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Little is known about the influence of Riluzole on serum neurofilament light chain (sNfL) levels, a biomarker of prognosis in amyotrophic lateral sclerosis (ALS), and variations with time of sNfL concentrations are controversial.Sera from ALS patients (n = 141) and controls (n = 33) were collected at inclusion (sNfL1) and second visit (sNfL2, mean delay 10.4 ± 8.7 months). sNfL levels, determined by single-molecule array, were compared between ALS and controls at both time points. sNfL concentration changes were compared between patients with Riluzole (w/Ril) at inclusion in the study and those who were treated by Riluzole following inclusion (w/o Ril). The factors influencing sNfL concentrations and changes were studied using linear regression and multivariate analysis.sNfL levels were higher in ALS patients than in controls at the two time points (p 0.00001). In ALS patients, sNfL concentrations were higher in females for both sNfL1 (p = 0.014) and sNfL2 (p 0.001). In the whole ALS group, sNfL levels were higher at sNfL2 than at sNfL1 (p 0.001). sNfL1 and sNfL2 concentrations were similar between the two ALS subgroups (w/ and w/o Ril). ALS functional rating scale-revised rate of decline and gender were the two main factors significantly influencing both sNfL1 and sNfL2 levels (p 0.01). However, only gender was shown to significantly influence sNfL changes with time (p = 0.003).In this study, sNfL levels increased with time in ALS patients and there was no difference between subjects already treated by Riluzole and those treated after sNfL1. Further studies with larger population samples and different sampling intervals are warranted to better determine the real potential of sNfL measurement as a tool to monitor treatment response in ALS.

The diagnosis of skin wound vitality is currently based on standard histology, but histological findings lack sensitivity in case of a short survival time. New reliable biomarkers of vitality are therefore strongly needed. We assessed the ability of 10 candidate cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α) to discriminate between vital and early post-mortem wounds.Twenty-four cadavers with a recent open skin wound ( 3 h) were included (20 men, 4 women, mean age = 51.0 ± 24.3 years). An early post-mortem wound was performed in an uninjured skin area, and both wounds were sampled at the autopsy (post-mortem interval (PMI) = 66.3 ± 28.3 h). Needle-puncture sites related to resuscitation cares were included as very early post-mortem wounds (n = 6). In addition to standard histology, cytokines levels were simultaneously measured in each sample using a multiplex sandwich immunoassay, then normalized on healthy skin levels. A quantitative evaluation of IL-8-positive cells in ante- and post-mortem wound samples was also performed.In the training set of samples (n = 72), cytokine levels were significantly higher in vital wounds (mean age = 47 ± 53 min) than in post-mortem wounds (mean PMI = 6.9 ± 9.0 h) (p 0.2), except for two cytokines (IFN-γ and IL-2). IL-8 was the best discriminatory cytokine (Se = 54%, Sp = 100%, AUC = 0.79), while a multivariate model combining IL-4 and IL12p70 was a bit more discriminant (Se = 55%, Sp = 100%, AUC = 0.84). In the validation set (n = 72), the discriminatory power of the cytokines and the predictive model was slightly lower, with IL-8 remaining the best cytokine (Se = 46%, Sp = 96%, AUC = 0.75). The predictive model remained highly specific (Sp = 100%). Both the cytokines and the predictive model allowed the iatrogenic injuries to be correctly classified as post-mortem wounds. Standard histology and immunohistochemistry showed 21% sensitivity and a specificity of 79% and 100%, respectively. Only two iatrogenic wounds could be properly categorized histologically.This study suggests that cytokines could be useful biomarkers of skin wound vitality and that the immunoassay method could be more sensitive than immunohistochemistry to identify wounds with a short survival time. Further research is underway to confirm these preliminary data.

Tau proteins are recognized biomarkers of neurodegeneration and neuronal damage in the cerebrospinal fluid (CSF). It has also been suggested that these CSF proteins could increase post-mortem due to neuronal death. The aim of this study was to investigate the changes in CSF total and phosphorylated tau (p-tau) levels in the early post-mortem interval (PMI), to determine whether these proteins could be relevant biomarkers of time since death.Tau and p-tau levels were measured by ELISA in lumbar and cisternal CSF samples from 82 corpses (46 men, 36 women, mean age: 72.4 ± 15.2 years) with a PMI 12 h. Forty-eight of them were considered neurologically healthy at the time of death. Rectal and tympanic temperatures were also measured in 37 individuals, and two validated temperature-based methods of PMI estimation were applied (Henssge's nomogram and Baccino's method).CSF tau and p-tau levels were significantly increased, with respective median values of 3315 pg/mL and 68.5 pg/mL in the whole cohort, while lower but still increased levels were observed in neurologically healthy patients. Sub-occipital punctures systematically provided higher tau and p-tau values (p 0.0001). Despite a great inter-individual variability, the concentrations of both biomarkers were positively correlated with the early PMI, with the highest correlation for cisternal p-tau (r = 0.50, p 0.0001 in the whole cohort; r = 0.58, p = 0.0003 in the neurologically healthy patients). Higher levels of CSF biomarkers were observed for PMI 6 h versus PMI ≤ 6 h, the discriminatory power of the biomarkers being higher in the subgroup of neurologically healthy patients. Based on cut-off values obtained by ROC curve analysis, the CSF biomarkers could rectify or adjust the time interval provided by the temperature-based methods in a significant number of cases. A predictive model combining tympanic temperature and cisternal tau values was found to be particularly accurate to assign individuals according to their PMI (≤ or 6 h), with a Se of 83% and a Sp of 100% (AUC = 0.95).Our findings suggest that CSF tau and p-tau proteins could serve as potential biomarkers of time since death, in association with tympanic temperature. The practical applicability of such an integrated approach has to be assessed by further studies.

International audience; To improve the etiological diagnosis of neurodegenerative dementias like Alzheimer's disease (AD) or frontotemporal dementia (FTD), we evaluated the value of individual and combined measurements of the following relevant cerebrospinal fluid (CSF) biomarkers: Tau, 181p-Tau, Aβ38, Aβ40, Aβ42, sAβPPα, and sAβPPβ. This study conducted in two centers included patients with FTD (n = 34), AD (n = 52), as well as a control group of persons without dementia (CTRL, n = 42). Identical clinical criteria and pre-analytical conditions were used while CSF biomarkers were measured using commercial single and multiplex quantitative immunoassays. Thorough statistical analyses, including ROC curves, logistic regressions, and decision trees, were performed. We validated in AD the specific increase of p-Tau levels and the decrease of Aβ42 levels, two biological hallmarks of this disease. Tau concentrations were highest in AD and intermediate in FTD when compared to CTRL. The most interesting results were obtained by focusing on amyloid biomarkers as we found out in FTD a significant decrease of sAβPPβ, Aβ38, and Aβ40 levels. Aβ38 in particular was the most useful biomarker to differentiate FTD subjects from the CTRL population. Combining p-Tau and Aβ38 led us to correctly classifying FTD patients with sensitivity at 85% and specificity at 82%. Significant changes in amyloid biomarkers, particularly for Aβ38, are therefore seen in FTD. This could be quite useful for diagnosis purposes and it might provide additional evidence on the interrelationship between Tau and AβPP biology which understanding is essential to progress towards optimal therapeutic and diagnostic approaches of dementia.

International audience; Detection of autoantibodies, which are involved in tissue injury and/or the reporters from the immune system of various pathologic events, has an important potential for diagnosis, prognosis, disease staging and treatment selection. This explains the interest for new proteomics technologies, such as the high-density protein microarray used here, that allow a high-throughput, multiplexed and sensitive detection of specific autoantibodies. So far, most of the research has been performed on blood. In this note, we focus on the cerebrospinal fluid in an attempt to address autoimmune events associated with neurological disorders. Importantly, the cerebrospinal fluid is quite different from the blood in terms of protein composition and concentration. We had therefore to adapt the available blood protocols. We present here the result of our optimization that will be useful to carry out full scale immunological studies of the cerebrospinal fluid using high-density protein microarrays.

International audience; Background: Proteomics analysis of human cerebrospinal fluid (CSF) is a major tool for identifying novel biomarkers for neurological diseases. However, the complexity and wide dynamic range of CSF represent a major challenge for detecting specific low-abundance biomarkers. One way to overcome this problem is to rely on different pre-fractionation techniques. However, the most relevant technique remains to be determined. Methods: This study compared three different well-known pre-fractionation methods: immuno-depletion of major proteins (Seppro® IgY14), hydrophobic solid phase extraction (Oasis® HLB), and lipophilic sorbent concentration (Liposorb™). Unfractionated and pre-fractionated CSF was digested with trypsin and analyzed by RP-LC-MS/MS with an OrbitrapTM mass spectrometer. We documented the number of peptides detected and sets of proteins identified. Experiments were repeated to minimize pre-analytical and analytical variability.Results: Compared to unfractionated CSF, the OASIS® HLB fractionated CSF method showed a significant 28% increase in the total number of proteins identified, while the Liposorb™ capture resulted in a significant 46% decrease. Interestingly, results based on the number of peptides detected were different. We also evaluated the capacity of these pre-fractionation methods to detect different proteins in terms of their molecular weight, isoelectrophoretic point (IEP) or nature. Each of these pre-fractionation methods identified a specific subset of proteins, when compared to unfractionated CSF, and/or other methods. This was particularly obvious for the lipophilic sorbent, which allowed the detection of many lipoproteins.Conclusion: Direct analysis of digested CSF led to the identification of several proteins despite matrix complexity. As expected, single pre-fractionation methods that can be included in simple and cost-effective workflows, yielded significant differences in terms of number, or range of proteins identified. This suggests that a single pre-fractionation method cannot cover the full range of protein species present in a complex sample

Background: The use of dried blood spots on filter paper is well documented as an affordable and practical alternative to classical venous sampling for various clinical needs. This technique has indeed many advantages in terms of collection, biological safety, storage, and shipment. Amyloid β (A β ) peptides are useful cerebrospinal fluid (CSF) biomarkers for Alzheimer disease diagnosis. However, A β determination is hindered by preanalytical difficulties in terms of sample collection and stability in tubes. Methods: We compared the quantification of A β peptides (1 – 40, 1 – 42, and 1 – 38) by simplex and multiplex ELISA, following either a standard operator method (liquid direct quantification) or after spotting CSF onto dried matrix paper card. Results: The use of dried matrix spot (DMS) overcame preanalytical problems and allowed the determination of A β concentrations that were highly commutable (BlandAltman) with those obtained using CSF in classical tubes. Moreover, we found a positive and significant correlation (r 2 = 0.83, Pearson coefficient p = 0.0329) between the two approaches. Conclusions: This new DMS method for CSF represents an interesting alternative that increases the quality and efficiency in preanalytics. This should enable the better exploitation of A β analytes for Alzheimer ’ s diagnosis.

Cerebrospinal fluid (CSF) biomarkers are now widely used for diagnosis of Alzheimer disease (AD) in atypical clinical forms, for differential and early diagnosis, or for stratification of patients in clinical trials. Among these biomarkers, different forms of amyloid peptides (Aβ) produced by the cleavage of a transmembrane precursor protein called APP (amyloid precursor protein) have a major role. Aβ peptides exist in different length the most common ones having 40 (Aβ40), 42 (Aβ42), or 38 (Aβ38) amino acids in length. APP processing by gamma-secretase releases also an amino-terminal secreted fragment called sAβPP-beta while an alternative nonamyloidogenic cleavage of APP, through an alpha-secretase, liberates another fragment called sAβPP-alpha. To decipher the molecular and pathological mechanisms leading to the production and the detection of these entities is essential for the comprehension and the prevention of AD. In this report, we present the results of the multiplex measurement of CSF Aβ38, Aβ40, Aβ42, sAβPP-alpha, and sAβPP-beta in 60 patients mostly with dementia eventually segregated between neurochemical dementia diagnostic (NDD) positive and negative groups. The NDD classification was based on our routine Tau, P-tau(181), and Aβ(42) cutoff values. We confirmed previous findings regarding the correlation between sAβPP-alpha and sAβPP-beta, as well as the potential interest of these new biomarkers. We also studied the correlation between sAβPPs and Aβ peptides, as well as between Aβ peptides themselves. We observed a strong correlation between Aβ38 and sAβPP-beta which suggested that the production of this peptide was in direct relation with β secretase activities. We also reported a strong correlation between Aβ38 and Aβ40, while Aβ42 was correlated to these fragments only in nonpathological situations. These results enlighten the complex relationships between these molecular markers in both physiological and pathological situations. Our results are important for the further use of these analytes for AD diagnosis as well as for validating the cell biological hypotheses of APP processing and Aβ fragment production.