Publications by authors named "Constance Delaby"

37 Publications

The importance of an integrated genotype-phenotype strategy to unravel the molecular bases of titinopathies.

Neuromuscul Disord 2020 11 28;30(11):877-887. Epub 2020 Sep 28.

Laboratoire de Génétique Moléculaire, Centre Hospitalier Universitaire de Montpellier, LGMR, Université de Montpellier, France. Electronic address:

Next generation sequencing (NGS) has allowed the titin gene (TTN) to be identified as a major contributor to neuromuscular disorders, with high clinical heterogeneity. The mechanisms underlying the phenotypic variability and the dominant or recessive pattern of inheritance are unclear. Titin is involved in the formation and stability of the sarcomeres. The effects of the different TTN variants can be harmless or pathogenic (recessive or dominant) but the interpretation is tricky because the current bioinformatics tools can not predict their functional impact effectively. Moreover, TTN variants are very frequent in the general population. The combination of deep phenotyping associated with RNA molecular analyses, western blot (WB) and functional studies is often essential for the interpretation of genetic variants in patients suspected of titinopathy. In line with the current guidelines and suggestions, we implemented for patients with skeletal myopathy and with potentially disease causing TTN variant(s) an integrated genotype-transcripts-protein-phenotype approach, associated with phenotype and variants segregation studies in relatives and confrontation with published data on titinopathies to evaluate pathogenic effects of TTN variants (even truncating ones) on titin transcripts, amount, size and functionality. We illustrate this integrated approach in four patients with recessive congenital myopathy.
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http://dx.doi.org/10.1016/j.nmd.2020.09.032DOI Listing
November 2020

Cerebrospinal fluid A beta 1-40 peptides increase in Alzheimer's disease and are highly correlated with phospho-tau in control individuals.

Alzheimers Res Ther 2020 10 2;12(1):123. Epub 2020 Oct 2.

Univ Montpellier, INSERM, CHU Montpellier (CMRR), Montpellier, France.

Background: Amyloid pathology, which is one of the characteristics of Alzheimer's disease (AD), results from altered metabolism of the beta-amyloid (Aβ) peptide in terms of synthesis, clearance, or aggregation. A decrease in cerebrospinal fluid (CSF) level Aβ1-42 is evident in AD, and the CSF ratio Aβ42/Aβ40 has recently been identified as one of the most reliable diagnostic biomarkers of amyloid pathology. Variations in inter-individual levels of Aβ1-40 in the CSF have been observed in the past, but their origins remain unclear. In addition, the variation of Aβ40 in the context of AD studied in several studies has yielded conflicting results.

Methods: Here, we analyzed the levels of Aβ1-40 using multicenter data obtained on 2466 samples from six different cohorts in which CSF was collected under standardized protocols, centrifugation, and storage conditions. Tau and p-tau (181) concentrations were measured using commercially available in vitro diagnostic immunoassays. Concentrations of CSF Aβ1-42 and Aβ1-40 were measured by ELISA, xMAP technology, chemiluminescence immunoassay (CLIA), and mass spectrometry. Statistical analyses were calculated for parametric and non-parametric comparisons, linear regression, correlation, and odds ratios. The statistical tests were adjusted for the effects of covariates (age, in particular).

Results: Regardless of the analysis method used and the cohorts, a slight but significant age-independent increase in the levels of Aβ40 in CSF was observed in AD. We also found a strong positive correlation between the levels of Aβ1-40 and p-tau (181) in CSF, particularly in control patients.

Conclusions: These results indicate that an increase in the baseline level of amyloid peptides, which are associated with an increase in p-tau (181), may be a biological characteristic and possibly a risk factor for AD. Further studies will be needed to establish a causal link between increased baseline levels of Aβ40 and the development of the disease.
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http://dx.doi.org/10.1186/s13195-020-00696-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532565PMC
October 2020

Hepcidin and ferritin levels in restless legs syndrome: a case-control study.

Sci Rep 2020 07 17;10(1):11914. Epub 2020 Jul 17.

Sleep-Wake Disorders Unit, Department of Neurology, Gui-de-Chauliac Hospital, CHU Montpellier, Montpellier University, Montpellier, France.

The association between restless legs syndrome (RLS) and iron homeostasis remains unclear. We compared serum hepcidin and ferritin levels in patients with RLS and controls, and assessed their relationships with RLS phenotype, drug intake, and history of augmentation syndrome. 102 drug-free RLS patients (age 58.9 [24.5-77.2], 63 females) and 73 controls (age 56.8 [23.46-76.6], 45 females) underwent a polysomnography recording. Hepcidin levels were quantified by ELISA. 34 RLS patients had a second assessment after starting dopaminergic drugs. Ferritin level was low (< 50 µg/l) in 14.7% of patients and 25% of controls, with no between-group differences in the mean values. Hepcidin levels were higher in patients even after adjustment for confounding factors, and excluding participants with low ferritin levels. Ferritin and hepcidin levels were comparable before and after treatment, and between patients with (n = 17) and without history of augmentation. Ferritin and hepcidin levels correlated with age, body mass index, and periodic leg movements. Higher hepcidin levels were associated with older age, older age at RLS onset, less daytime sleepiness and familial RLS. In conclusion, serum hepcidin levels but not ferritin were higher in RLS patients regardless of treatment and history of augmentation. Serum hepcidin may be a more relevant biomarker of RLS than ferritin.
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http://dx.doi.org/10.1038/s41598-020-68851-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367854PMC
July 2020

Detection of amyloid beta peptides in body fluids for the diagnosis of alzheimer's disease: Where do we stand?

Crit Rev Clin Lab Sci 2020 03 29;57(2):99-113. Epub 2019 Oct 29.

INSERM U1183, Laboratoire de Biochimie-Protéomique Clinique, CHU de Montpellier, Université de Montpellier, Montpellier, France.

Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by progressive decline of cognitive abilities. Amyloid beta peptides (Aβ), Tau proteins and the phosphorylated form of the Tau protein, p-Tau, are the core pathological biomarkers of the disease, and their detection for the diagnosis of patients is progressively being implemented. However, to date, their quantification is mostly performed on cerebrospinal fluid (CSF), the collection of which requires an invasive lumbar puncture. Early diagnosis has been shown to be important for disease-modifying treatment, which is currently in development, to limit the progression of the disease. Nevertheless, the diagnosis is often delayed to the point where the disease has already progressed, and the tools currently available do not allow for a systematic follow-up of patients. Thus, the search for a molecular signature of AD in a body fluid such as blood or saliva that can be collected in a minimally invasive way offers hope. A number of methods have been developed for the quantification of core biomarkers, especially in easily accessible fluids such as the blood, that improve their accuracy, specificity and sensitivity. This review summarizes and compares these approaches, focusing in particular on their use for Aβ detection, the earliest biomarker to be modified in the course of AD. The review also discusses biomarker quantification in CSF, blood and saliva and their clinical applications.
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http://dx.doi.org/10.1080/10408363.2019.1678011DOI Listing
March 2020

The Sant Pau Initiative on Neurodegeneration (SPIN) cohort: A data set for biomarker discovery and validation in neurodegenerative disorders.

Alzheimers Dement (N Y) 2019 14;5:597-609. Epub 2019 Oct 14.

Department of Neurology, Sant Pau Memory Unit, Hospital de la Santa Creu i Sant Pau - IIB Sant Pau, Barcelona, Spain.

Introduction: The SPIN (Sant Pau Initiative on Neurodegeneration) cohort is a multimodal biomarker platform designed for neurodegenerative disease research following an integrative approach.

Methods: Participants of the SPIN cohort provide informed consent to donate blood and cerebrospinal fluid samples, receive detailed neurological and neuropsychological evaluations, and undergo a structural 3T brain MRI scan. A subset also undergoes other functional or imaging studies (video-polysomnogram, F-fluorodeoxyglucose PET, amyloid PET, Tau PET). Participants are followed annually for a minimum of 4 years, with repeated cerebrospinal fluid collection and imaging studies performed every other year, and brain donation is encouraged.

Results: The integration of clinical, neuropsychological, genetic, biochemical, imaging, and neuropathological information and the harmonization of protocols under the same umbrella allows the discovery and validation of key biomarkers across several neurodegenerative diseases.

Discussion: We describe our particular 10-year experience and how different research projects were unified under an umbrella biomarker program, which might be of help to other research teams pursuing similar approaches.
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http://dx.doi.org/10.1016/j.trci.2019.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804606PMC
October 2019

Biochemical markers of time since death in cerebrospinal fluid: A first step towards .

Crit Rev Clin Lab Sci 2019 06 6;56(4):274-286. Epub 2019 Jun 6.

b Laboratory of Biochemistry and Clinical Proteomics, Montpellier University Hospital, Institute for Regenerative Medicine and Biotherapy , Montpellier , France.

The accurate estimation of the time of death is a challenge in forensic medicine, as the methods routinely used to assess the postmortem interval (PMI) are far from being precise. Over the past decades, biochemical methods have been implemented on postmortem samples to improve the precision of PMI estimation. Studies have focussed on the biochemical profiles of closed compartment body fluids, as they are preserved longer than blood after death and are thus subject to confined postmortem chemical changes. Cerebrospinal fluid (CSF) has been considered a suitable fluid to investigate these changes, as it is found in large amounts and is easy to sample. Moreover, the main molecules found in CSF have known reference values in living subjects, unlike most other body fluids. In this literature review, we focus on the panel of biomarkers that have been studied in CSF based on their potential of offering information on the time of death. The interest in these biomarkers for casework and the research perspectives in this field are discussed. Integrating data from different methods, including biochemistry, for better estimation of the time of death would represent a step forward in the forensic field, paving the way for an innovative approach that we suggest to call
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http://dx.doi.org/10.1080/10408363.2019.1619158DOI Listing
June 2019

Impact of CSF storage volume on the analysis of Alzheimer's disease biomarkers on an automated platform.

Clin Chim Acta 2019 Mar 20;490:98-101. Epub 2018 Dec 20.

Sant Pau Memory Unit, Department of Neurology, Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas, CIBERNED, Spain. Electronic address:

Objectives: To assess the potential influence of the ratio between the storage tube surface area and the volume of cerebrospinal fluid (CSF) (surface/volume) on the quantifications of four Alzheimer's disease (AD) biomarkers on the Lumipulse G600II automated platform.

Methods: CSF samples of 10 consecutive patients were stored in 2 ml polypropylene tubes containing four different CSF volumes: 1.5 ml, 1 ml, 0.5 ml and 0.25 ml. Concentration of CSF Aβ1-42, Aβ1-40, t-Tau and p-Tau were measured in all aliquots using the LUMIPULSE G600II automated platform from Fujirebio.

Results: Levels of CSF Aβ1-42 and Aβ1-40 were lower in samples stored with lower volumes (higher surface/volume ratios). This decrease was partly compensated by using the ratio Aβ1-42/Aβ1-40. Quantification of t-Tau and p-Tau were not influenced by this pre-analytical condition.

Conclusion: The surface/volume ratio can potentially influence the results of amyloid AD biomarkers. It appears essential to take into account the surface/volume ratio of the storage tubes when quantifying CSF biomarkers in clinical routine.
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http://dx.doi.org/10.1016/j.cca.2018.12.021DOI Listing
March 2019

Hepcidin: immunoanalytic characteristics.

Ann Biol Clin (Paris) 2018 Dec;76(6):705-715

CHU de Montpellier, Institut de médecine régénératrice et de biothérapies (IRMB), Laboratoire de biochimie-protéomique clinique, CCBHM, Hôpital Saint Eloi, Montpellier, France, Faculté de médecine, Université de Montpellier, France, Inserm U1040, Montpellier, France.

Hepcidin has progressively become essential in clinical practice for the diagnosis and follow-up of a large spectrum of diseases. Anyway, its own biochemical and structural characteristics have complicated and delayed the acquisition of a standardized quantifying tool of the peptide.
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http://dx.doi.org/10.1684/abc.2018.1382DOI Listing
December 2018

Plasma and CSF biomarkers for the diagnosis of Alzheimer's disease in adults with Down syndrome: a cross-sectional study.

Lancet Neurol 2018 10 29;17(10):860-869. Epub 2018 Aug 29.

Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau-Biomedical Research Institute Sant Pau-Universitat Autònoma de Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

Background: Diagnosis of Alzheimer's disease in Down syndrome is challenging because of the absence of validated diagnostic biomarkers. We investigated the diagnostic performance of plasma and CSF biomarkers in this population.

Methods: We did a cross-sectional study of adults aged 18 years and older with Down syndrome enrolled in a population-based health plan in Catalonia, Spain. Every person with Down syndrome assessed in the health plan was eligible to enter the Down Alzheimer Barcelona Neuroimaging Initiative, and those with a plasma or CSF sample available were included in this study. Participants underwent neurological and neuropsychological examination and blood sampling, and a subset underwent a lumbar puncture. Adults with Down syndrome were classified into asymptomatic, prodromal Alzheimer's disease, or Alzheimer's disease dementia groups by investigators masked to biomarker data. Non-trisomic controls were a convenience sample of young (23-58 years) healthy people from the Sant Pau Initiative on Neurodegeneration. Amyloid-β (Aβ), Aβ, total tau (t-tau), 181-phosphorylated tau (p-tau; only in CSF), and neurofilament light protein (NfL) concentrations were measured in plasma with a single molecule array assay and in CSF with ELISA. Plasma and CSF biomarker concentrations were compared between controls and the Down syndrome clinical groups. Diagnostic performance was assessed with receiver operating characteristic curve analyses between asymptomatic participants and those with prodromal Alzheimer's disease and between asymptomatic participants and those with Alzheimer's disease dementia.

Findings: Between Feb 1, 2013, and Nov 30, 2017, we collected plasma from 282 participants with Down syndrome (194 asymptomatic, 39 prodromal Alzheimer's disease, 49 Alzheimer's disease dementia) and 67 controls; CSF data were available from 94 participants (54, 18, and 22, respectively) and all 67 controls. The diagnostic performance of plasma biomarkers was poor (area under the curve [AUC] between 0·53 [95% CI 0·44-0·62] and 0·74 [0·66-0·82]) except for plasma NfL concentrations, which had an AUC of 0·88 (0·82-0·93) for the differentiation of the asymptomatic group versus the prodromal Alzheimer's disease group and 0·95 (0·92-0·98) for the asymptomatic group versus the Alzheimer's disease dementia group. In CSF, except for Aβ concentrations (AUC 0·60, 95% CI 0·45-0·75), all biomarkers had a good performance in the asymptomatic versus prodromal Alzheimer's disease comparison: AUC 0·92 (95% CI 0·85-0·99) for Aβ, 0·81 (0·69-0·94) for t-tau, 0·80 (0·67-0·93) for p-tau, and 0·88 (0·79-0·96) for NfL. Performance of the CSF biomarkers was optimal in the asymptomatic versus Alzheimer's disease dementia comparison (AUC ≥0·90 for all except Aβ [0·59, 0·45-0·72]). Only NfL concentrations showed a strong correlation between plasma and CSF biomarker concentrations in participants with Down syndrome (rho=0·80; p<0·0001).

Interpretation: Plasma NfL and CSF biomarkers have good diagnostic performance to detect Alzheimer's disease in adults with Down syndrome. Our findings support the utility of plasma NfL for the early detection of Alzheimer's disease in Down syndrome in clinical practice and clinical trials.

Funding: Institute of Health Carlos III, Fundació La Marató de TV3, Fundació Bancaria Obra Social La Caixa, Fundació Catalana Síndrome de Down, and Fundació Víctor Grífols i Lucas.
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http://dx.doi.org/10.1016/S1474-4422(18)30285-0DOI Listing
October 2018

Relevance of Aβ42/40 Ratio for Detection of Alzheimer Disease Pathology in Clinical Routine: The PLM Scale.

Front Aging Neurosci 2018 28;10:138. Epub 2018 May 28.

Laboratoire de Biochimie Protéomique Clinique, Institute of Regenerative Medicine and Biotherapies, Centre Hospitalier Universitaire de Montpellier, Montpellier, France.

Cerebrospinal fluid (CSF) biomarkers (Aβ peptides and tau proteins) improved the diagnosis of Alzheimer's disease (AD) in research and clinical settings. We previously described the PLM-scale (Paris-Lille-Montpellier study), which combines Aβ42, tau, and phosphorylated ptau(181) biomarkers in an easy to use and clinically relevant way. The purpose of this work is to evaluate an optimized PLMscale (PLM ratio scale) that now includes the Aβ42/Aβ40 ratio to detect AD versus non-AD (NAD) participants in clinical routine of memory centers. Both scales were compared using 904 participants with cognitive impairment recruited from two independent cohorts (Mtp-1 and Mtp-2). The CSF Aβ42/Aβ40 ratio was measured systematically in Mtp-1, and only on biologically discordant cases in Mtp-2. Two different ELISA kit providers were also employed. The distribution of AD and NAD patients and the discrepancies of biomarker profiles were computed. Receiver Operating Characteristic curves were used to represent clinical sensitivity and specificity for AD detection. The classification of patients with the net reclassification index (NRI) was also evaluated. Nine hundred and four participants (342 AD and 562 NAD) were studied; 400 in Mtp-1 and 504 in Mtp-2. For AD patients, the mean CSF Aβ42 and CSF Aβ42/40 ratio was 553 ± 216 pg/mL and 0.069 ± 0.022 pg/mL in Mtp-1 and 702 ± 335 pg/mL and 0.045 ± 0.020 pg/mL in Mtp-2. The distribution of AD and NAD differed between the PLM and the PLM scales ( < 0.0001). The percentage AD well-classified (class 3) increased with PLM from 38 to 83% in Mpt-1 and from 33 to 53% in Mpt-2. A sharp reduction of the discordant profiles going from 34 to 16.3% and from 37.5 to 19.8%, for Mtp-1 and Mtp-2 respectively, was also observed. The AUC of the PLM scale was 0.94 in Mtp-1 and 0.87 in Mtp-2. In both cohorts, the PLM outperformed CSF Aβ42 or Aβ42/40 ratio. The diagnostic performance was improved with the PLM with an NRI equal to 44.3% in Mtp-1 and 28.8% in Mtp-2. The integration of the Aβ42/Aβ40 ratio in the PLM scale resulted in an easy-to-use tool which reduced the discrepancies in biologically doubtful cases and increased the confidence in the diagnosis in memory center.
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http://dx.doi.org/10.3389/fnagi.2018.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985301PMC
May 2018

Nano-flow vs standard-flow: Which is the more suitable LC/MS method for quantifying hepcidin-25 in human serum in routine clinical settings?

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Jun 10;1086:110-117. Epub 2018 Apr 10.

University of Montpellier, CHU Montpellier, IRMB, LBPC, Montpellier F-34000, France. Electronic address:

Hepcidin-25 peptide is a biomarker which is known to have considerable clinical potential for diagnosing iron-related diseases. Developing analytical methods for the absolute quantification of hepcidin is still a real challenge, however, due to the sensitivity, specificity and reproducibility issues involved. In this study, we compare and discuss two MS-based assays for quantifying hepcidin, which differ only in terms of the type of liquid chromatography (nano LC/MS versus standard LC/MS) involved. The same sample preparation, the same internal standards and the same MS analyzer were used with both approaches. In the field of proteomics, nano LC chromatography is generally known to be more sensitive and less robust than standard LC methods. In this study, we established that the performances of the standard LC method are equivalent to those of our previously developed nano LC method. Although the analytical performances were very similar in both cases. The standard-flow platform therefore provides the more suitable alternative for accurately determining hepcidin in clinical settings.
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http://dx.doi.org/10.1016/j.jchromb.2018.04.003DOI Listing
June 2018

Association between serum hepcidin level and restless legs syndrome.

Mov Disord 2018 04 8;33(4):618-627. Epub 2018 Feb 8.

CHU Montpellier, Institut de Recherche en Biothérapie, hôpital St Eloi, Laboratoire de Biochimie Protéomique Clinique et CCBHM, INSERM U1040, Montpellier, France.

Background: To better understand the role of iron homeostasis dysregulation in restless legs syndrome, we compared serum hepcidin and ferritin levels in drug-free patients with primary restless legs syndrome and healthy controls and studied the relationship between hepcidin level and restless legs syndrome severity.

Methods: One hundred and eight drug-free patients with primary restless legs syndrome (65 women; median age, 61.5 years) and 45 controls (28 women; median age, 53.9 years) were enrolled. Inclusion criteria were: normal ferritin level (>50 ng/mL) and absence of iron disorders, chronic renal or liver failure, and inflammatory or neurological diseases. Each subject underwent a thorough clinical examination and a polysomnography assessment. Serum hepcidin-25 was quantified using a validated mass spectrometry method. Restless legs syndrome severity was evaluated according to the International Restless Legs Syndrome Study Group.

Results: Despite no group difference between normal ferritin levels and demographic features, serum hepcidin level and hepcidin/ferritin ratio were higher in patients than in controls. Hepcidin level and hepcidin/ferritin ratio, but not ferritin level, were positively correlated with periodic leg movements during sleep and wakefulness in the whole sample. Hepcidin level seem to be associated with restless legs syndrome severity in a complex U-shaped relationship, without relationship with age at restless legs syndrome onset, positive family history, sleep and depressive symptoms, genetic background, and polysomnographic measurements. No relationship was found between ferritin level and restless legs syndrome severity.

Conclusion: In drug-free patients with primary restless legs syndrome, hepcidin level is higher than in controls and may be associated with restless legs syndrome clinical severity. This result emphasizes the complex peripheral iron metabolism deregulation in restless legs syndrome, opening potential perspectives for a personalized approach with a hepcidin antagonist. © 2018 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.27287DOI Listing
April 2018

Relevance of Follow-Up in Patients with Core Clinical Criteria for Alzheimer Disease and Normal CSF Biomarkers.

Curr Alzheimer Res 2018 ;15(7):691-700

University Lille, Inserm U1171, Centre Memoire de Ressources et de Recherche & CNR-MAJ, CHU Lille, F-59000 Lille, France.

Background: Few patients with a normal cerebrospinal fluid (CSF) biomarker profile fulfill the clinical criteria for Alzheimer disease (AD).

Objective: The aim of this study was to test the hypothesis of misdiagnoses for these patients.

Method: Patients from the e-PLM centers fulfilling the core clinical criteria for probable AD dementia or mild cognitive impairment due to AD (AD-MCI), with normal CSF Aβ1-42, T-tau and P-tau biomarkers and clinical follow-up, were included. Clinical and imaging data were reviewed by an independent board, from baseline (visit with clinical evaluation and CSF analysis) to the end of the follow-up, for a final diagnosis.

Results: In the e-PLM cohort of 1098 AD patients with CSF analysis, 37 (3.3%) patients (20 with AD dementia core clinical criteria and 17 with AD-MCI core clinical criteria) had normal CSF biomarker profile and a clinical follow-up. All patients presented with episodic memory impairment and 27 (73%) had medial temporal lobe atrophy on MRI-scan. After a median follow-up of 36 months (range 7-74), the final diagnosis was AD MCI or dementia for 9 (24%) patients, and unlikely due to AD for 28 (76%) patients. A misdiagnosis was corrected in 18 (49%) patients (mood disorders, non-AD degenerative dementia, vascular cognitive impairment, alcohol cognitive disorders, temporal epilepsy and hippocampal sclerosis), and 10 (27%) patients had cognitive disorders of undetermined etiology.

Conclusion: AD diagnosis (MCI or dementia) with normal CSF biomarkers is a rare condition. A clinical follow- up is particularly recommended to consider an alternative diagnosis.
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http://dx.doi.org/10.2174/1567205015666180110113238DOI Listing
May 2019

Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model.

Haematologica 2017 02 10;102(2):260-270. Epub 2016 Nov 10.

INSERM U1149/ERL CNRS 8252, Centre de Recherche sur l'Inflammation Paris Montmartre, 75018 Paris, France

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.
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http://dx.doi.org/10.3324/haematol.2016.151621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286934PMC
February 2017

Added value of hepcidin quantification for the diagnosis and follow-up of anemia-related diseases.

Ann Biol Clin (Paris) 2017 Feb;75(1):9-18

Institut de médecine régénératrice et de biothérapies (IRMB)-Laboratoire de biochimie-protéomique clinique-CCBHM, Hôpital Saint Eloi, CHRU de Montpellier, Montpellier, France, Université de Montpellier, France, Inserm U1040, Montpellier, France.

Iron homeostasis is based on a strict control of both intestinal iron absorption and iron recycling through reticulo-endothelial system. Hepcidin controls the iron fluxes in order to maintain sufficient iron levels for erythropoietic activities, hemoproteins synthesis or enzymes function, but also to limit its toxic accumulation throughout the body. Hepcidin expression is regulated by various stimuli: inflammation and iron stimulate the production of the peptide, while anemia, erythropoiesis and hypoxia repress its production. Regulation of hepcidin expression is not so simple in complex pathological situations such as hemolytic anemia, cancer or chronic inflammation. Serum hepcidin quantification in association with the diagnostic tests currently available is quite promising for the diagnosis or the follow-up of anemia in those conditions. This study is part of the working group « Clinical interests of hepcidin quantification » of the Société française de biologie clinique.
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http://dx.doi.org/10.1684/abc.2016.1208DOI Listing
February 2017

Looking for new biomarkers of skin wound vitality with a cytokine-based multiplex assay: preliminary study.

Ann Biol Clin (Paris) 2017 Feb;75(1):53-60

Laboratoire de biochimie - protéomique clinique, Institut de médecine régénératrice et de biothérapies, Hôpital St Éloi, CHU de Montpellier, Université de Montpellier, France.

Determination of skin wound vitality is an important issue in forensic practice. No reliable biomarker currently exists. Quantification of inflammatory cytokines in injured skin with MSD technology is an innovative and promising approach. This preliminary study aims to develop a protocol for the preparation and the analysis of skin samples. Samples from ante mortem wounds, post mortem wounds, and intact skin ("control samples") were taken from corpses at the autopsy. After an optimization of the pre-analytical protocol had been performed in terms of skin homogeneisation and proteic extraction, the concentration of TNF-α was measured in each sample with the MSD approach. Then five other cytokines of interest (IL-1β, IL-6, IL-10, IL-12p70 and IFN-γ) were simultaneously quantified with a MSD multiplex assay. The optimal pre-analytical conditions consist in a proteic extraction from a 6 mm diameter skin sample, in a PBS buffer with triton 0,05%. Our results show the linearity and the reproductibility of the TNF-α quantification with MSD, and an inter- and intra-individual variability of the concentrations of proteins. The MSD multiplex assay is likely to detect differential skin concentrations for each cytokine of interest. This preliminary study was used to develop and optimize the pre-analytical and analytical conditions of the MSD method using injured and healthy skin samples, for the purpose of looking for and identifying the cytokine, or the set of cytokines, that may be biomarkers of skin wound vitality.
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http://dx.doi.org/10.1684/abc.2016.1214DOI Listing
February 2017

Quantification of hepcidin-25 in human cerebrospinal fluid using LC-MS/MS.

Bioanalysis 2017 Feb 20;9(4):337-347. Epub 2017 Jan 20.

Laboratoire de Biochimie et de Protéomique Clinique - Institut de Médecine Régénérative et Biothérapies (LBPC-IRMB), CHU de Montpellier, 34250 Montpellier, France.

Aim: Hepcidin, the main iron metabolism regulator, can be detected in various biological fluids. Here, we describe a quantitative method of LC-MS/MS to quantify the 25 amino acid isoform of hepcidin (hepcidin-25) in human cerebrospinal fluid (CSF). Results & methodology: Samples were prepared through protein precipitation followed by solid phase extraction (SPE) and injected into a triple-quadrupole mass spectrometer. Validation of our method included determination of LOQ (0.1 ng/ml), repeatability, intermediate precision, recovery and linearity (up to 25 ng/ml). Hepcidin-25 was subsequently quantified in 36 human CSF samples and its concentration ranged from 0.21 to 3.54 ng/ml.

Conclusion: This is the first time that hepcidin-25 can be reliably quantified in human CSF. This may open interesting perspectives for the management of iron-related neurological disorders.
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http://dx.doi.org/10.4155/bio-2016-0240DOI Listing
February 2017

Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.

Clin Chim Acta 2016 Feb 19;454:33-8. Epub 2015 Dec 19.

CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France. Electronic address:

Background: Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping.

Methods: Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti human ApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping.

Results: Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance.

Conclusion: Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.
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http://dx.doi.org/10.1016/j.cca.2015.12.020DOI Listing
February 2016

Central Nervous System and Peripheral Inflammatory Processes in Alzheimer's Disease: Biomarker Profiling Approach.

Front Neurol 2015 24;6:181. Epub 2015 Aug 24.

Laboratoire de Biochimie-Protéomique Clinique, Institute for Regenerative Medicine and Biotherapy (IRMB), CHU de Montpellier and Université Montpellier , Montpellier , France.

Brain inflammation is one of the hallmarks of Alzheimer disease (AD) and a current trend is that inflammatory mediators, particularly cytokines and chemokines, may represent valuable biomarkers for early screening and diagnosis of the disease. Various studies have reported differences in serum level of cytokines, chemokines, and growth factors in patients with mild cognitive impairment or AD. However, data were often inconsistent and the exact function of inflammation in neurodegeneration is still a matter of debate. In the present work, we measured the expression of 120 biomarkers (corresponding to cytokines, chemokines, growth factors, and related signaling proteins) in the serum of 49 patients with the following diagnosis distribution: 15 controls, 14 AD, and 20 MCI. In addition, we performed the same analysis in the cerebrospinal fluid (CSF) of 20 of these patients (10 AD and 10 controls). Among the biomarkers tested, none showed significant changes in the serum, but 13 were significantly modified in the CSF of AD patients. Interestingly, all of these biomarkers were implicated in neurogenesis or neural stem cells migration and differentiation. In the second part of the study, 10 of these putative biomarkers (plus 4 additional) were quantified using quantitative multiplex ELISA methods in the CSF and the serum of an enlarged cohort composed of 31 AD and 24 control patients. Our results confirm the potential diagnosis interest of previously published blood biomarkers, and proposes new ones (such as IL-8 and TNFR-I). Further studies will be needed to validate these biomarkers which could be used alone, combined, or in association with the classical amyloid and tau biomarkers.
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http://dx.doi.org/10.3389/fneur.2015.00181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547499PMC
September 2015

Cerebrospinal fluid amyloid-β 42/40 ratio in clinical setting of memory centers: a multicentric study.

Alzheimers Res Ther 2015 1;7(1):30. Epub 2015 Jun 1.

Biochimie-Protéomique Clinique - IRB - CCBHM, INSERM U1040, Université de Montpellier, Montpellier, France.

Introduction: The cerebrospinal fluid (CSF) biomarkers amyloid-β (Aβ), tau and phosphorylated tau (p-tau181) are now used for the diagnosis of Alzheimer's disease (AD). Aβ40 is the most abundant Aβ peptide isoform in the CSF, and the Aβ 42/40 ratio has been proposed to better reflect brain amyloid production. However, its additional value in the clinical setting remains uncertain.

Methods: A total of 367 subjects with cognitive disorders who underwent a lumbar puncture were prospectively included at three French memory centers (Paris-North, Lille and Montpellier; the PLM Study). The frequency of positive, negative and indeterminate CSF profiles were assessed by various methods, and their adequacies with the diagnosis of clinicians were tested using net reclassification improvement (NRI) analyses.

Results: On the basis of local optimum cutoffs for Aβ42 and p-tau181, 22% of the explored patients had indeterminate CSF profiles. The systematic use of Aβ 42/40 ratio instead of Aβ42 levels alone decreased the number of indeterminate profiles (17%; P = 0.03), but it failed to improve the classification of subjects (NRI = -2.1%; P = 0.64). In contrast, the use of Aβ 42/40 ratio instead of Aβ42 levels alone in patients with a discrepancy between p-tau181 and Aβ42 led to a reduction by half of the number of indeterminate profiles (10%; P < 0.001) and was further in agreement with clinician diagnosis (NRI = 10.5%; P = 0.003).

Conclusions: In patients with a discrepancy between CSF p-tau181 and CSF Aβ42, the assessment of Aβ 42/40 ratio led to a reliable biological conclusion in over 50% of cases that agreed with a clinician's diagnosis.
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http://dx.doi.org/10.1186/s13195-015-0114-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450486PMC
June 2015

A diagnostic scale for Alzheimer's disease based on cerebrospinal fluid biomarker profiles.

Alzheimers Res Ther 2014 26;6(3):38. Epub 2014 Jun 26.

CHU de Montpellier and Université Montpellier I, IRMB, CCBHM, Laboratoire de Biochimie Protéomique Clinique, 80 Avenue Augustin Fliche, 34295 Montpellier, France ; Centre Mémoire Ressources Recherche Languedoc-Roussillon, CHU de Montpellier, Hôpital Gui de Chauliac, Montpellier, and Université Montpellier I, Montpellier, France.

Introduction: The relevance of the cerebrospinal fluid (CSF) biomarkers for the diagnosis of Alzheimer's disease (AD) and related disorders is clearly established. However, the question remains on how to use these data, which are often heterogeneous (not all biomarkers being pathologic). The objective of this study is to propose to physicians in memory clinics a biologic scale of probabilities that the patient with cognitive impairments has an Alzheimer's disease (AD) pathologic process.

Methods: For that purpose, we took advantage of the multicenter data of our Paris-North, Lille, and Montpellier (PLM) study, which has emerged through the initial sharing of information from these memory centers. Different models combining the CSF levels of amyloid-β 42, tau, and p-tau(181) were tested to generate categories of patients with very low (<10%), low (<25%), high (>75%), and very high predictive values (>90%) for positive AD. In total, 1,273 patients (646 AD and 627 non-AD) from six independent memory-clinic cohorts were included.

Results: A prediction model based on logistic regressions achieved a very good stratification of the population but had the disadvantages of needing mathematical optimization and being difficult to use in daily clinical practice. Remarkably, a simple and intuitive model based on the number (from zero to three) of three pathologic CSF biomarkers resulted in a very efficient predictive scale for AD in patients seen in memory clinics. The scale's overall predictive value for AD for the different categories were as follows: class 0, 9.6% (95% confidence interval (CI), 6.0% to 13.2%); class 1, 24.7% (95% CI, 18.0% to 31.3%); class 2, 77.2% (95% CI, 67.8% to 86.5%); and class 3, 94.2% (95% CI, 90.7% to 97.7%). In addition, with this scale, significantly more patients were correctly classified than with the logistic regression. Its superiority in model performance was validated by the computation of the net reclassification index (NRI). The model was also validated in an independent multicenter dataset of 408 patients (213 AD and 195 non-AD).

Conclusions: In conclusion, we defined a new scale that could be used to facilitate the interpretation and routine use of multivariate CSF data, as well as helping the stratification of patients in clinical research trials.
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http://dx.doi.org/10.1186/alzrt267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255520PMC
December 2014

Systemic delivery of siRNA down regulates brain prion protein and ameliorates neuropathology in prion disorder.

PLoS One 2014 14;9(2):e88797. Epub 2014 Feb 14.

Institut de Médecine Régénératrice et de Biothérapie (I.M.R.B.), Physiopathologie, diagnostic et thérapie cellulaire des affections neurodégénératives -Institut National de la Santé et de la Recherche Médicale Université Montpellier 1 U1040 Centre Hospitalo-Universitaire de Montpellier, Université Montpellier 1, Montpellier, France ; Institut de Génétique Humaine, Centre National de la Recherche Scientifique- UPR1142, Montpellier, France.

One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrP(C) level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088797PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925167PMC
October 2014

Impact of harmonization of collection tubes on Alzheimer's disease diagnosis.

Alzheimers Dement 2014 Oct 23;10(5 Suppl):S390-S394.e2. Epub 2013 Oct 23.

Service de Neurobiologie, Hospices Civils de Lyon, Université Lyon 1-CNRS UMR5292-INSERM U1028, Lyon, France.

Objective: The objective of this study was to analyze differences in biomarker outcomes before and after harmonization of cerebrospinal fluid (CSF) collection tubes in Alzheimer's disease (AD) diagnosis.

Methods: We analyzed data from French memory centers that switched from different CSF collection tubes to a common one. A total of 1966 patients were included in the study. CSF concentrations of β-amyloid 1-42 (Aβ42), total tau, and phosphorylated tau (p-tau181) were measured in each center using the same commercial enzyme-linked immunoabsorbent assay (ELISA) kits. The diagnostic value of CSF biomarkers according to the type of tube used was then assessed using different cutoffs.

Results: The predictive value of Aβ42 was highly affected by the type of collection tube used. The optimal cutoff value for p-tau181 appeared not to be affected by the type of collection tube whereas that of total tau was slightly changed. New optimal cutoff values were then computed.

Conclusions: In a routine clinical environment, the selection of the collection tube and biomarker cutoff value makes a major difference in AD biological diagnosis. The use of a common collection tube among different centers will reduce the risk of misdiagnosis and incorrect patient stratification.
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http://dx.doi.org/10.1016/j.jalz.2013.06.008DOI Listing
October 2014

Development and validation of dried matrix spot sampling for the quantitative determination of amyloid β peptides in cerebrospinal fluid.

Clin Chem Lab Med 2014 May;52(5):649-55

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 (Aβ) peptides are useful cerebrospinal fluid (CSF) biomarkers for Alzheimer disease diagnosis. However, Aβ determination is hindered by preanalytical difficulties in terms of sample collection and stability in tubes.

Methods: We compared the quantification of Aβ 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 Aβ concentrations that were highly commutable (Bland-Altman) with those obtained using CSF in classical tubes. Moreover, we found a positive and significant correlation (r2=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 Aβ analytes for Alzheimer's diagnosis.
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http://dx.doi.org/10.1515/cclm-2013-0611DOI Listing
May 2014

Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice.

Mamm Genome 2013 Dec;24(11-12):427-38

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.
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http://dx.doi.org/10.1007/s00335-013-9479-6DOI Listing
December 2013

Current and future use of "dried blood spot" analyses in clinical chemistry.

Clin Chem Lab Med 2013 Oct;51(10):1897-909

The analysis of blood spotted and dried on a matrix (i.e., "dried blood spot" or DBS) has been used since the 1960s in clinical chemistry; mostly for neonatal screening. Since then, many clinical analytes, including nucleic acids, small molecules and lipids, have been successfully measured using DBS. Although this pre-analytical approach represents an interesting alternative to classical venous blood sampling, its routine use is limited. Here, we review the application of DBS technology in clinical chemistry, and evaluate its future role supported by new analytical methods such as mass spectrometry.
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http://dx.doi.org/10.1515/cclm-2013-0228DOI Listing
October 2013

Hepcidin regulates intrarenal iron handling at the distal nephron.

Kidney Int 2013 Oct 24;84(4):756-66. Epub 2013 Apr 24.

1] INSERM U773, Centre de Recherche Biomédicale Bichat-Beaujon, Université Paris Diderot, Site Bichat, Paris, France [2] Université Paris Diderot, Site Bichat, Paris, France.

Hepcidin, the key regulatory hormone of iron homeostasis, and iron carriers such as transferrin receptor1 (TFR1), divalent metal transporter1 (DMT1), and ferroportin (FPN) are expressed in kidney. Whether hepcidin plays an intrinsic role in the regulation of renal iron transport is unknown. Here, we analyzed the renal handling of iron in hemochromatosis Hepc(-/-) and Hjv(-/-) mouse models, as well as in phenylhydrazine (PHZ)-treated mice. We found a marked medullary iron deposition in the kidneys of Hepc(-/-) mice, and iron leak in the urine. The kidneys of Hepc(-/-) mice exhibited a concomitant decrease in TFR1 and increase in ferritin and FPN expression. Increased FPN abundance was restricted to the thick ascending limb (TAL). DMT1 protein remained unaffected despite a significant decrease of its mRNA level, suggesting that DMT1 protein is stabilized in the absence of hepcidin. Treatment of kidney sections from Hepc(-/-) mice with hepcidin decreased DMT1 protein, an effect confirmed in renal cell lines where hepcidin markedly decreased (55)Fe transport. In the kidneys of Hjv(-/-) mice exhibiting low hepcidin expression, the iron overload was similar to that in the kidneys of Hepc(-/-) mice. However, in PHZ mice, iron accumulation resulting from hemoglobin leak was detected in the proximal tubule. Thus, kidneys exhibit a tissue-specific handling of iron that depends on the extra iron source. Hepcidin may control the expression of iron transporters to prevent renal iron overload.
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http://dx.doi.org/10.1038/ki.2013.142DOI Listing
October 2013

Subcellular localization of iron and heme metabolism related proteins at early stages of erythrophagocytosis.

PLoS One 2012 30;7(7):e42199. Epub 2012 Jul 30.

INSERM, U773, Centre de Recherche Bichat Beaujon CRB3, Paris, France.

Background: Senescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined.

Methodology/principal Findings: We took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes.

Conclusions/significance: Our study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042199PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408460PMC
January 2013

[Serum hepcidin assay in 2011: where do we stand?].

Ann Biol Clin (Paris) 2012 Jul-Aug;70(4):377-86

INSERM U773, Centre de recherche biomédicale Bichat Beaujon CRB3, Université Paris Diderot Paris 7, UFR de Médecine site Bichat, Paris, France.

The characterization of hepcidin and of its role in iron metabolism in 2001 has entirely modified our understanding of the pathogenesis of iron-linked disorders. Clinical applications related to hepcidin are numerous, and involve iron-overload diseases, anemia, renal disease, chronic inflammation or cancer. Thus, new avenues have emerged from the discovery of this 25 amino acid peptide and numerous diagnostic and therapeutic tools have been developed and described in the past 10 years. In particular, various methods for accurate quantification of hepcidin in urine and serum have been published but development of a reliable assay is quite a challenging task because, in particular, of inherent properties of the peptide's structure and its low immunogenicity. Thus, the different methods described so far show high variability, in terms of quantitative value of hepcidin measured, specificity and sensitivity. Comparison of these methods has been recently described in a "Round Robin" study, in order to harmonise hepcidin assays and to improve and standardise the available detection/quantification tests. This article focuses on the molecular mechanisms of hepcidin regulation and reviews the various methods of hepcidin quantification, describing the advantages and limitations of each one of them.
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http://dx.doi.org/10.1684/abc.2012.0725DOI Listing
January 2013