Jean-Baptiste Oudart

Pleural Effusion in a Patient with Multiple Myeloma

A 62-year-old woman presenting with dyspnea on exertion was admitted to our hospital. Her medical history included type 2 diabetes and κ light chain multiple myeloma (MM) diagnosed 3 years ago. The results of a basic biochemical examination performed 2 weeks earlier were normal. Serum protein electrophoresis revealed hypoproteinemia (65 g/L; reference interval, 68–73 g/L), associated with decreased γ globulins (4 g/L; reference interval, 9–15 g/L) but without a detectable paraprotein band. An assay for serum free light chain showed decreased κ light chains (0.5 mg/L; reference interval, 3.3–19.4 mg/L) and λ light chains (<0.3 mg/L; reference interval, 5.7–26.3 mg/L); the κ/λ ratio could not be accurately determined because of the low concentration of λ light chains. An examination of a bone marrow aspirate taken 3 months earlier showed dystrophic plasma cells accounting for 50% of the nucleated cells. On admission, laboratory tests revealed normal values for hemoglobin (142 g/L; reference interval, 130–180 g/L), white blood cells (6.7 × 109/L; reference interval, 4–10 × 109/L), platelets (211 × 109/L; reference interval, 150–400 × 109/L), and creatinine [38 μmol/L (0.4 mg/dL); reference interval, 45–90 μmol/L (0.5–1.0 mg/dL)], but the tests also revealed mild hypocalcemia [2.14 mmol/L (8.6 mg/dL); reference interval, 2.20–2.60 mmol/L (8.8–10.4 mg/dL)]. A chest radiograph and spiral thoracic computed tomography showed a large right-sided pleural effusion. A sample of the pleural fluid had a total protein concentration of 38 g/L and a white blood cell count of 20.5 × 109/L, with 100% lymphoid cells. The results of bacterial and mycobacterial cultures were negative. Protein electrophoresis evaluations of serum, urine, and the …

Analytical methods for measuring collagen XIX in human cell cultures, tissue extracts, and biological fluids

Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed real-time PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis.

Endostatin Level in Cerebrospinal Fluid of Patients with Alzheimer's Disease

The aim of this study was to measure the level of endostatin, a fragment of collagen XVIII that accumulates in the brain of patients with Alzheimers disease (AD), in the cerebrospinal fluids (CSF) of patients with neurodegenerative diseases. The concentrations of total protein, endostatin, amyloid-β1-42 peptide, tau, and hyperphosphorylated tau proteins were measured by enzyme-linked immunosorbent assay in CSF of patients with AD (n = 57), behavioral frontotemporal dementia (bvFTD, n = 22), non AD and non FTD dementia (nAD/nFTD, n = 84), and 45 subjects without neurodegenerative diseases. The statistical significance of the results was assessed by Mann-Whitney and Kruskal and Wallis tests, and by ROC analysis. The concentration of endostatin in CSF was higher than the levels of the three markers of AD both in control subjects and in patients with neurodegenerative diseases. The endostatin/amyloid-β1-42 ratio was significantly increased in patients with AD (257%, p < 0.0001) and nAD/nFTD (140%, p < 0.0001) compared to controls. The endostatin/tau protein ratio was significantly decreased in patients with AD (-49%, p < 0.0001) but was increased in bvFTD patients (89%, p < 0.0001) compared to controls. In the same way, the endostatin/hyperphosphorylated tau protein ratio was decreased in patients with AD (-21%, p = 0.0002) but increased in patients with bvFTD (81%, p = 0.0026), compared to controls. The measurement of endostatin in CSF and the calculation of its ratio relative to well-established AD markers improve the diagnosis of bvFTD patients and the discrimination of patients with AD from those with bvFTD and nAD/nFTD.

Homocitrulline as marker of protein carbamylation in hemodialyzed patients

BACKGROUND Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. METHODS Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n=108) just before (M0) and six months (M6) after the initiation of HD therapy. RESULTS Mean HCit concentrations reached 1000μmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r=0.58) and carbamylated hemoglobin (r=0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. CONCLUSION HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.

The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvβ3 integrin interaction.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

Evaluation of Lumipulse® G1200 for the measurement of six tumor markers: Comparison with AIA® 2000

Tumor marker assays are daily practiced, for screening and follow up of cancers. Interassay precision is an important parameter for the interpretation of the kinetics of the markers, in order to conclude to the efficiency or failure of treatment. The aim of this study was to compare two automated Immunoassay analyzers, Lumipulse® G1200 and AIA® 2000. Both analyzers used an immunoassay system but with different antibodies. Six tumor markers commonly used were studied: AFP, PSA, CA 19-9, CA 15-3, CA 125 and CEA. 253 samples have been collected over a period of one month and analyzed by both analyzers. Regression of Passing-Badblock and Bland-Altman diagram were used to analyze the results for AFP (n=36), PSA (n=39), CA-125 (n=40), CA 15-3 (n=40), CA 19-9 (n=46) and CEA (n=52) were performed. Analytical performances of Lumipulse® G1200 highlighted the good inter-run and intra-run precision of the analyzer. We obtained a good correlation coefficient between Lumipulse G1200® and AIA 2000®, >0.96 for most markers except CA 19-9 which provided a correlation coefficient significantly lower than that obtained with other markers. The concordance for all markers was >94% except for CA 19-9 (83.7%). This study showed a good correlation between the two analyzers and, therefore, a transfer from one analyzer to the other is possible for the different markers studied. However, we found here the classical difficulty to transfer this type of analysis, due to the absence of method standardization. This difficulty was particularly illustrated by CA19-9.

Milky Pleural Fluid

A 90-year-old man was admitted for progressive dyspnea. His medical history included hypertension and chronic myeloid leukemia diagnosed 10 years before and treated with dasatinib. Physical examination revealed pitting edema of the legs and dullness to percussion in the right chest. Laboratory test results were clinically relevant for white blood cells 7.7 × 109/L (reference interval 4–10 × 109/L), platelets 162 × 109/L (150–400 × 109/L), hemoglobin 10.5 g/dL (13–18 g/dL), total protein 7.1 g/dL (6.3–8.1 g/dL), cholesterol 154 mg/dL (116–201 mg/dL) [3.99 mmol/L (3–5.2 mmol/L)], triglycerides 122 mg/dL (27–151 mg/dL) [1.38 mmol/L (0.3–1.71 mmol/L)], creatinine 192 μmol/L (45–90 μmol/L), and lactate dehydrogenase (LDH) activity 504 U/L (200–450 U/L). Chest radiograph showed a large right-sided pleural effusion; thoracentesis yielded milky pleural fluid with total protein 4.3 g/dL, LDH activity 291 U/L, cholesterol 66 mg/dL (1.7 mmol/L), and triglycerides 259 mg/dL (2.9 mmol/L). Bacterial cultures had no growth. After 12 h at 4 °C, the pleural fluid had a white ring at the top of the sample (Fig. 1). Fig. 1. The patients pleural fluid after 12 hours at 4 °C. The first step in evaluation of a pleural effusion is to differentiate exudative and transudative effusions; this plays a key role in determining the etiology of the effusion. Both exudative and transudative fluids are characterized by translocation of excess fluid in the pleural cavity. The main difference is the functional integrity of the pleural membranes. Transudative effusions are defined by unaltered pleural membranes and characterized by increased hydrostatic pressure or decreased oncotic pressure. Exudative effusions are characterized by altered pleural membranes associated with increased permeability. The main etiologic …

Interference of M-paraprotein in automated urea assays

a Jean-Baptiste Oudart and Vichita Ok contributed equally to this work. *Corresponding author: Dr. Laurent Ramont , Laboratoire Central de Biochimie, CHU de Reims, Rue Serge Kochman, 51092 Reims Cedex, France, Phone: + 33 326783181, Fax: + 33 326788539, E-mail: lramont@chu-reims.fr Jean-Baptiste Oudart, Vichita Ok, Caroline Faucon, Laure Zucchini, Andrada Chiron and Fran ç ois-Xavier Maquart: CHU de Reims, Central Laboratory of Biochemistry, Reims, France Jean-Baptiste Oudart, Fran ç ois-Xavier Maquart and Laurent Ramont: University of Reims Champagne-Ardenne, FRE CNRS/URCA N ° 3481, Reims, France

Conformation-dependent binding of a Tetrastatin peptide to α v β 3 integrin decreases melanoma progression through FAK/PI 3 K/Akt pathway inhibition

Tetrastatin, a 230 amino acid sequence from collagen IV, was previously demonstrated to inhibit melanoma progression. In the present paper, we identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin in vivo and in vitro on melanoma cell proliferation, migration and invasion. We demonstrated that QS-13 binds to SK-MEL-28 melanoma cells through the αvβ3 integrin using blocking antibody and β3 integrin subunit siRNAs strategies. Relevant QS-13 conformations were extracted from molecular dynamics simulations and their interactions with αVβ3 integrin were analyzed by docking experiments to determine the binding areas and the QS-13 amino acids crucial for the binding. The in silico results were confirmed by in vitro experiments. Indeed, QS-13 binding to SK-MEL-28 was dependent on the presence of a disulfide-bound as shown by mass spectroscopy and the binding site on αVβ3 was located in close vicinity to the RGD binding site. QS-13 binding inhibits the FAK/PI3K/Akt pathway, a transduction pathway that is largely involved in tumor cell proliferation and migration. Taken together, our results demonstrate that the QS-13 peptide binds αvβ3 integrin in a conformation-dependent manner and is a potent antitumor agent that could target cancer cells through αVβ3.

Unexpected M-protein in an Anticoagulated Patient

A 78-year-old woman treated with dabigatran for atrial fibrillation was admitted for acute renal failure. Coagulation investigations showed an overdose of dabigatran, requiring injection of the antidote, idarucizumab. Fig. 1 shows urine immunofixation electrophoresis (IFE)3 (Hydragel 4IF, Sebia) performed 2 days after initiation of treatment with idarucizumab. Fig. 1. Urine IFE performed 2 days after idarucizumab injection. 1. What is the mechanism of action of idarucizumab? …