Arnaud Coudreuse

Risk of Alzheimer's disease biological misdiagnosis linked to cerebrospinal collection tubes.

Tau proteins and amyloid-β (Aβ) peptides are the current recognized cerebrospinal fluid (CSF) biomarkers used as an aid in the diagnosis of Alzheimers disease (AD). However, there is no consensus on their clinical use due to non-qualified cut-off values, probably related to the observed high pre-analytical and analytical variability. Standardized pre-analytical protocols have therefore been proposed. Importantly, these recommend the use of polypropylene collection/sampling tubes while, to date, no broad comparison of these types of tubes has been conducted. In this study, we first compared, as part of a real clinical workflow, the impact of four different collection tubes on the CSF concentration of Aβ peptides (Aβ42, Aβ40) and total (hTau) and phosphorylated (P-Tau181P) tau proteins measured using routine ELISA kits. We then extended this study to 11 polypropylene tubes used by different clinical laboratories, and investigated their plastic polymer composition using differential scanning calorimetry and Fourier Transformed Infrared spectroscopy. Significant concentration variations linked solely to the use of different types of tubes were observed. This was particularly marked for Aβ peptides, with >50% disparity occurring in less than five minutes. Polymer composition analysis revealed that most polypropylene tubes were in fact copolymers with at least polyethylene. There was no clear correlation between tube composition and pre-analytical behavior. Our results show that the use of polypropylene tubes does not guarantee satisfactory pre-analytical behavior. They also point to collection/sampling tubes being a major pre-analytical source of variability that could impact the significance of AD biological diagnosis.

AF fluoropolymer for optical use: spectroscopic and surface energystudies; comparison with other fluoropolymers

Four fluoropolymers are studied by Infra-Red, Raman and solid state NMR; one of them (Teflon AF) is optically transparent thanks to the presence of large ether cycles which prevent crystallization. Detailed investigations of the different spectra lead to the interpretation of the different lines characteristic of the constitutive fluoro-based sites.

Cerebrospinal Fluid Collection Tubes: A Critical Issue for Alzheimer Disease Diagnosis

To the Editor: Total tau protein (hTau),1 its phosphorylated isoform (p-Tau181P), and Aβ1–42 peptides are the currently accepted cerebrospinal fluid (CSF) biomarkers used as aids in the diagnosis of Alzheimer disease (1). Although polypropylene (PP) was previously reported as the best material for CSF collection tubes (2), heterogeneity in CSF Aβ1–42 values was observed with different PP sampling tubes (3). Because the recommendation to use PP tubes did not lead to standardization of clinical cutoff values (4), we decided to fully address this issue by comparing various types of tubes within an actual clinical work flow and by analyzing the material of different commercially available PP tubes. In the framework of an ethically approved study, we collected CSF samples from 12 patients directly (from the lumbar puncture needle) into 2 PP tubes [BD catalog no. 352096 (BD-PP); Sarstedt catalog no. 62.610.201 (ST-PP)], 1 hemolysis polyethylene tube [Fisher Scientific catalog no. ref.W1773X (HE-PE)], and 1 polystyrene tube [BD catalog no. 352095 (BD-PS)]. CSF biomarker concentrations were measured in parallel in these 4 types of tubes with Innogenetics INNOTEST® kits. We extended this analysis by comparing the results obtained with 11 different commercially available collection tubes labeled as “PP” for a series of fresh (unfrozen) …

Surface treatment of polycarbonate films aimed at biomedical application

Aiming to encapsulate pancreatic islets, a biocompatible polycarbonate membrane (What-man) was treated with plasma argon in order to improve its surface properties. The argon plasma treatment decreased the hydrophobicity of the membrane by fixing polyvinylpyrrolidone (PVP) at the surface. The water angle contact decreased from 47° to 20° after this treatment, while the structure and pore diameter were preserved. The treatment also increased significantly the water permeability from 62 ± 8 ml/min to 200 ± 29 ml/min (P < 0.001). ToF-SIMS analyses revealed that the argon plasma treatment of the membrane allowed the installation of an uniform PVP layer at the surface. The concentration equilibrum in glucose was reached after 8 h diffusion for the treated membrane, while it was only 32.4 ± 8.6% (P < 0.01) for the untreated membrane. The biocompatibility of the polycarbonate membrane was assessed after one month of implantation in rats and proved to be unaffected by the surface treatment. In conclusion, the present study provided sufficient information to establish a relationship between the physicochemical modifications of the PVP-plasma-treated polycarbonate membrane and the improvement in its permeability.

Elaboration of nano-structured grafted polymeric surface

The surface grafting of multi-polymeric materials can be achieved by grafting as components such as polymers poly(N-isopropylacrylamide) and/or surfactant molecules (hexatrimethylammonium bromide, polyoxyethylene sorbitan monolaurate). The chosen grafting techniques, i.e. plasma activation followed by coating, allow a large spectrum of functional groups that can be inserted on the surface controlling the surface properties like adhesion, wettability and biocompatibility. The grafted polypropylene surfaces were characterized by contact angle analyses, XPS and AFM analyses. The influence of He plasma activation, of the coating parameters such as concentrations of the various reactive agents are discussed in terms of hydrophilic character, chemical composition and morphologic surface heterogeneity. The plasma pre-activation was shown inevitable for a permanent polymeric grafting. PNIPAM was grafted alone or with a mixture of the surfactant molecules. Depending on the individual proportion of each component, the grafted surfaces are shown homogeneous or composed of small domains of one component leading to a nano-structuration of the grafted surface.

Surface Engineering and Cell Adhesion

Cell adhesion is a multi-process phenomenon involving physical, physico-chemical and biological mechanisms. The complexity of interfaces is the reason why progress in the theory of cell adhesion has been slow. Greater understanding of interaction mechanisms has been enhanced by complete knowledge of supports and of biological components, in particular the extracellular matrix, membrane walls, cell multiplication processes and apoptosis. The construction of novel surfaces with strongly hydrophilic or ultrahydrophobic properties has allowed new theoretical advances, while at the same time offering numerous and varied technological applications. These include: • Bioadhesion with mechanical anchoring using ubiquitous surface roughness and deformability of certain micro-organisms. • Physico-chemical bioadhesion or repellence resulting mainly from the energy characteristics of support surfaces. • Processes of sorting and guidance by biomolecules present at the support–biofilm interface, generating biochemical responses that can induce cell multiplication or degeneration (as in cancer), or cell death.

Are the Interactions between Recombinant Prion Proteins and Polymeric Surfaces Related to the Hydrophilic/Hydrophobic Balance?

New non-fouling tubes are developed and their influence on the adhesion of neuroproteins is studied. Recombinant prion proteins are considered as a single component representative of hydrophobic proteins. Samples are stored for 24 h at 4 °C in tubes coated with two different coatings: poly(N-isopropylacrylamide) as a hydrophilic surface and a plasma-fluorinated coating as a hydrophobic one. The protein adhesion is monitored by ELISA tests, XPS and confocal microscopy. It appears that the highest recovery of recombinant prion protein in the liquid phase is obtained with the hydrophilic surface while the hydrophobic character of the storage tube induces an important amount of biological loss. However, the recovery is not complete even for tubes coated with poly(N-isopropylacrylamide).

Sampling Tube, a Critical Factor in Alzheimer’s Disease Biomarker Standardization

Background: In 2006, frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are linked by the discovery of the cytoplasmic inclusions of TAR DNA-binding protein-43, TDP-43. TDP-43 is a highly conserved protein comprised of 414 residues with two significant RNA recognition motifs (RRM) and a glycine-rich C-terminal region. It is normally located in the nucleus and involved in gene transcriptions such as suppression of HIV transcription through binding to TAR DNA. The pathogenic mechanism of TDP-43 proteinopathy is largely unknown. The current understanding of TDP-43 proteinopathy involves change of subcellular location from the nucleus to cytoplasm and the formation of TDP-43 inclusions. TDP-43 inclusions isolated from brain tissue of these patients are characterized by ubiquitination, with hyperphosphorylation and N-terminal truncation. Methods: To elucidate the mechanisms of TDP-43 proteinopathies, we generated recombinant full length TDP-43 from E. coil and subjected to analytical size-exclusion chromatography, dynamic light scattering analysis, dot blotting, circular dichroism, DNA binding, and cytotoxicity to characterize the recombinant TDP-43. Results: TDP-43 readily formed large molecular weight aggregates after purification which was eluted in the void volume in the analytical size exclusion chromatography. Dynamic light scattering analysis showed the particle dominantly populated at w30 nm in diameter. Interestingly, the purified TDP-43 from HEK293 cell culture also existed as large oligomers which were able to react with specific anti-amyloid oligomer antibody, indicative of common epitopes between TDP-43 oligomer and amyloid oligomers. These species were resistant to general chemical denaturations and highly helical examined by far-UV circular dichroism spectroscopy. Fluorescence titration studies showed that TDP-43 oligomer was not able to bind to TAR DNA. Under electron and atomic force microscopy, TDP-43 oligomers adopted pore or channel-like structures. The species induced neuritic degeneration in retinoic acid-differentiated Neuro2a cells, suggestive of neurotoxicity. Conclusions: Our data demonstrate that the recombinant full length TDP-43 formed structurally stable and helical amyloid-like oligomers with pore-like morphology. Overall, our results established a possible pathogenic mechanism for TDP-43 proteinopathies which may be related to amyloidosis.

Study of the Adhesion of Neurodegenerative Proteins on Plasma-Modified and Coated Polypropylene Surfaces

Abstract The inner polymeric surface of an ELISA titration well is plasma-modified and coated with different surfactant molecules. The titration of neurodegenerative proteins markers (prion, Tau and β-synuclein), previously demonstrated as more efficient with such modified tubes, is related to the adhesion behaviour of these proteins and their corresponding capture antibodies. The adhesion process is studied in terms of anchoring and specific mechanisms. The proteins and antibodies binding onto such modified surfaces is related to the substrate hydrophilic character calculated from the angle contact measure, to the polymer surface charge measured through the streaming potential determination at different pH and the inner surface roughness determined from AFM images. Furthermore, the influence of the blocking agent used during the ELISA titration is also studied.

Increasing the Detection Limit of the Parkinson Disorder through a Specific Surface Chemistry Applied onto Inner Surface of the Titration Well

The main objective of this paper was to illustrate the enhancement of the sensitivity of ELISA titration for neurodegenerative proteins by reducing nonspecific adsorptions that could lead to false positives. This goal was obtained thanks to the association of plasma and wet chemistries applied to the inner surface of the titration well. The polypropylene surface was plasma-activated and then, dip-coated with different amphiphilic molecules. These molecules have more or less long hydrocarbon chains and may be charged. The modified surfaces were characterized in terms of hydrophilic—phobic character, surface chemical groups and topography. Finally, the coated wells were tested during the ELISA titration of the specific antibody capture of the α-synuclein protein. The highest sensitivity is obtained with polar (Θ = 35°), negatively charged and smooth inner surface.