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Titchmarsh L.,University of Cambridge | Zeh C.,Centers for Disease Control and Prevention | Verpoort T.,Macopharma | Allain J.-P.,University of Cambridge | Lee H.,University of Cambridge
Journal of Clinical Microbiology | Year: 2015

In order to limit the interference of HIV-1 cellular nucleic acids in estimating viral load (VL), the feasibility of leukodepletion of a small whole-blood (WB) volume to eliminate only leukocyte cell content was investigated, using a selection of filters. The efficacy of leukocyte filtration was evaluated by counting, CD45 quantitative PCR, and HIV-1 DNA quantification. Plasma HIV-1 was tested by real-time reverse transcription (RT)-PCR. A specific, miniaturized filter was developed and tested for leukocyte and plasma virus retention, WB sample dilution, and filtration parameters in HIV-1-spiked WB samples. This device proved effective to retain >99.9% of white blood cells in 100 μl of WB without affecting plasma VL. The Samba sample preparation chemistry was adapted to use a leukodepleted WB sample for VL monitoring using the point-of-care Samba-1 semiautomated system. The clinical performance of the assay was evaluated by testing 207 consecutive venous EDTA WB samples from HIV-1-infected patients attending a CD4 testing clinic. Most patients were on antiretroviral treatment (ART), but their VL status was unknown. Compared to the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, the new Samba assay had a concordance of 96.5%. The use of the Samba system with a VL test for WB might contribute to HIV-1 ART management and reduce loss-to-follow-up rates in resource-limited settings. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


Menssen A.,Charité - Medical University of Berlin | Menssen A.,Leibniz Institute for Molecular Pharmacology | Haupl T.,Charité - Medical University of Berlin | Sittinger M.,Charité - Medical University of Berlin | And 3 more authors.
BMC Genomics | Year: 2011

Background: Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data.Results: The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein.Conclusions: Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes. © 2011 Menssen et al; licensee BioMed Central Ltd.


PubMed | U.S. Center for Disease Control and Prevention, Macopharma and University of Cambridge
Type: Journal Article | Journal: Journal of clinical microbiology | Year: 2015

In order to limit the interference of HIV-1 cellular nucleic acids in estimating viral load (VL), the feasibility of leukodepletion of a small whole-blood (WB) volume to eliminate only leukocyte cell content was investigated, using a selection of filters. The efficacy of leukocyte filtration was evaluated by counting, CD45 quantitative PCR, and HIV-1 DNA quantification. Plasma HIV-1 was tested by real-time reverse transcription (RT)-PCR. A specific, miniaturized filter was developed and tested for leukocyte and plasma virus retention, WB sample dilution, and filtration parameters in HIV-1-spiked WB samples. This device proved effective to retain >99.9% of white blood cells in 100 l of WB without affecting plasma VL. The Samba sample preparation chemistry was adapted to use a leukodepleted WB sample for VL monitoring using the point-of-care Samba-1 semiautomated system. The clinical performance of the assay was evaluated by testing 207 consecutive venous EDTA WB samples from HIV-1-infected patients attending a CD4 testing clinic. Most patients were on antiretroviral treatment (ART), but their VL status was unknown. Compared to the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, the new Samba assay had a concordance of 96.5%. The use of the Samba system with a VL test for WB might contribute to HIV-1 ART management and reduce loss-to-follow-up rates in resource-limited settings.


PubMed | Medical University of Graz, Austrian Center of Industrial Biotechnology, Macopharma and University of Natural Resources and Life Sciences, Vienna
Type: Journal Article | Journal: New biotechnology | Year: 2014

Bacterial contamination of platelet concentrates (PCs) can lead to fatal transfusion transmitted diseases and is the most abundant infectious risk in transfusion medicine. The storage conditions of PCs provide a good environment for bacterial growth. The detection of these contaminations at an early stage is therefore important to avoid the transfusion of contaminated samples. In this study, bioresponsive polymer (BRP) systems were used for the detection of microorganisms in PCs. The backbone of the polymer consisted of labelled protein (casein), which was demonstrated to be degraded by pure proteases as models and by extracellular enzymes released by contaminating microorganisms. The concomitant colour change was easily visible to the naked eye. To enhance stability, the protein was cross-linked with glycidyl methacrylate (GMA). The cross-linked polymer was easier to handle but was less sensitive than the non-cross-linked material. A contamination of a PC with 10CFU/mL S. aureus was detectable after 24 hours. The visible colour reaction was quantified as a E value according to the CIELab concept. A E value of 21.8 was already reached after 24 hours. Hence, this simple but effective system could prevent transfusion of a contaminated PC.


PubMed | Red Cross, MacoPharma International GmbH and MacoPharma
Type: Journal Article | Journal: Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie | Year: 2016

The THERAFLEX UV-Platelets system uses shortwave ultraviolet C light (UVC, 254 nm) to inactivate pathogens in platelet components. Plasma carryover influences pathogen inactivation and platelet quality following treatment. The plasma carryover in the standard platelets produced by our institution are below the intended specification (<30%).A pool and split study was carried out comparing untreated and UVC-treated platelets with <30% plasma carryover (n = 10 pairs). This data was compared to components that met specifications (>30% plasma). The platelets were tested over storage for in vitro quality.Platelet metabolism was accelerated following UVC treatment, as demonstrated by increased glucose consumption and lactate production. UVC treatment caused increased externalization of phosphatidylserine on platelets and microparticles, activation of the GPIIb/IIIa receptor (PAC-1 binding), and reduced hypotonic shock response. Platelet function, as measured with thrombelastogram, was not affected by UVC treatment. Components with <30% plasma were similar to those meeting specification with the exception of enhanced glycolytic metabolism.This in vitro analysis demonstrates that treatment of platelets with <30% plasma carryover with the THERAFLEX UV-Platelets system affects some aspects of platelet metabolism and activation, although in vitro platelet function was not negatively impacted. This study also provides evidence that the treatment specifications of plasma carryover could be extended to below 30%.


Lavergne M.,ABCell Bio | Lavergne M.,French Institute of Health and Medical Research | Vanneaux V.,Laboratoire Of Therapie Cellulaire | Delmau C.,French Institute of Health and Medical Research | And 4 more authors.
Cell Proliferation | Year: 2011

Adult peripheral blood (PB) endothelial progenitor cells (EPC) are produced in the bone marrow and are able to integrate vascular structures in sites of neoangiogenesis. EPCs thus represent a potential therapeutic tool for ischaemic diseases. However, use of autologous EPCs in cell therapy is limited by their rarity in adult PB. Cord blood (CB) contains more EPCs than PB, and they are functional after expansion. They form primary colonies that give rise to secondary colonies, each yielding more than 10 7 cells after few passages. The number of endothelial cells obtained from one unit of CB is compatible with potential clinical application. EPC colonies can be securely produced, expanded and cryopreserved in close culture devices and endothelial cells produced in these conditions are functional as shown in different in vitro and in vivo assays. As CB EPC-derived endothelial cells would be allogeneic to patients, it would be of interest to prepare them from ready-existing CB banks. We show that not all frozen CB units from a CB bank are able to generate EPC colonies in culture, and when they do so, number of colonies is lower than that obtained with fresh CB units. However, endothelial cells derived from frozen CB have the same phenotypical and functional properties than those derived from fresh CB. This indicates that CB cryopreservation should be improved to preserve integrity of stem cells other than haematopoietic ones. Feasibility of using CB for clinical applications will be validated in porcine models of ischaemia. © 2011 Blackwell Publishing Ltd.


Gerard E.,Catholic University of Louvain | Bessy E.,MacoPharma | Henard G.,MacoPharma | Verpoort T.,MacoPharma | Marchand-Brynaert J.,Catholic University of Louvain
Journal of Biomedical Materials Research - Part B Applied Biomaterials | Year: 2012

For clinical indications and according to European standards, a depletion of the leukocytes from whole blood has to be realized before transfusion to a patient. In this study, the surface of a layer of meltblown oxygen-plasma treated polypropylene nonwoven (O2-PP), making part of the composition of leukodepletion filters, was modified with bioactive molecules to enhance the adhesion of leukocytes. These synthetic small molecules (called peptidomimetics) mimic the "Leu-Asp-Val" (LDV) tripeptide sequence recognized by the α4β1 integrin, a receptor expressed on leukocytes. Their activity, as ligands of the α 4β1 integrin, was confirmed through cell adhesion assays. The peptidomimetics were covalently immobilized on O2-PP nonwoven via activation of the hydroxyl- and carboxyl-functions displayed on the polymer surface with trifluoro-triazine reagent, or were simply deposited on the O2-PP surface. The treated materials were characterized by X-ray photoelectron spectroscopy, wettability, and morphological analyses, before and after steam sterilization. Experimental filters composed of 10 layers of O 2-PP nonwovens and a last layer modified with the peptidomimetics were evaluated, using whole blood filtration assays, for their leukodepletion efficiency, the recovery of red cells and platelets and the waste of blood, with an objective to design new filters fulfilling practical and medical criteria. © 2012 Wiley Periodicals, Inc.


Gerard E.,Catholic University of Louvain | Bessy E.,MacoPharma | Henard G.,MacoPharma | Ducoroy L.,MacoPharma | And 2 more authors.
Journal of Polymer Science, Part A: Polymer Chemistry | Year: 2011

The surface of meltblown poly(butylene terephthalate) (PBT) nonwoven was modified by photochemistry using the photolinker O-succinimidyl 4-azido-2,3,5,6-tetrafluorobenzoate for the introduction of activated ester functions and then coupling of molecular probes or biomolecules. Approximately 4000 pmol of (L)-4,5-[ 3H]-lysine was fixed per PBT sample (1.13 cm 2) and measured by liquid scintillation counting. The method consisted in a two-step process: (a) coating of the clip (0.05 mg/sample) on the fibrous surface of the PBT followed by UV irradiation (30 min, 254 nm) and (b) coupling of amine-terminated molecules (10 -3 M in phosphate buffer-CH 3CN [1/1, v/v], 20 h). Moreover, about 2000 pmol of 3H-lysine can be immobilized on the PBT surface after UV irradiation (without clip) by aminolysis reactions with the created oxygenated functions. The derivatizations (via the clip and UV irradiation only) were stable after long-term heating at 100 °C in water or under steam-sterilization conditions. They induce neither modifications of the nonwoven morphology nor cytotoxicity. This method was applied for the grafting of peptides Gly-Arg-Gly-Asp-Ser and Gly-Gly-Gly-Gly-Gly to perform blood filtration experiments and to retain the leukocytes. © 2011 Wiley Periodicals, Inc.


Lescoutra-Etchegaray N.,CEA Fontenay-aux-roses | Sumian C.,Macopharma | Culeux A.,CEA Fontenay-aux-roses | Durand V.,CEA Fontenay-aux-roses | And 3 more authors.
Transfusion | Year: 2014

Background Five cases of variant Creutzfeldt-Jakob disease (vCJD) infections were attributed to infusion of contaminated blood components, turning to real the interhuman transmissibility of this prion disease from asymptomatic carriers. Preventive policies rely on exclusion from blood donation and benefit of leukoreduction initially implemented against leukotropic viruses. In the absence of available antemortem diagnostic tests, the updated prevalence of silent vCJD infections (1/2000 in the United Kingdom) urges the necessity to enforce blood safety with more efficient active measures able to remove the remaining infectivity. Study Design and Methods Several affinity resins were demonstrated to reduce high levels of brain-spiked infectivity from human leukoreduced red blood cells (L-RBCs). One was integrated in a device adapted to field constraints (volumes, duration) of human transfusion. We assessed here the ability of the resulting removal filter, termed P-Capt, to remove infectivity from human L-RBC units spiked with scrapie-infected hamster brain (≥10,000 infectious units/mL), through inoculation of hamsters with pre- and post-blood filtration samples. Results Incubation periods of recipient animals suggest around a 3-log removal of brain-derived prion infectivity by filtration through the P-Capt. Conclusion On brain-derived spiked infectivity, the P-Capt filter provided a performance similar to the resin packed in columns used for initial proof-of-concept studies, suggesting an appropriate scale-up to efficiently remove infectivity from an individual human blood bag. According to the ability of resin to completely remove apparent endogenous infectivity from hamster leukoreduced blood, the implementation of such a filter, now commercially available, might seriously improve blood safety toward prions. © 2013 American Association of Blood Banks.


Analysis of archived appendix samples reveals that one in 2000 individuals in the United Kingdom may carry the infectious prion protein associated with variant Creutzfeldt-Jakob disease (vCJD), raising questions about the risk of transfusion transmission from apparently healthy carriers. Blood leukoreduction shows limited efficiency against prions. Therefore, in absence of antemortem diagnostic tests, prion removal filters, including the P-Capt filter were designed to improve blood transfusion safety.We evaluated the performances of two filters, the P-Capt and one prototype (PMC#005), with blood-borne infectivity in two independent experiments. Blood was drawn twice from prion-infected macaques. Corresponding RBCCs were prepared according to two different procedures: in StudyA, the leukoreduction step was followed by the filtration through the P-Capt. In StudyB, the leukoreduction and prion removal were performed simultaneously through the PMC#005. For each study, two groups of three animals were transfused twice with samples before or after filtration.Among the six macaques transfused with nonfiltered samples, five developed neurologic signs but only four exhibited peripheral detectable protease-resistant prion protein (PrPres) accumulation. In StudyA, the three animals transfused with P-Capt-filtered samples remain asymptomatic and devoid of PrPres in lymph node biopsies 6years after the transfusion. In StudyB, one animal transfused with PMC#005-filtered samples developed vCJD.After 5 to 6 years of progress, this ongoing study provides encouraging results on the prion blood removal performances of the P-Capt filter in macaques, an utmost relevant model for human prion diseases.

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