 lumbar puncture (all cohorts), 2 or more cognitive assessments (ADNI and WISC), and imaging (ADNI only) performed on 2 separate occasions. Data were analyzed on August 2022 to April 2023. Exposures: Baseline CSF Aß42/40 and phosphorylated tau (p-tau)181; cognitive tests (ADNI: modified preclinical Alzheimer cognitive composite [mPACC]; WISC: modified 3-test PACC [PACC-3]). Exposures in the ADNI cohort included [18F]-florbetapir amyloid positron emission tomography (PET), magnetic resonance imaging (MRI), [18F]-fluorodeoxyglucose PET (FDG-PET), and cross-sectional tau-PET (ADNI: [18F]-flortaucipir, WISC: [18F]-MK6240). Main Outcomes and Measures: Primary outcomes were the prevalence of CSF AT biomarker profiles and continuous longitudinal global cognitive outcome and imaging biomarker trajectories in A-T+ vs A-T- groups. Secondary outcomes included cross-sectional tau-PET. Results: A total of 7679 individuals (mean [SD] age, 71.0 [8.4] years; 4101 male [53%]) were included in the UGOT cohort, 970 individuals (mean [SD] age, 73 [7.0] years; 526 male [54%]) were included in the ADNI cohort, and 519 individuals (mean [SD] age, 60 [7.3] years; 346 female [67%]) were included in the WISC cohort. The prevalence of an A-T+ profile in the UGOT cohort was 4.1% (95% CI, 3.7%-4.6%), being less common than the other patterns. Longitudinally, no significant differences in rates of worsening were observed between A-T+ and A-T- profiles for cognition or imaging biomarkers. Cross-sectionally, A-T+ had similar tau-PET uptake to individuals with an A-T- biomarker profile. Conclusion and Relevance: Results suggest that the CSF A-T+ biomarker profile was found in approximately 5% of lumbar punctures and was not associated with a higher rate of cognitive decline or biomarker signs of disease progression compared with biomarker-negative individuals.

Analytical and clinical validation of a blood progranulin ELISA in frontotemporal dementias.

OBJECTIVES: Heterozygous mutations in the granulin (GRN) gene may result in haploinsufficiency of progranulin (PGRN), which might lead to frontotemporal dementia (FTD). In this study, we aimed to perform analytical and clinical validation of a commercial progranulin kit for clinical use. METHODS: Analytical validation parameters including assay precision, selectivity, measurement range, dilution linearity, interferences and sample stability were tested according to previously described procedures. For clinical validation, PGRN levels were measured in plasma from 32 cognitively healthy individuals, 52 confirmed GRN mutation carriers, 25 C9orf72 mutation carriers and 216 patients with different neurodegenerative diseases of which 70 were confirmed as non-mutation carriers. RESULTS: Among the analytical validation parameters, assay precision and repeatability were very stable (coefficients of variation <7 %). Spike recovery was 96 %, the measurement range was 6.25-400 µg/L and dilution linearity ranged from 1:50-1:200. Hemolysis did not interfere with progranulin levels, and these were resistant to freeze/thaw cycles and storage at different temperatures. For the clinical validation, the assay was capable of distinguishing GRN mutation carriers from controls and non-GRN mutation carriers with very good sensitivity and specificity at a cut-off of 57 µg/L (97 %, 100 %, respectively). CONCLUSIONS: In this study, we demonstrate robust analytical and diagnostic performance of this commercial progranulin kit for implementation in clinical laboratory practice. This easy-to-use test allows identification of potential GRN mutation carriers, which may guide further evaluation of the patient. This assay might also be used to evaluate the effect of novel PGRN-targeting drugs and therapies.

Cerebrospinal fluid biomarkers of axonal and synaptic degeneration in a population-based sample.

BACKGROUND: Neurofilament light (NfL) and neurogranin (Ng) are promising candidate AD biomarkers, reflecting axonal and synaptic damage, respectively. Since there is a need to understand the synaptic and axonal damage in preclinical Alzheimer's disease (AD), we aimed to determine the cerebrospinal fluid (CSF) levels of NfL and Ng in cognitively unimpaired elderly from the Gothenburg H70 Birth Cohort Studies classified according to the amyloid/tau/neurodegeneration (A/T/N) system. METHODS: The sample consisted of 258 cognitively unimpaired older adults (age 70, 129 women and 129 men) from the Gothenburg Birth Cohort Studies. We compared CSF NfL and Ng concentrations in A/T/N groups using Student's T-test and ANCOVA. RESULTS: CSF NfL concentration was higher in the A-T-N+ group (p=0.001) and the A-T+N+ group (p=0.006) compared with A-T-N-. CSF Ng concentration was higher in the A-T-N+, A-T+N+, A+T-N+, and A+T+N+ groups (p<0.0001) compared with A-T-N-. We found no difference in NfL or Ng concentration in A+ compared with A- (disregarding T- and N- status), whereas those with N+ had higher concentrations of NfL and Ng compared with N- (p<0.0001) (disregarding A- and T- status). CONCLUSIONS: CSF NfL and Ng concentrations are increased in cognitively normal older adults with biomarker evidence of tau pathology and neurodegeneration.

Neurofilament light oligomers in neurodegenerative diseases: quantification by homogeneous immunoassay in cerebrospinal fluid.

Background: Neurofilament light (NfL) is a widely used biomarker for neurodegeneration. NfL is prone to oligomerisation, but available assays do not reveal the exact molecular nature of the protein variant measured. The objective of this study was to develop a homogeneous ELISA capable of quantifying oligomeric NfL (oNfL) in cerebrospinal fluid (CSF). Methods: A homogeneous ELISA, based on the same capture and detection antibody (NfL21), was developed and used to quantify oNfL in samples from patients with behavioural variant frontotemporal dementia (bvFTD, n=28), non-fluent variant primary progressive aphasia (nfvPPA, n=23), semantic variant PPA (svPPA, n=10), Alzheimer's disease (AD, n=20) and healthy controls (n=20). The nature of NfL in CSF, and the recombinant protein calibrator, was also characterised by size exclusion chromatography (SEC). Results: CSF concentration of oNfL was significantly higher in nfvPPA (p<0.0001) and svPPA patients (p<0.05) compared with controls. CSF oNfL concentration was also significantly higher in nfvPPA compared with bvFTD (p<0.001) and AD (p<0.01) patients. SEC data showed a peak fraction compatible with a full-length dimer (~135 kDa) in the in-house calibrator. For CSF, the peak was found in a fraction of lower molecular weight (~53 kDa), suggesting dimerisation of NfL fragments. Conclusions: The homogeneous ELISA and SEC data suggest that most of the NfL in both the calibrator and human CSF is present as a dimer. In CSF, the dimer appears to be truncated. Further studies are needed to determine its precise molecular composition.

Plasma neurofilament light significantly decreases following treatment in Lyme neuroborreliosis and is not associated with persistent symptoms.

BACKGROUND AND PURPOSE: Currently there is an unmet need for a highly standardized blood biomarker test to monitor treatment response in Lyme neuroborreliosis (LNB). Differentiating between active or past infection is challenged by the relatively high frequency of persistent symptoms after the end of antibiotic treatment (estimated 15%-20%), the variable clinical course and the long-lasting Borrelia burgdorferi antibodies. The aim was therefore to evaluate plasma neurofilament light chain (pNfL) as a marker for disease activity in LNB. METHODS: This was a prospective cohort of definite LNB (N = 36) with blood samples and clinical evaluation including Glasgow Outcome Score at treatment initiation and 3 and 6 months' follow-up. Consecutive plasma was retrospectively analysed for the content of neurofilament light chain by Quanterix® kits (Simoa® NF-light Kit). RESULTS: Plasma neurofilament light chain significantly decreased between treatment initiation and the 3-month follow-up (median 83 pg/ml vs. median 14 pg/ml (25 pairs), p < 0.0001). No significant change was observed between 3 and 6 months' follow-up (median 14 pg/ml vs. median 12 pg/ml (21 pairs), p = 0.33). At treatment initiation 90% had pNfL above the age-defined reference compared to only 23% and 7% respectively at 3 and 6 months' follow-up. Decreases in pNfL were mirrored by increasing Glasgow Outcome Score. Reporting persistent symptoms at the 6-month follow-up was not associated with pNfL (relative change from reference or actual values) at baseline or at 6 months' follow-up. CONCLUSION: Plasma neurofilament light chain decreases following antibiotic treatment in LNB and is not associated with reporting persistent symptoms. It was therefore speculated that it may prove useful as a treatment response biomarker in LNB.

Assessing the commutability of candidate reference materials for the harmonization of neurofilament light measurements in blood.

OBJECTIVES: Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. METHODS: Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. RESULTS: The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman's ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. CONCLUSIONS: The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein.

Establishment of reference values for plasma neurofilament light based on healthy individuals aged 5-90 years.

The recent development of assays that accurately quantify neurofilament light, a neuronal cytoskeleton protein, in plasma has generated a vast literature supporting that it is a sensitive, dynamic, and robust biomarker of neuroaxonal damage. As a result, efforts are now made to introduce plasma neurofilament light into clinical routine practice, making it an easily accessible complement to its cerebrospinal fluid counterpart. An increasing literature supports the use of plasma neurofilament light in differentiating neurodegenerative diseases from their non-neurodegenerative mimics and suggests it is a valuable biomarker for the evaluation of the effect of putative disease-modifying treatments (e.g. in multiple sclerosis). More contexts of use will likely emerge over the coming years. However, to assist clinical interpretation of laboratory test values, it is crucial to establish normal reference intervals. In this study, we sought to derive reliable cut-offs by pooling quantified plasma neurofilament light in neurologically healthy participants (5-90 years) from eight cohorts. A strong relationship between age and plasma neurofilament light prompted us to define the following age-partitioned reference limits (upper 95th percentile in each age category): 5-17 years = 7 pg/mL; 18-50 years = 10 pg/mL; 51-60 years = 15 pg/mL; 61-70 years = 20 pg/mL; 70 + years = 35 pg/mL. The established reference limits across the lifespan will aid the introduction of plasma neurofilament light into clinical routine, and thereby contribute to diagnostics and disease-monitoring in neurological practice.

Neurofilament Light in Cerebrospinal Fluid is Associated With Disease Staging in European Lyme Neuroborreliosis.

Background: Drivers of differences in disease presentation and symptom duration in Lyme neuroborreliosis (LNB) are currently unknown. Objectives: We hypothesized that neurofilament light (NfL) in cerebrospinal fluid (CSF) would predict disease location and sequelae in a historic LNB cohort. Design: Using a cross-sectional design and archived CSF samples from 185 patients diagnosed with LNB, we evaluated the content of NfL in the total cohort and in a subgroup of 84 patients with available clinical and paraclinical information. Methods: Individuals were categorized according to disease location: a. Central nervous system (CNS) with stroke (N=3), b. CNS without stroke (N=11), c. Peripheral nervous system (PNS) with cranial nerve palsy (CNP) (N=40) d. PNS without CNP (N=30). Patients with hospital follow-up more than 6 months after completed antibiotic therapy were categorized as having LNB associated sequelae (N=15). Results: At diagnosis concentration of NfL exceeded the upper reference level in 60% (105/185), especially among individuals above 30 years. Age-adjusted NfL was not found to be associated with symptom duration. Age-adjusted NfL was significantly higher among individuals with CNS involvement. Category a. (stroke) had significantly higher NfL concentrations in CSF compared to all other categories, category b. (CNS involvement without stroke) had significantly higher values compared to the categories of PNS involvement. We found no significant difference between the categories with PNS involvement (with or without CNP). Significantly higher NfL was found among patients with follow-up in hospital setting. Conclusion: Comparison of NfL concentrations between the 4 groups of LNB disease manifestations based on clinical information revealed a hierarchy of neuron damage according to disease location and suggested evolving mechanisms with accelerated injury especially when disease is complicated by stroke. Higher values of NfL among patients with need of follow-up in hospital setting suggest NfL could be useful to identify rehabilitative needs.

Galectin-3 is elevated in CSF and is associated with Aß deposits and tau aggregates in brain tissue in Alzheimer's disease.

Galectin-3 (Gal-3) is a beta-galactosidase binding protein involved in microglial activation in the central nervous system (CNS). We previously demonstrated the crucial deleterious role of Gal-3 in microglial activation in Alzheimer's disease (AD). Under AD conditions, Gal-3 is primarily expressed by microglial cells clustered around Aß plaques in both human and mouse brain, and knocking out Gal-3 reduces AD pathology in AD-model mice. To further unravel the importance of Gal-3-associated inflammation in AD, we aimed to investigate the Gal-3 inflammatory response in the AD continuum. First, we measured Gal-3 levels in neocortical and hippocampal tissue from early-onset AD patients, including genetic and sporadic cases. We found that Gal-3 levels were significantly higher in both cortex and hippocampus in AD subjects. Immunohistochemistry revealed that Gal-3+ microglial cells were associated with amyloid plaques of a larger size and more irregular shape and with neurons containing tau-inclusions. We then analyzed the levels of Gal-3 in cerebrospinal fluid (CSF) from AD patients (n = 119) compared to control individuals (n = 36). CSF Gal-3 levels were elevated in AD patients compared to controls and more strongly correlated with tau (p-Tau181 and t-tau) and synaptic markers (GAP-43 and neurogranin) than with amyloid-ß. Lastly, principal component analysis (PCA) of AD biomarkers revealed that CSF Gal-3 clustered and associated with other CSF neuroinflammatory markers, including sTREM-2, GFAP, and YKL-40. This neuroinflammatory component was more highly expressed in the CSF from amyloid-ß positive (A+), CSF p-Tau181 positive (T+), and biomarker neurodegeneration positive/negative (N+/-) (A + T + N+/-) groups compared to the A + T-N- group. Overall, Gal-3 stands out as a key pathological biomarker of AD pathology that is measurable in CSF and, therefore, a potential target for disease-modifying therapies involving the neuroinflammatory response.

Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid.

OBJECTIVES: The core cerebrospinal fluid (CSF) biomarkers; total tau (tTau), phospho-tau (pTau), amyloid ß 1-42 (Aß 1-42), and the Aß 1-42/Aß 1-40 ratio have transformed Alzheimer's disease (AD) research and are today increasingly used in clinical routine laboratories as diagnostic tools. Fully automated immunoassay instruments with ready-to-use assay kits and calibrators has simplified their analysis and improved reproducibility of measurements. We evaluated the analytical performance of the fully automated immunoassay instrument LUMIPULSE G (Fujirebio) for measurement of the four core AD CSF biomarkers and determined cutpoints for AD diagnosis. METHODS: Comparison of the LUMIPULSE G assays was performed with the established INNOTEST ELISAs (Fujirebio) for hTau Ag, pTau 181, ß-amyloid 1-42, and with V-PLEX Plus Aß Peptide Panel 1 (6E10) (Meso Scale Discovery) for Aß 1-42/Aß 1-40, as well as with a LC-MS reference method for Aß 1-42. Intra- and inter-laboratory reproducibility was evaluated for all assays. Clinical cutpoints for Aß 1-42, tTau, and pTau was determined by analysis of three cohorts of clinically diagnosed patients, comprising 651 CSF samples. For the Aß 1-42/Aß 1-40 ratio, the cutpoint was determined by mixture model analysis of 2,782 CSF samples. RESULTS: The LUMIPULSE G assays showed strong correlation to all other immunoassays (r>0.93 for all assays). The repeatability (intra-laboratory) CVs ranged between 2.0 and 5.6%, with the highest variation observed for ß-amyloid 1-40. The reproducibility (inter-laboratory) CVs ranged between 2.1 and 6.5%, with the highest variation observed for ß-amyloid 1-42. The clinical cutpoints for AD were determined to be 409 ng/L for total tau, 50.2 ng/L for pTau 181, 526 ng/L for ß-amyloid 1-42, and 0.072 for the Aß 1-42/Aß 1-40 ratio. CONCLUSIONS: Our results suggest that the LUMIPULSE G assays for the CSF AD biomarkers are fit for purpose in clinical laboratory practice. Further, they corroborate earlier presented reference limits for the biomarkers.

The global Alzheimer's Association round robin study on plasma amyloid ß methods.

INTRODUCTION: Blood-based assays to measure brain amyloid beta (Aß) deposition are an attractive alternative to the cerebrospinal fluid (CSF)-based assays currently used in clinical settings. In this study, we examined different blood-based assays to measure Aß and how they compare among centers and assays. METHODS: Aliquots from 81 plasma samples were distributed to 10 participating centers. Seven immunological assays and four mass-spectrometric methods were used to measure plasma Aß concentrations. RESULTS: Correlations were weak for Aß42 while Aß40 correlations were stronger. The ratio Aß42/Aß40 did not improve the correlations and showed weak correlations. DISCUSSION: The poor correlations for Aß42 in plasma might have several potential explanations, such as the high levels of plasma proteins (compared to CSF), sensitivity to pre-analytical sample handling and specificity, and cross-reactivity of different antibodies. Different methods might also measure different pools of plasma Aß42. We, however, hypothesize that greater correlations might be seen in future studies because many of the methods have been refined during completion of this study.

Amyotrophic lateral sclerosis transcriptomics reveals immunological effects of low-dose interleukin-2.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis.

Circulating granulocyte colony-stimulating factor and functional outcome after ischemic stroke: an observational study.

Objectives: While granulocyte colony-stimulating factor (G-CSF) has shown beneficial effects in experimental ischemic stroke (IS), these effects have not been reproduced clinically. Small-to-medium-sized observational studies have reported varying associations for G-CSF with stroke severity and post-stroke functional outcome, prompting their investigation in a larger study.Methods: Endogenous serum G-CSF (S-GCSF) was measured in the acute phase and after 3 months in patients with IS (N = 435; 36% females; mean age, 57 years) from the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS). Stroke severity was scored according to the National Institutes of Health Stroke Scale (NIHSS), and the modified Rankin Scale (mRS) assessed functional outcomes at 3-month and 2-year post-stroke. Correlation and logistic regression analyses with confounder adjustments assessed the relationships.Results: The acute S-GCSF level was 23% higher than at 3-month post-stroke (p < 0.001). Acute G-CSF correlated weakly with stroke severity quintiles (r = 0.12, p = 0.013) and with high-sensitivity C-reactive protein (r = 0.29, p < 0.001). The association between S-GCSF (as quintiles, q) and poor functional outcome at 3 months (mRS 3-6; S-GCSF-q5 vs. S-GCSF-q1, age- and sex-adjusted odds ratio: 4.27, 95% confidence interval: 1.82-9.99; p = 0.001) withstood adjustment for cardiovascular risk factors and stroke subtype, but not additional correction for stroke severity. Post-stroke changes in S-GSCF and absolute 3-month S-GCSF were not associated with 3-month or 2-year functional outcomes.Discussion: Early post-stroke S-GCSF is increased in severe IS and associated with 3-month poor functional outcomes. The change in S-GCSF and the 3-month S-GCSF appear to be less-important, and S-GCSF likely reflects inflammation in large infarctions.

Measurement batch differences and between-batch conversion of Alzheimer's disease cerebrospinal fluid biomarker values.

INTRODUCTION: Batch differences in cerebrospinal fluid (CSF) biomarker measurement can introduce bias into analyses for Alzheimer's disease studies. We evaluated and adjusted for batch differences using statistical methods. METHODS: A total of 792 CSF samples from 528 participants were assayed in three batches for 12 biomarkers and 3 biomarker ratios. Batch differences were assessed using Bland-Altman plot, paired t test, Pitman-Morgan test, and linear regression. Generalized linear models were applied to convert CSF values between batches. RESULTS: We found statistically significant batch differences for all biomarkers and ratios, except that neurofilament light was comparable between batches 1 and 2. The conversion models generally had high R 2 except for converting P-tau between batches 1 and 3. DISCUSSION: Between-batch conversion allows harmonized CSF values to be used in the same analysis. Such method may be applied to adjust for other sources of variability in measuring CSF or other types of biomarkers.

Lumbar and ventricular CSF concentrations of extracellular matrix proteins before and after shunt surgery in idiopathic normal pressure hydrocephalus.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is a reversible CNS disease characterized by disturbed cerebrospinal fluid (CSF) dynamics. Changes in the extracellular matrix (ECM) composition might be involved in the pathophysiology of iNPH. The aim of this study was to explore possible differences between lumbar and ventricular CSF concentrations of the ECM markers brevican and neurocan, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and their relation to clinical symptoms in iNPH patients before and after shunt surgery. METHODS: Paired lumbar and ventricular CSF was collected from 31 iNPH patients, before and four months after shunt surgery. CSF was analysed for concentrations of tryptic peptides originating from brevican and neurocan using a mass spectrometry-based panel, and for MMP-1, -2, -9, -10 and TIMP-1 using fluorescent or electrochemiluminescent immunoassays. RESULTS: Brevican and neurocan peptide levels were not influenced by CSF origin, but MMP-1, -2, -10 and TIMP-1 were increased (p ≤ 0.0005), and MMP-9 decreased (p ≤ 0.0003) in lumbar CSF compared with ventricular CSF. There was a general trend of ECM proteins to increase following shunt surgery. Ventricular TIMP-1 was inversely correlated with overall symptoms (rho = - 0.62, p < 0.0001). CSF concentrations of the majority of brevican and neurocan peptides were increased in iNPH patients with a history of cardiovascular disease (p ≤ 0.001, AUC = 0.84-0.94) compared with those without. CONCLUSION: Levels of the CNS-specific proteins brevican and neurocan did not differ between the lumbar and ventricular CSF, whereas the increase of several CNS-unspecific MMPs and TIMP-1 in lumbar CSF suggests contribution from peripheral tissues. The increase of ECM proteins in CSF following shunt surgery could indicate disturbed ECM dynamics in iNPH that are restored by restitution of CSF dynamics.

TMEM106B and CPOX are genetic determinants of cerebrospinal fluid Alzheimer's disease biomarker levels.

INTRODUCTION: Neurofilament light (NfL), chitinase-3-like protein 1 (YKL-40), and neurogranin (Ng) are biomarkers for Alzheimer's disease (AD) to monitor axonal damage, astroglial activation, and synaptic degeneration, respectively. METHODS: We performed genome-wide association studies (GWAS) using DNA and cerebrospinal fluid (CSF) samples from the EMIF-AD Multimodal Biomarker Discovery study for discovery, and the Alzheimer's Disease Neuroimaging Initiative study for validation analyses. GWAS were performed for all three CSF biomarkers using linear regression models adjusting for relevant covariates. RESULTS: We identify novel genome-wide significant associations between DNA variants in TMEM106B and CSF levels of NfL, and between CPOX and YKL-40. We confirm previous work suggesting that YKL-40 levels are associated with DNA variants in CHI3L1. DISCUSSION: Our study provides important new insights into the genetic architecture underlying interindividual variation in three AD-related CSF biomarkers. In particular, our data shed light on the sequence of events regarding the initiation and progression of neuropathological processes relevant in AD.