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Fabry disease is a frequent lysosomal storage disorder secondary to the deficiency of alpha-galactosidase A enzyme. This X-linked genetic disease realizes progressive and systemic manifestations that affect both male and female. Fabry disease may present as "classical", as "late-onset" or "non-classical" forms. Symptoms and organ involvements of classical Fabry disease are acral pain crisis, cornea verticillata, hypertrophic cardiomyopathy, stroke and chronic kidney disease with proteinuria. Other common symptoms are often poorly recognized, such as gastrointestinal or ear involvements. In classical Fabry disease, symptoms first appear during childhood or during teenage years in males, but later in females. Patients with non-classical or late-onset Fabry disease have delayed manifestations or a single-organ involvement. Diagnosis is therefore difficult when classical organ involvements are missing, in paucisymptomatic patients or in late-onset forms. Recognition of Fabry disease is important because effective treatments are available. They have to be prescribed early. In male, diagnosis is made with alpha-galactosidase A enzyme activity dosage in leukocyte, that is very low or null in classical forms and under 30 percent in late-onset forms. Diagnosis is more challenging in females who may express normal residual enzyme activity. Other plasmatic biomarkers, such as lyso-globotriaosylceramide are interesting, especially in females. In this review, we aimed to summarize main clinical manifestations of Fabry disease to know when to evoke Fabry disease and propose a practical diagnosis algorithm to know how to diagnose.

Alkaline phosphatase activity is a parameter included in biochemical liver test. These isoenzymes are of various cellular origin inducing physiological variations on age and sex. The alkaline phosphatase activity standardization as well as numerous international studies have made it possible to standardize the pediatric reference values. The hyperphosphatasemia etiologies are very well know but the hypophosphatasemia are hardly explored and can allow the diagnosis of pathologies including hypophosphatasia, a rare treatable disease.

The metabolism of homocysteine is complex and involves many enzymes as well as vitamin-derived cofactors. Any dysregulation of this metabolism may lead to hyperhomocysteinemia (HHCy) which is responsible for many clinical disorders including thromboembolic events. HHCy may result from very different etiologies and is generally classified into three groups according to homocysteine concentrations: moderate ( 100 μmol/L). Major HHCy cases are generally due to monogenic defects of key enzymes involved in homocysteine metabolism, such as cystathionine-β-synthase or 5,10-methylene-tetrahydrofolate reductase, or to any defect in vitamin B12 absorption, transport or metabolism. By contrast, moderate and intermediate HHCy tend to result from so-called “secondary” etiologies (e.g. tobacco, drugs, alcohol, vitamin deficiencies or pathological contexts). Here we describe the case of a patient with an unusually high plasma homocysteine concentration (1562 μmol/L) which was only explained by a combination of such secondary etiologies, among them chronic renal failure, hypothyroidism, the homozygous C677T MTHFR variant, a novel heterozygous variant of the MSR gene, and a vitamin deficiency. In addition, this patient exhibited a spectacular decline in homocysteine concentrations (returning to normal) after betaine and vitamin administration. In conclusion, this case highlights that major HHCy may also result from the combination of secondary etiologies, with vitamin deficiency as a triggering factor.

Recently, pre-analytical, analytical, and post-analytical issues have been addressed to implement biofluid FTIR spectroscopy as a novel diagnostic tool in the clinical setting. Although hemolysis, icterus, and hyperlipidemia are known to interfere with colorimetric and turbidimetric biochemical methods, there are no data on their impact on serum/plasma FTIR spectra. This study aimed at investigating the impact of hemoglobin, bilirubin, and triglycerides concentrations on plasma spectral analysis. Plasma samples with high concentrations of hemoglobin, conjugated bilirubin, or triglycerides were studied. To mimic the various concentrations observed in clinical setting, samples were diluted using normal plasma and analyzed using high-throughput FTIR spectroscopy. Hemolytic, icteric, and hyperlipidemic plasma spectra were compared with control plasma spectra. Unsupervised analysis of all spectra was performed using principal component analysis. The comparison between control and hemolytic plasmas did not show spectral differences in the range of hemoglobin concentrations observed in spurious or pathological hemolysis. By contrast, spectra from lipidemic plasmas had different spectral profiles compared with control plasma, exhibiting increased absorbance in lipid bands. Differences in the same spectral regions were observed in spectra from icteric plasma, which may be explained by the hyperlipidemia associated with cholestasis. PCA did not discriminate between control and hemolytic plasmas up to 1 g/L hemoglobin but confirmed the interference of bilirubin and triglycerides concentrations on spectral classification. Our results show that hemolysis does not have an impact on the plasma spectral profile except for high concentrations of hemoglobin rarely observed in clinical practice, whereas icterus and hyperlipidemia constitute significant confounding factors. Graphical abstract.

BACKGROUND: Urea cycle disorders (UCD) are rare diseases that usually affect neonates or young children. During decompensations, hyperammonemia is neurotoxic, leading to severe symptoms and even coma and death if not treated rapidly. AIMS: Description of a cohort of patients with adult onset of UCDs. METHODS: Multicentric, retrospective and descriptive study of French adult patients with a diagnosis after 16 years of age of UCDs due to a deficiency in one of the 6 enzymes (arginase, ASL, ASS, CPS1, NAGS, OTC) or the two transporters (ORNT1 or citrin). RESULTS: Seventy-one patients were included (68% female, 32% male). The diagnosis was made in the context of (i) a metabolic decompensation (42%), (ii) family history (55%), or (iii) chronic symptoms (3%). The median age at diagnosis was 33 years (range 16-86). Eighty-nine percent of patients were diagnosed with OTC deficiency, 7% CPS1 deficiency, 3% HHH syndrome and 1% argininosuccinic aciduria. For those diagnosed during decompensations (including 23 OTC cases, mostly female), 89% required an admission in intensive care units. Seven deaths were attributed to UCD - 6 decompensations and 1 epilepsy secondary to inaugural decompensation. CONCLUSION: This is the largest cohort of UCDs diagnosed in adulthood, which confirms the triad of neurological, gastrointestinal and psychiatric symptoms during hyperammonemic decompensations. We stress that females with OTC deficiency can be symptomatic. With 10% of deaths in this cohort, UCDs in adults remain a life-threatening condition. Physicians working in adult care must be aware of late-onset presentations given the implications for patients and their families. This article is protected by copyright. All rights reserved.

Background and study aims White bile is defined as a colorless fluid occasionally found in the biliary tract of patients with bile duct obstruction. Its significance is not clearly established. Our objective was to analyze the prognostic value of white bile in a series of patients with biliary obstruction due to biliary or pancreatic cancer. Patients and methods The study was conducted on a series of consecutive patients with malignant obstructive jaundice. They all underwent endoscopic retrograde cholangiopancreatography with collection of bile and biliary stent insertion. White bile was defined as bile duct fluid with bilirubin level Results Seventy-three patients were included (32 pancreatic cancers, 41 bile duct cancers). Thirty-nine (53.4 %) had white bile. The mean bile duct bilirubin level in this group was 4.2 ± 5.9 µmol/L vs 991 ± 1039 µmol/L in patients with colored bile (P Conclusion White bile in patients with inoperable malignant biliary obstruction is an independent factor of poor survival.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Organic acid chromatography allows the identification of several hundred compounds and the quantification of the main molecules of interest. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from the French society for inborn errors of metabolism (SFEIM) recommends an approach to accredit organic acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Acylcarnitine profile allows the identification and quantification of more than thirty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit acylcarnitine profile. Validation parameters and recommendations are discussed in this specific framework.

International audience; In this study, we investigated the effect of [N-(5-chloro-2-hydroxyphenyl)-l-aspartato] chlorogallate (GS2), a new water soluble gallium complex, on cell invasion and on the expression and activity of matrix metalloproteinases (MMPs) in human metastatic HT-1080 fibrosarcoma and MDA-MB 231 breast carcinoma cells. The effect on cell invasion was studied using a modified Boyden chamber coated with a type-I collagen. We analyzed the effect of GS2 on MMP-2, MMP-9, and MMP-14 via zymography and enzymatic assay using high affinity fluorogenic substrates. The expression of MMP mRNA was analyzed via qRT-PCR. GS2 induced a decrease in cell invasion. A dose-dependent inhibition effect was observed on the activities of MMP-2, MMP-9, and MMP-14 with the IC50 values of 168, 82, and 20 μM, respectively. A decrease in the expression of MMP-14 mRNA was observed in both cell lines, whereas the expression of MMP-2 and MMP-9 mRNA was decreased only in the MDA-MB231 cells. Data obtained for the expression of MMP-14 mRNA were confirmed via Western blotting. In fact, MMP-14 expression was decreased in the presence of GS2. Overall, these data show that GS2 is a promising compound for anti-invasive and anticancer therapy.

Infrared spectroscopy is a rapid, easy-to-operate, label-free and therefore cost-effective technique. Many studies performed on biofluids (eg, serum, plasma, urine, sputum, bile and cerebrospinal fluid) have demonstrated its promising application as a clinical diagnostic tool. Given all these characteristics, infrared spectroscopy appears to be an ideal candidate to be implemented into the clinics. However, before considering its translation, a clear effort is needed to standardise protocols for biofluid spectroscopic analysis. To reach this goal, careful investigations to identify and track errors that can occur during the pre-analytical phase is a crucial step. Here, we report for the first time, results of investigations into pre-analytical factors that can affect the quality of the spectral data acquired on serum and plasma, such as the impact of long-term freezing time storage of samples as well as the month-to-month reproducibility of the spectroscopic analysis. The spectral data discrimination has revealed to be majorly impacted by a residual water content variation in serum and plasma dried samples.

International audience; Background: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methods: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.Results: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.Conclusions: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

We report the case of an alcoholic patient with severe pancytopenia with low plasma folate level but normal erythrocyte folates and cobalamin levels. The bone marrow smear concluded to a pancytopenia due to folates and/or cobalamin deficiency. Severe pancytopenia due to acute plasma folate deficiency can be observed despite normal erythrocyte folates level which reflects the organism's folates store.

// Charles Saby 1 , Emilie Buache 1 , Sylvie Brassart-Pasco 2 , Hassan El Btaouri 3 , Marie-Pierre Courageot 3 , Laurence Van Gulick 1 , Roselyne Garnotel 2 , Pierre Jeannesson 1 and Hamid Morjani 1 1 Centre National de la Recherche Scientifique (CNRS), Unite Mixte de Recherche (UMR) Matrice Extracellulaire et Dynamique Cellulaire, Universite de Reims Champagne-Ardenne, Unite de Formation et de Recherche (UFR) Pharmacie, Reims, France 2 Centre National de la Recherche Scientifique (CNRS), Unite Mixte de Recherche (UMR) Matrice Extracellulaire et Dynamique Cellulaire, Universite de Reims Champagne-Ardenne, Unite de Formation et de Recherche (UFR) Medecine, Reims, France 3 Centre National de la Recherche Scientifique (CNRS), Unite Mixte de Recherche (UMR) Matrice Extracellulaire et Dynamique Cellulaire, Universite de Reims Champagne-Ardenne, Unite de Formation et de Recherche (UFR) Sciences Exactes et Naturelles, Reims, France Correspondence to: Hamid Morjani, email: // Keywords : type I collagen, aging, cancer, discoidin domain receptor 2, cell proliferation, Gerotarget Received : July 16, 2015 Accepted : April 10, 2016 Published : April 18, 2016 Abstract Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D). We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen. In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21 CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21 CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

Affecting more than 30% of the Western population, nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and can lead to multiple complications, including nonalcoholic steatohepatitis (NASH), cancer, hypertension, and atherosclerosis. Insulin resistance and obesity are described as potential causes of NAFLD. However, we surmised that factors such as extracellular matrix remodeling of large blood vessels, skin, or lungs may also participate in the progression of liver diseases. We studied the effects of elastin-derived peptides (EDPs), biomarkers of aging, on NAFLD progression. We evaluated the consequences of EDP accumulation in mice and of elastin receptor complex (ERC) activation on lipid storage in hepatocytes, inflammation, and fibrosis development. The accumulation of EDPs induces hepatic lipogenesis (i.e., SREBP1c and ACC), inflammation (i.e., Kupffer cells, IL-1β, and TGF-β), and fibrosis (collagen and elastin expression). These effects are induced by inhibition of the LKB1-AMPK pathway by ERC activation. In addition, pharmacological inhibitors of EDPs demonstrate that this EDP-driven lipogenesis and fibrosis relies on engagement of the ERC. Our data reveal a major role of EDPs in the development of NASH, and they provide new clues for understanding the relationship between NAFLD and vascular aging.

International audience

Maintenance of mesenchymal stem cells (MSCs) requires a tissue-specific microenvironment (i.e., niche), which is poorly represented by the typical plastic substrate used for two-dimensional growth of MSCs in a tissue culture flask. The objective of this study was to address the potential use of collagen-based medical devices (HEMOCOLLAGENE®, Saint-Maur-des-Fossés, France) as mimetic niche for MSCs with the ability to preserve human MSC stemness in vitro. With a chemical composition similar to type I collagen, HEMOCOLLAGENE® foam presented a porous and interconnected structure (>90%) and a relative low elastic modulus of around 60 kPa. Biological studies revealed an apparently inert microenvironment of HEMOCOLLAGENE® foam, where 80% of cultured human MSCs remained viable, adopted a flattened morphology, and maintained their undifferentiated state with basal secretory activity. Thus, three-dimensional HEMOCOLLAGENE® foams present an in vitro model that mimics the MSC niche with the capacity to support viable and quiescent MSCs within a low stiffness collagen I scaffold simulating Wharton’s jelly. These results suggest that haemostatic foam may be a useful and versatile carrier for MSC transplantation for regenerative medicine applications.

This study aimed at determining whether FTIR spectroscopy is able to distinguish bile samples from patients with and without malignant biliary strictures. Bile samples were collected in 19 patients with malignant biliary strictures and 38 with benign biliary diseases during endoscopic procedures. FTIR spectra were acquired on dried drops of whole bile, aqueous and organic phases obtained after lipid extraction. Data were analyzed by principal component analysis and by the support vector machine classification using a leave-n-out cross validation procedure. This was applied to the whole set of spectra and the mean and median spectra of each patient. By leaving one patient out, the classifier allowed discriminating patients with and without malignant biliary strictures with a sensitivity between 82% and 95% and a specificity between 85% and 100%. Using a randomized leave-n -out cross-validation with n = 2, 5 and 10 patients, the sensitivity decreased slightly by about 5 to 10% while the specificity remained stable, suggesting the robustness of the classifier. FTIR spectroscopy combined with chemometrics therefore shows potential to differentiate bile from patients with and without malignant biliary strictures. Although promising, the results of this pilot study cannot be generalized and needs to be confirmed in a larger population.

International audience; AIMS: Elastin is degraded during vascular ageing and its products, elastin-derived peptides (EP), are present in the human blood circulation. EP binds to the elastin receptor complex (ERC) at the cell surface, composed of elastin-binding protein (EBP), a cathepsin A and a neuraminidase 1. Some in vitro functions have clearly been attributed to this binding, but the in vivo implications for arterial diseases have never been clearly investigated. METHODS AND RESULTS: Here, we demonstrate that chronic doses of EP injected into mouse models of atherosclerosis increase atherosclerotic plaque size formation. Similar effects were observed following an injection of a VGVAPG peptide, suggesting that the ERC mediates these effects. The absence of phosphoinositide 3-kinase γ (PI3Kγ) in bone marrow-derived cells prevented EP-induced atherosclerosis development, demonstrating that PI3Kγ drive EP-induced arterial lesions. Accordingly, in vitro studies showed that PI3Kγ was required for EP-induced monocyte migration and ROS production and that this effect was dependent upon neuraminidase activity. Finally, we showed that degradation of elastic lamellae in LDLR(-/-) mice fed an atherogenic diet correlated with atherosclerotic plaque formation. At the same time, the absence of the cathepsin A-neuraminidase 1 complex in cells of the haematopoietic lineage abolished atheroma plaque size progression and decreased leucocytes infiltration, clearly demonstrating the role of this complex in atherogenesis and suggesting the involvement of endogenous EP. CONCLUSION: Altogether, this work identifies EP as an enhancer of atherogenesis and defines the Neuraminidase 1/PI3Kγ signalling pathway as a key mediator of this function in vitro and in vivo.

L’histoire de la spectrometrie de masse debute au crepuscule du XIXe siecle lorsque Sir Joseph J. Thompson, au laboratoire Cavendish de l’Universite de Cambridge decouvre l’electron en 1897. Cette decouverte le mena au debut du XXe siecle a construire le premier spectrometre de masse, appele a l’epoque le spectrographe parabolique (figure 1), qui lui permit de determiner les rapports masse/charge ionique, qui lui valut en 1906 le prix Nobel de physique [1].Le professeur [...]

Several proof-of-concept studies on the vibrational spectroscopy of biofluids have demonstrated that the methodology has promising potential as a clinical diagnostic tool. However, these studies also show that there is a lack of a standardised protocol in sample handling and preparation prior to spectroscopic analysis. One of the most important sources of analytical errors is the pre-analytical phase. For the technique to be translated into clinics, it is clear that a very strict protocol needs to be established for such biological samples. This study focuses on some of the aspects of the pre-analytical phase in the development of the high-throughput Fourier Transform Infrared (FTIR) spectroscopy of some of the most common biofluids such as serum, plasma and bile. Pre-analytical considerations that can impact either the samples (solvents, anti-coagulants, freeze–thaw cycles…) and/or spectroscopic analysis (sample preparation such as drying, deposit methods, volumes, substrates, operators dependence…) and consequently the quality and the reproducibility of spectral data will be discussed in this report.

The extracellular matrix critically controls cancer cell behavior by inducing several signaling pathways through cell membrane receptors. Besides conferring structural properties to tissues around the tumor, the extracellular matrix is able to regulate cell proliferation, survival, migration, and invasion. Among these receptors, the integrins family constitutes a major class of receptors that mediate cell interactions with extracellular matrix components. Twenty years ago, a new class of extracellular matrix receptors has been discovered. These tyrosine kinase receptors are the two discoidin domain receptors DDR1 and DDR2. DDR1 was first identified in the Dictyostelium discoideum and was shown to mediate cell aggregation. DDR2 shares highly conserved sequences with DDR1. Both receptors are activated upon binding to collagen, one of the most abundant proteins in extracellular matrix. While DDR2 can only be activated by fibrillar collagen, particularly types I and III, DDR1 is mostly activated by type I and IV collagens. In contrast with classical growth factor tyrosine kinase receptors which display a rapid and transient activation, DDR1 and DDR2 are unique in that they exhibit delayed and sustained receptor phosphorylation upon binding to collagen. Recent studies have reported differential expression and mutations of DDR1 and DDR2 in several cancer types and indicate clearly that these receptors have to be taken into account as new players in the different aspects of tumor progression, from non-malignant to highly malignant and invasive stages. This review will discuss the current knowledge on the role of DDR1 and DDR2 in malignant transformation, cell proliferation, epithelial to mesenchymal transition, migratory, and invasive processes, and finally the modulation of the response to chemotherapy. These new insights suggest that DDR1 and DDR2 are new potential targets in cancer therapy.

Anticancer drug resistance is a multifactorial process that includes acquired andde novodrug resistances. Acquired resistance develops during treatment, whilede novoresistance is the primary way for tumor cells to escape chemotherapy. Tumor microenvironment has been recently shown to be one of the important factors contributing tode novoresistance and called environment-mediated drug resistance (EMDR). Two forms of EMDR have been described: soluble factor-mediated drug resistance (SFM-DR) and cell adhesion-mediated drug resistance (CAM-DR). Anthracyclines, among the most potent chemotherapeutic agents, are widely used in clinics against hematopoietic and solid tumors. Their main mechanism of action relies on the inhibition of topoisomerase I and/or II and the induction of apoptosis. Beyond this well-known antitumor activity, it has been recently demonstrated that anthracyclines may display potent anti-invasive effects when used at subtoxic concentrations. In this paper, we will describe two particular modes of EMDR by which microenvironment may influence tumor-cell response to one of these anthracyclines, doxorubicin. The first one considers the influence of type I collagen on the antimigratory effect of doxorubicin (CAM-DR). The second considers the protection of tumor cells by thrombospondin-I against doxorubicin-induced apoptosis (SFM-DR).

Matrix metalloproteinase (MMP) family proteins play diverse roles in many aspects of cellular processes such as osteoblastic differentiation. Besides, mechanical forces that occur in 3D collagen gel promote the osteoblastic phenotype and accelerate matrix mineralization. Although MMPs have been involved in bone differentiation, the proteolytic cascades triggered by mechanical forces are still not well characterized. In this study, we have investigated the contribution of both proteolytic cascades, MMP-3/MMP-1 and MMP-2/MMP-13/MT1-MMP in the differentiation of human osteoblasts cultured in a floating type I collagen lattice (FL) versus an attached collagen lattice (AL). Compared to AL, contraction of human osteoblasts-populated FL led to a fast (1 day) induction of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteoprotegerin (OPG), and Runx-2 expression. At day 4, osteocalcin (OC) overexpression preceded the formation of calcium-containing nodule formation as assessed by X-ray analyses. MMP-1 and MMP-3 were produced to similar extent by cells cultured in FL and AL, whereas contraction of collagen lattices triggered both mRNA overexpression of MMP-2, MMP-13, and MT1-MMP (i.e., MMP-14), and their activation as evidenced by Western blotting or zymographic analyses. Down-regulating MT1-MMP expression or activity either by siRNA transfection or supplementation of culture medium with TIMP-1 or TIMP-2 highlighted the contribution of that enzyme in OC, ALP, and OPG expression. MMP-2 and MMP-13 were more directly involved in BSP expression. So, these results suggest that the main proteolytic cascade, MMP-2/MMP-13/MT1-MMP, and more particularly, its initial regulator MT1-MMP is involved in osteoblast differentiation through mechanical forces.

To implement neuroprotective strategies in newborns, sensitive and specific biomarkers are needed for identifying those who are at risk for brain damage. We evaluated the effectiveness of matrix metalloproteinases (MMPs) and their naturally occurring tissue inhibitors of metalloproteinases (TIMPs) in predicting neonatal encephalopathy (NE) damage in newborns.Plasma MMP-9 and TIMP-1 levels were upregulated as early as 1 h after the HI insult but not did not show such elevations after other types of injury (ibotenate-induced excitotoxicity, hypoxia, lipopolysaccharide-induced inflammation), and brain levels reflected this increase soon thereafter. We confirmed these results by carrying out plasma MMP-9 and TIMP-1 measurements in human newborns with NE. In these infants, protein levels of MMP-9 and TIMP-1 were found to be elevated during a short window up to 6 h after birth.This feature is particularly useful in identifying newborns in need of neuroprotection. A second peak observed 72 h after birth is possibly related to the second phase of energy failure after a HI insult. Our data, although preliminary, support the use of MMP-9 and TIMP-1 as early biomarkers for the presence and extent of perinatal brain injury in human term newborns.We first used a mouse model of neonatal HI injury to explore mechanistic aspects such as the time course of these markers after the hypoxia-ischemia event, and the correlation between the levels of these candidate markers in brain and plasma.

Les peptides d'elastine (kE) augmentent l'expression des MMP3 et MMP1 dans les fibroblastes gingivaux humains cultives en 2D sur plastique alors qu'ils n'ont aucune influence sur la production d'uPA, des MMPs2, 13, 14 et des TIMPs1 et 2. L'effet de kE sur la production de MMP3 depend du recepteur membranaire S-Gal tandis qu'il est inhibe par le lactose et reproduit par des peptides contenant la sequence VGVAPG capable de se fixer sur S-Gal. Afin d'evaluer l'implication des peptides d'elastine dans la collagenolyse, des cultures cellulaires en 3D ou lattis de collagene attache sont utilises : kE ou le plasminogene (Plgn) seuls n'induisent pas de facon significative une collagenolyse des lattis de collagene. Toutefois, kE, en presence de Plgn, augmente de facon synergique la degradation du collagene et l'activation des MMPs. Au sein des lattis en presence de peptides d'elastine, la collagenolyse serait dependante d'une sur-expression de la cascade proteolytique MMP3/MMP1 par les fibroblastes gingivaux humains, associee a une sous-expression des TIMP1 et de TIMP2. (Med Buccale Chir Buccale 2009 ; 15 : 75-85).

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the α1 chain C-terminal telopeptide in type I collagen-induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post-translational binding of cyanate to lysine e-NH2 groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen-induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6-amino-hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the α1CB6 collagen peptide, was involved in this mechanism. A recombinant α1CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen-mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.

The apicomplexan Toxoplasma gondii , an obligate intracellular parasite, can infect humans and a wide range of vertebrates. Following oral infection, the parasite invades tissues by crossing non-permissive biological barriers such as the placenta or the blood–brain barrier. But the molecular mechanisms underlying migration of T. gondii remain poorly characterized. The crossing of various basal membranes and infiltration into the extracellular matrix by T. gondii could involve matrix metalloproteinases (MMPs). We demonstrated a decrease in proMMP-2 and proMMP-9 secretion by THP-1 cells at 24 and 48 h post invasion with regulation at the mRNA level throughout infection. This down regulation was associated with a decrease in TIMP-2 secretion and an inhibition of its expression. Moreover, results showed an activation of MT1-MMP; its expression was regulated after 6, 24, and 48 h.

// Marie Guilbert 1,* , Blandine Roig 2,* , Christine Terryn 3 , Roselyne Garnotel 1 , Pierre Jeannesson 1 , Ganesh D. Sockalingum 1,3 , Michel Manfait 1 , Francois Perraut 2 , Jean-Marc Dinten 2 , Anne Koenig 2 and Olivier Piot 1,3 1 MeDIAN-Biophotonique et Technologies pour la Sante, Universite de Reims Champagne-Ardenne, CNRS UMR 7369 MEDyC, UFR de Pharmacie, SFR CAP Sante, Reims, France 2 CEA, LETI, Minatec campus, Grenoble, France 3 Plate-forme Imagerie Cellulaire et Tissulaire, Universite de Reims Champagne-Ardenne, Reims, France * These authors have contributed equally to this work Correspondence to: Olivier Piot, email: // Keywords : Type I collagen aging, 3D matrix, confocal reflectance microscopy, diffuse reflectance spectroscopy, ATR infrared imaging, Gerotarget Received : September 07, 2015 Accepted : January 30, 2016 Published : February 14, 2016 Abstract During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

Classic galactosemia is an autosomal recessive metabolic disease involving the galactose pathway, caused by the deficiency of galactose-1-phosphate uridyltransferase. Galactose accumulation induces in newborns many symptoms, such as liver disease, cataracts, and sepsis leading to death if untreated. Neonatal screening is developed and applied in many countries using several methods to detect galactose or its derived product accumulation in blood or urine. High-throughput FTIR spectroscopy was investigated as a potential tool in the current screening methods. IR spectra were obtained from blood plasma of healthy, diabetic, and galactosemic patients. The major spectral differences were in the carbohydrate region, which was first analysed in an exploratory manner using principal component analysis (PCA). PCA score plots showed a clear discrimination between diabetic and galactosemic patients and this was more marked as a function of the glucose and galactose increased concentration in these patients’ plasma respectively. Then, a support vector machine leave-one-out cross-validation (SVM-LOOCV) classifier was built with the PCA scores as the input and the model was tested on median, mean and all spectra from the three population groups. This classifier was able to discriminate healthy/diabetic, healthy/galactosemic, and diabetic/galactosemic patients with sensitivity and specificity rates ranging from 80% to 94%. The total accuracy rate ranged from 87% to 96%. High-throughput FTIR spectroscopy combined with the SVM-LOOCV classification procedure appears to be a promising tool in the screening of galactosemia patients, with good sensitivity and specificity. Furthermore, this approach presents the advantages of being cost-effective, fast, and straightforward in the screening of galactosemic patients.

Tandem mass spectrometry allows to screen many inherited metabolic diseases within a single blood spot. It is possible to screen newborns for amino and organic acid, fatty acid oxidation disorders and lysosomal diseases. Tandem mass spectrometry neonatal screening is not yet realized in France in 2014.

Metabolomics is defined as an integrative approach consisting in the comprehensive analysis of all of the small molecules of a biological system (the "metabolome"). The main objective of metabolomics in medecine is to discover metabolic biomarkers for diseases. Mass spectrometry (MS) coupled to liquid or gas chromatography is amongst major analytical tools used in metabolomics. However, the holistic approach used in metabolomics requires very good performances of the analytical system (chromatographic column and MS equipment) and the use of non-conventional validation strategies. Metabolomics workflow can be divided in three main steps: sample preparation, MS data acquisition and processing, and statistical analysis. Processing of the "raw" data (obtained after MS acquisition) is mostly required to normalise chromatographic conditions and to carry out accurate quantification of MS features. Features resulting from this processing may be identified later. The statistical analyses include typically multivariate techniques such as supervised and non-supervised methods. Supervised methods make use of the response variable (e.g., case/control) for model construction while non-supervised methods do not use this piece of information. When the study is focused on a particular set of metabolites, targeted metabolomics could be an interesting alternative to the holistic approach since it may allow absolute quantitation and be associated with a reduced cost.

Background Multiple sulfatase deficiency is a rare inherited metabolic disorder caused by mutations in the SUMF1 gene. The disease remains poorly known, often leading to a late diagnosis. This study aimed to provide improved knowledge of the disease, through complete clinical, biochemical, and molecular descriptions of a cohort of unrelated patients. The main objective was to identify prognostic markers, both phenotypic and genotypic, to accelerate the diagnosis and improve patient care. Methods The phenotypes of ten unrelated patients were fully documented at the clinical and biochemical levels. The long-term follow-up of each patient allowed correlations of the phenotypes to the disease outcomes. Each patient’s molecular defects were also identified. Site-directed mutagenesis was used to individually express the mutants and assess their stability. Characterisation of the protein mutants was completed by in silico analyses based on sequence comparisons and structural models. Results The most severe cases were characterised by the presence of non-neurological symptoms as well as the occurrence of psychomotor regression before 2 years of age. Nine novel SUMF1 mutations were identified. Clinically severe forms were often associated with SUMF1 mutations that strongly affected the protein stability and/or catalytic function as predicted from in silico and western blot analyses. Conclusions This detailed clinical description and follow-up of a cohort of patients, together with the molecular characterisation of their underlying defects, contribute to improved knowledge of multiple sulfatase deficiency. Predictors of a bad prognosis were the presence of several non-neurological symptoms and the onset of psychomotor regression before 2 years of age. No strict correlation existed between in vitro residual sulfatase activity and disease severity. Genotype–phenotype correlations related to previously reported mutants were strengthened. These and previous observations allow not only improved prediction of the disease outcome but also provision of appropriate care for patients, in the expectation of specific treatment development. Electronic supplementary material The online version of this article (doi:10.1186/s13023-015-0244-7) contains supplementary material, which is available to authorized users.

Le depistage des maladies metaboliques en pediatrie comprend le depistage neonatal systematique de cinq maladies rares et graves, chez tous les nouveau-nes. Les evolutions technologiques recentes de la spectrometrie de masse en tandem permettent egalement le diagnostic de plus de vingt maladies hereditaires du metabolisme, a partir d’une seule tache de sang deposee sur papier.

Carbamylation by urea-derived cyanate is a posttranslational modification of proteins increasing during chronic renal insufficiency, which alters structural and functional properties of proteins and modifies their interactions with cells. We report here the major structural alterations of type I collagen induced by carbamylation. Biophysical methods revealed that carbamylated collagen retained its triple-helical structure, but that slight changes destabilized some regions within the triple helix and decreased its ability to polymerize into normal fibrils. These changes were associated with the incapacity of carbamylated collagen to stimulate polymorphonuclear neutrophil oxidative functions. This process involved their interaction with LFA-1 integrin, but no subsequent p(125)FAK phosphorylation. Carbamylation of collagen might alter interactions between collagen and inflammatory cells in vivo and interfere with the normal remodeling of extracellular matrix, thus participating in the pathophysiological processes occurring during renal insufficiency.Carbamylation by urea-derived cyanate is a posttranslational modification of proteins increasing during chronic renal insufficiency, which alters structural and functional properties of proteins and modifies their interactions with cells. We report here the major structural alterations of type I collagen induced by carbamylation. Biophysical methods revealed that carbamylated collagen retained its triple-helical structure, but that slight changes destabilized some regions within the triple helix and decreased its ability to polymerize into normal fibrils. These changes were associated with the incapacity of carbamylated collagen to stimulate polymorphonuclear neutrophil oxidative functions. This process involved their interaction with LFA-1 integrin, but no subsequent p(125)FAK phosphorylation. Carbamylation of collagen might alter interactions between collagen and inflammatory cells in vivo and interfere with the normal remodeling of extracellular matrix, thus participating in the pathophysiological processes occurring during renal insufficiency.

Plasma homocysteine is a modifiable cardiovascular risk factor related to the extent of both coronary and carotid atherosclerosis (1)(2)(3)(4). Plasma homocysteine has been shown to predict the occurrence of cardiac events and mortality in patients with coronary atherosclerosis (5)(6)(7). The predictive value of homocysteine on restenosis after percutaneous coronary intervention (PCI) has been debated (8)(9)(10). Recent evidence, however, indicated that the pre-PCI homocysteine plasma concentration was an independent predictor of death, nonfatal myocardial infarction (MI), and target lesion revascularization (11). Cardiac troponins provide prognostic information in patients with acute coronary syndrome (ACS) (12). Several studies have demonstrated that PCI induces MI as assessed by increases in cardiac troponins, particularly in the case of ACS (13)(14)(15). Furthermore, increased cardiac troponin concentrations after PCI are associated with poor clinical outcome (16)(17)(18)(19). We therefore hypothesized that the pre-PCI plasma homocysteine concentration could be related to the occurrence of MI after PCI, as assessed by changes in plasma cardiac troponin I (cTnI) concentration. Consecutive admissions for nonemergency PCI were studied prospectively. All patients had a stenosis >70% in 1 or more coronary arteries. Two groups were examined: patients with stable angina (SA) pain and those with ACS. The SA pain group had myocardial ischemia during exercise stress testing (n = 29). The ACS group included patients admitted with unstable angina without a subsequent increase in troponin concentrations (n = 28) and those with a definite MI with a documented cTnI increase and electrocardiogram changes that had occurred 7–14 days previously (n = 39). Patients with inflammatory diseases, as well as those being treated with corticosteroids or nonsteroidal antiinflammatory or immunosuppressive drugs, were excluded to minimize potential bias. This study complies …

L'invasion d'une cellule hote est essentielle pour la pathogenicite de Toxoplasma gondii, parasite protozoaire mire-cellulaire obligatoire. Dans cette etude, nous evoluions la capacite des tachyzoites de T gondii a declencher la phosphorylation des differentes mitogen-activated protein kinases (MAPK) dans une lignee de cellules myelomonocytaires humaines THP1. Le pic de phosphorylation des extracellular-signal-regulated kinase (ERK1/2), P38 et c-Jun-NH2 terminal kinase (JNKs) se produit entre 10 et 60 minutes et la specificite de l'activation des MAPKs a pu etre montree par utilisation d'inhibiteurs specifiques de ERKs, P38 et JNKs. tes voies de signalisation passant par les MAP kinases cellulaires, mais aussi parasitaires paraissent jouer un role critique dans les processus d'invasion de T. gondii.

Fibroblasts cultivated in tridimensional collagen lattices exhibit a downregulation of protein synthesis, related to decreased ribosomal RNA (rRNA) content and half life, when compared to monolayer cultivated cells. The involvement in this process of nucleophosmin/B23, a nucleolar phosphoprotein with ribonuclease properties, was checked. We compared production of nucleophosmin/B23 in monolayer and collagen lattice cultured fibroblasts. A significant increase of nucleophosmin/B23 mRNA levels was noticed in lattice-cultured fibroblasts vs monolayers (+154%, p < 0.05). A concomitant enhancement of nucleolar nucleophosmin/B23 content was found (+112%, p < 0.001). Simultaneously, ribonuclease activity contained in nucleolar extracts from collagen lattice-cultured fibroblasts was significantly increased (+54%, p < 0.01). These data demonstrate that extracellular collagen matrix induces the overexpression of nucleophosmin/B23, and suggest that the regulation of protein syntheses in collagen lattice cultures may be explained, at least partly, by an increased degradation of neosynthesized rRNAs dependent on nucleophosmin.

International audience; Familial transmission of chromosome 6 duplications is rare. We report on the first observation of a maternally-inherited pure segmental 6q duplication split into two segments, 6q15q16.3 and 6q16.3q21, and associated with obesity. Obesity has previously been correlated to chromosome 6 q-arm deletion but has not yet been assessed in duplications. The aim of this study was to characterize the structure of these intrachromosomal insertional translocations by classic cytogenetic banding, array-CGH, FISH, M-banding and genotyping using microsatellites and SNP array analysis, in a mother and four offspring. The duplicated 6q segments, 9.75 Mb (dup 1) and 7.05 Mb (dup 2) in size in the mother, were inserted distally into two distinct chromosome 6q regions. They were transmitted to four offspring. A son and a daughter inherited the two unbalanced insertions and displayed, like the mother, an abnormal phenotype with facial dysmorphism, intellectual disability, and morbid obesity. Curiously, two daughters with a normal phenotype inherited only the smaller segment, 6q16.3q21. The abnormal phenotype was associated with the larger proximal 6q15q16.3 duplication. We hypothesize a mechanism for this exceptional phenomenon of recurrent reduction and transmission of the duplication during meiosis in a family. We expect the interpretation of our findings to be useful for genetic counseling and for understanding the mechanisms underlying these large segmental 6q duplications and their evolution.

Le depistage de la phenylcetonurie est effectue, en France au 3e jour de vie, sur carton Guthrie, par mesure de la concentration en phenylalanine. Nous avons entrepris d’examiner, lors de cette etude, le diagnostic final des patients ayant une hyperphenylalaninemie lors du depistage neonatal au laboratoire. Sur une periode de 11 ans, du 1er fevrier 2002 au 31 janvier 2013, tous les nouveau-nes ayant une concentration de phenylalanine augmentee (> 180 μmol/L) ont ete identifies et la cause de cette elevation a ete notee. Sur les 165 113 nouveau-nes depistes, une hyperphenylalaninemie a ete identifiee chez 90 patients lors du depistage neonatal au laboratoire. Durant cette periode, 35 % des cas etaient dus a une phenylcetonurie classique ou a une hyperphenylalaninemie. Dans 4,4 % des cas, les concentrations augmentees etaient dues a d’autres pathologies (deficit en biopterines, galactosemie, leucinose). Cependant, 48,9 % des concentrations elevees n’ont pas ete confirmees par un second prelevement et 11 % etaient des enfants decedes rapidement a quelques jours de vie. La valeur predictive positive (VPP) du test avec un seuil de positivite a > 180 μmol/L etait de 40 %. Notre etude a montre que le seuil de positivite de 180 μmol/L propose par l’Association francaise pour le depistage et la prevention des handicaps de l’enfant (AFDPHE) permet un depistage exhaustif des cas de phenylcetonurie ainsi que des hyperphenylalaninemies moderees permanentes et transitoires. L’utilisation d’un seuil plus eleve, bien qu’ayant l’avantage d’augmenter la VPP du test, nous aurait fait omettre certains cas a suivre.

Dans de nombreux laboratoires de biologie medicale, en particulier dans les CHU, sont mis au point des examens ne faisant pas l’objet de methodes commercialisees. Ces examens sont realises a l’aide de reactifs prepares dans le laboratoire a partir de produits de base ou de produits de « recherche » (produits chimiques, anticorps, enzymes, sondes …). Ces reactifs ne sont pas des dispositifs medicaux pour le diagnostic in vitro (DMDIV) et n’ont pas d’obligation de marquage [...]

Metabolic functions of fibroblasts are tightly regulated by the extracellular environment. When cultivated in tridimensional collagen lattices, fibroblasts exhibit a lowered activity of protein synthesis, especially concerning extracellular matrix proteins. We have previously shown that extracellular collagen impaired the processing of ribosomal RNA (rRNA) in nucleoli by generating changes in the expression of nucleolar proteins and a premature degradation of neosynthesized rRNA. In this study, we have investigated whether inhibiting the synthesis of fibrillarin, a major nucleolar protein with decreased expression in collagen lattices, could mimic the effects of extracellular matrix. Monolayer-cultured fibroblasts were transfected with anti-fibrillarin antisense oligodeoxynucleotides, which significantly decreased fibrillarin content. Downregulation of fibrillarin expression inhibited procollagen secretion into the extracellular medium, without altering total collagen production. No changes of pro1(I)collagen mRNA expression or proline hydroxylation were found. A concomitant intracellular retention of collagen and its chaperone protein HSP47 was found, but no effect on the production of other extracellular matrix macromolecules or remodelling enzymes was observed. These data show that collagen processing depends on unknown mechanisms, involving proteins primarily located in the nucleolar compartment with other demonstrated functions, and suggest specific links between nucleolar machinery and extracellular matrix.

Glycation and glycoxidation processes, which are increased in diabetes mellitus, are generally considered causative mechanisms of long-term complications. With reference to our previous studies, type-I and -IV collagens could induce differentially the adhesion and stimulation of polymorphonuclear leucocytes (PMNs). As PMNs play a role in sustained diabetic oxidative stress, the present study was designed to determine whether in vitro glycoxidation of these macromolecules could alter PMN adhesion, activation and migration. The adhesion of PMNs to in vitro-glycoxidized collagens was significantly increased when compared with control collagens: +37% (P < 0.05) and +99% (P < 0.01) for collagens I and IV, respectively. Glycoxidized type-I collagen increased the chemotactic properties of PMNs without significant stimulatory effect on respiratory burst, whereas pre-incubation of PMNs with glycoxidized type-I collagen induced a priming on subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine. Glycoxidation of type-IV collagen suppressed its inhibitory effect on further PMN stimulation or migration. Collectively, these results indicate that glycoxidation of two major extracellular-matrix collagens considerably alters their ability to modulate PMN migration and production of reactive oxygen species. This imbalance in PMN metabolism may be a major event in the increased oxidative status that characterizes diabetes mellitus.