Preparation of cell therapy products: contribution of closed systems]

A phase I clinical trial is being currently performed in our institution, aiming at evaluating the feasibility and toxicity related to the administration of Herpes Simplex-thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using sampling and transfer pack bags, culture bags and a sterile connection device which avoids opening the culture system. This closed system allows safe and reproducible ex vivo preparation of gene-modified primary T-lymphocytes for clinical use.     

Les développements de la thérapie cellulaire de l''an 2000

For the past thirty years, hematology has switched from the concept of bone marrow transplantation to the concept of hematopoietic stem cell (HSC) transplantation, from allograft to autograft, from non-manipulated graft to hyper-selection, from hematopoietic cellular therapy to immunotherapy. Indications of these transplantations are now more clear for malignant diseases and are ongoing for auto-immune diseases. A better knowledge of the HSC allows the control of their proliferation and differentiation, opening the field of ex vivo expansion. Very recently, new stem cells have been identified, establishing that a differentiated cell retain its totipotency: a nervous system cell can differentiate into HSC, which will further give hematopoiesis, mesenchymental cells or hepatocytes. New tools are under development: human ES cells, biomaterials, functionalized materials, opening the field of cellular engineering in the year 2000.     

Advantages of hydrophobic culture bags over flasks for the generation of monocyte-derived dendritic cells for clinical applications

Dendritic cells (DC), potent antigen presenting cells capable of activating both na?ve and primed T cells, are currently being pursued clinically in the development of cancer vaccines. Variations in the literature regarding DC source, culture conditions, maturation state, dose, and route of immunization make comparisons of clinical trial data difficult. In order to define and optimize the culture conditions for DC generation, we have performed a careful comparison of two culture methods, as well as different methods of DC maturation. Our studies demonstrate that high viability DC can be produced and matured in gas permeable hydrophobic culture bags. These cells express surface molecules characteristic of DC and have superior yield, viability, and function to cells cultured in plastic tissue culture flasks. These results suggest that hydrophobic culture bags are ideal for the preparation of clinical DC vaccines, as DC can be generated, antigen-loaded, and matured in a closed system, a scheme we have found to be superior to previously described flask culture methods.     

Conduite in vitro d'une étude de biocompatibilité pour la confection d'un substitut vasculaire bioartificiel de petit calibre

Le travail présenté rapporte une démarche, à savoir les différentes étapes d'une évaluation dans la perspective de l'élaboration d'un substitut vasculaire artificiel hybride, concept qui prévoit d'associer dans une même unité fonctionnelle une composante artificielle d'origine synthétique ou naturelle et une composante cellulaire: l'endothélium vasculaire. Les résultats des essais cliniques les plus récents rapportés dans la littérature, à condition que le revêtement endothélial du substitut soit complet au moment de l'implantation, permettent d'envisager l'utilisation de telles structures cellularisées. Les différentes étapes de cette étude comportent: (1) la sélection d'un matériau propice à l'endothélialisation in vitro par des études de cytocompatibilité, à l'aide de cellules endothéliales en culture; (2) l'étude du phénotype de la composante biologique endothéliale au contact des matériaux sélectionnés (mesures de la production et de la fonctionnalité de deux protéines-clé de la régulation de la balance hémostatique, quantification d'une molécule d'adhésion exprimée à la membrane de la cellule endothéliale, intervenant dans le recrutement des leucocytes en cas de processus inflammatoire; (3) l'étude du devenir du néo-endothélium obtenu, sous contraintes de cisaillement, par quantification de la radioactivité après radiomarquage in situ de la monocouche cellulaire.     

Évaluation par PIXE dans des coupes minces de la migration de certains éléments relargués par des implants métalliques

Migration evaluation in thin samples by PIXE of some metallic elements displaced from prosthesis.The metallic prosthesis used in orthopaedic surgery undergo degradations some years after their implantation. This phenomenon induces metallic element transfers in surrounding tissues. From an anatomical body, we have taken out implanted hip prosthesis and the surrounding tissues; we have determined the composition of the implanted prosthesis by spark spectrometry. Using a protocol adapted for each sample, we manufacture slides parallel to the femur axis. We determine the concentration of the displaced metallic elements versus depth. We have used the so-called PIXE method (Particle Induced X-rays Emission), based on the X-rays spectroscopy, to analyse our samples. The inducing particles were 3 MeV proton, the beam was 50 μm in diameter and had an intensity of 3 nA. Preliminary results show the migration of metallic elements from prosthesis to soft tissues.     

Conception, élaboration et caractérisation de matériaux bioactifs

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves grafting of adhesion peptides as RGD peptides (R: arginine; G: glycine; D: aspartic acid) to materials on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This review gives an overview of RGD modified materials, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on materials. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed. We have tried to relate one of numerous scientifics adventures initiated by Charles Baquey within our laboratory. This review is dedicated to him for his enthusiasm in the development of project and for his wish of always leading of a professional blooming of his students.     

Réingénierie des activités des services de biochimie et d'hématologie : Étude d'application aux activités de garde et de pré-analytiqueReengineering of Biochemistry and Haemotology services activity

French hospitals are confronted to more and more economic restrictions, aiming at reducing Health expenses. Care centers involved laboratories to counter this limitation, since these departments are expensive, althought they are essentials. Reorganization of the laboratories, pooling of their activities, automation and computerization increase was implemented in some establishments. Environment regulations required persons in charge to conceive new organization to deal with. Lariboisière—Fernand Widal group hospital wishes to set up such a project, within the framework of a restructuration of its medical and technical platform. For this, medical duty activities will be gathered and the pre-analytical phase of the biochemistry and hematology laboratories will be automated.     

Étude rhéo-acoustique de la rupture d''agrégates de particules en suspension dans un champ de cisaillement Application à la désagrégation des globules rouges

Rheo-acoustic study of particle aggregate break-up in a shear flow. Application to red blood cell aggregates.Shear-induced disruption of reversible flocs in a concentrated suspension is investigated by ultrasound backscattering in the low shear regime. Fractal flocs are considered as non-Brownian scatterers much smaller than the wavelength, with acoustic properties close to those of the surrounding medium, so that the attenuation of the coherent field is weak and multiple scattering can be neglected. The concept of variance in local particle volume fraction is used to deduce a first order expression of the ultrasound scattering coefficient (cross-section per unit volume) for Rayleigh scatterers in a dense suspension. On the basis of a scaling law for the shear-induced disruption of aggregates, the shear stress dependence of the ultrasonic scattered intensity from a dense suspension of flocs is derived. In a second part, the shear break-up of hardened red blood cell aggregates is investigated in a plane—plane flow geometry by ultrasound scattering. Rheo-acoustical experiments are analysed within the framework of the self consistent field approximation and the scaling laws currently used in microrheological models. Finally, the ability of ultrasonic, light reflectometry and viscometry methods to provide quantitative information about red blood cell aggregation and membrane adhesiveness is discussed.     

L'adhérence des érythrocytes à l'endothélium vasculaire

Blood cells are in continuous contact with the vascular endothelium. Endothelial cell culture, intravital videomicroscopy allowed the investigation of blood cell-endothelium interactions in dynamic conditions.In the various diseases, diabetes mellitus, sickle cell anemia and malaria, erythrocytes have an increased adhesion to endothelial cells. The presence of advanced glycation end products (AGE) on erythrocytes of diabetics is responsible for their binding to the receptor RAGE present on the endothelium. The AGE-RAGE binding provokes an oxidant stress and induces the expression of the adhesion molecule. Furthermore, erythrocyte AGE induce an increase in vascular permeability. In sickle cell anemia, the increased adhesiveness and the sickling of red blood cells are responsible for thrombosis. Plasmodium falciparum infestation of erythrocytes induces knob formation at the cell surface and the P. falciparum protein binding to CD36, ICAM-1 and thrombospondin present on the endothelium, and facilitates the parasite dissemination. © 2999 Éditions scientifiques et médicales Elsevier SAS     

Immunomodulation in the treatment of haematological malignancies

Despite the continuous advances in immunology and cancer biology, haematological malignancies are often incurable. Conventional chemotherapy and radiation are efficacious for some lymphoma and leukaemia, however relapse and progressive disease often occurs. The evidence that the immune system can play an essential role in controlling cancer progression provide a basis for the development of active therapies, such as immunization, aimed to evoke or amplify a tumour-specific immune response. However, the inability of the patient’s own immune system to mount effective responses against tumour antigens is a major limit of vaccination approaches. The adoptive transfer of effectors of the adaptive immune system is an attractive strategy to circumvent the limitations of autologous immune responses. Donor lymphocyte infusion and the transfer of monoclonal antibodies (MoAbs) have been the first forms of adoptive therapy approved for clinical use and are still fundamental components of immunotherapy of haematological malignancies. Due to the continuous characterization of tumour-specific antigen, the development of tumour-tailored therapies that exploit the specificity of antibodies and T cell receptors (TCRs) is progressing rapidly. This review highlights the current advances in the field of adoptive immunotherapy of haematological malignancies, starting by elucidating the ongoing progress in passive transfer of MoAbs. We will also discuss recent advances in the adoptive transfer with tumour-specific high avidity T cells, which can be generated ex vivo by the transfer of gene constructs encoding single chain antibodies or TCRs, thus redirecting T cell specificity to selected tumour antigens. The ability to produce gene-modified T cells of desired specificity and defined functional activity may improve in the future T cell based immunotherapy of cancer.     

Ex vivo expansion of megakaryocyte precursors from umbilical cord blood CD34 cells in a closed liquid culture system.

Umbilical cord blood (UCB) provides a rich source of stem cells for transplantation after myeloablative therapy. One major disadvantage of UCB transplantation is delayed platelet engraftment. We propose to hasten platelet engraftment by expanding the number of megakaryocyte (MK) precursors (CD34/CD41 cells) through cytokine stimulation within a closed, pre-clinical liquid culture system. Clinical engraftment data suggest a 5- to 10-fold increase in MK precursors in a UCB unit can accelerate platelet engraftment, so this was our goal. Thirteen UCB samples from full-term births were Ficoll-separated and frozen for subsequent use. On thawing, the mononuclear cell population was positively selected for CD34(+) expression. The cells were cultured in gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: recombinant human interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin. MK lineage cell expansion was assessed using mononuclear cell count and flow cytometry (CD34/41, CD41, CD34/61, and CD61 expression) on days 7, 11, and 14. Optimal expansion of CD34/41 and CD41 cells was observed at day 11, with a median 6-fold and 33-fold increase in the starting cell doses, respectively. CD34/61 and CD61 cell expansion at day 11 was 7-fold and 14-fold, respectively. MK precursors can be successfully expanded from CD34(+) UCB cells in a closed liquid culture system using interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin to a level that should have a clinical impact in the transplantation setting. Our ex vivo expansion technique needs to be further optimized before it can be used in a pilot UCB transplantation trial.     

Gene transfer into nonhuman primate hematopoietic stem cells: implications for gene therapy

Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation abilities. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. Using these models, clinically relevant levels (around 10% or even more) of gene-modified cells in peripheral blood have recently been achieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction, and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus or feline endogenous retrovirus envelope. Other strategies including the use of lentiviral vectors and in vivo selective expansion of gene-modified cells with the drug resistance gene or selective amplifier gene (also designated the molecular growth switch) are now being tested to further increase the fraction of gene-modified cells using nonhuman primate models. In addition to the high gene transfer efficiency, high-level and long-term expression of transgenes in human HSCs and their progeny is also required for effective HSC gene therapy. For this purpose, other backbones of retroviral vectors such as the murine stem cell virus and cis-DNA elements, such as the ss-globin locus control region and the chromatin insulator, also need to be tested in nonhuman primate models. Nonhuman primate studies will continue to provide an important framework for human HSC gene therapy. Well-designed nonhuman primate studies will also offer unique insights into the HSCs, immune system, and transplantation biology characteristic of large animals.     

Caractérisation de la microarchitecture trabéculaire osseuse par analyse de texture

Les analyses de texture visent à une caractérisation indirecte de la microarchitecture trabéculaire osseuse sur des images bidimensionnelles (2D) en projection, soit en niveaux de gris sans seuillage, soit seuillées. Les techniques d'analyse peuvent répondre à des méthodes statistiques, fractales, morphologiques ou d'anisotropie. Les images sont obtenues par tomodensitométrie, résonance magnétique ou radiographie conventionnelle, in vitro ou in vivo. La radiographie in vivo peut être réalisée a divers sites, surtout le calcanéum, et doit être très standardizée pour une meilleure reproducibilité. Les analyses fractales sur radiographies ont montré leur corrélation à la microarchitecture tridimensionnelle (3D), à la resistance mécanique, et leur capacité, à distinguer des groupes d'ostéoporotiques de groupes témoins, parfois de façon plus puissante que la densitométrie osseuse. Leur potentiel de développement clinique est très prometteur.     

T lymphocytes as targets of gene transfer with Moloney-type retroviral vectors

Peripheral T lymphocytes are a target of choice for many gene therapeutic strategies. Retrovirus-mediated transduction allows genomic integration and long-term expression of transgenes in target cells. Over many years, low transduction efficiency into primary T lymphocytes has limited clinical application of existing protocols. Recently, gene transfer rates > 50% have been achieved facilitating clinical studies. More attention is thus being focused on the ability of gene-modified cells to carry out innate as well as conferred functions in vivo and the influence of culture conditions, retroviral vector and host response thereon.     

Réseaux de neurones et électroencéphalogramme : fructueuse associationNeural networks and electroencephalogram: fruitful association

For the last two decades, neural networks have been progressively used in the field of biomedical research. Among them, electroencephalography largely benefited from these tools. In this paper, we are presenting the main characteristics of neural networks. Those networks are tools which are nicely suitable to the electroencephalogram processing. A large bibliography provides the reader a broad view of the works performed in this field and can be used to begin new studies.     

Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.     

Processus de mise en conformité de l''arrêté de périnatalité du 25 avril 2000

Application guide to neonatology regulations.Pregnancy safety, delivery, and care of the mother and baby have been a great concern lately, and these issues have mobilized health professionals. Regulations, means and obligation of results require an organization and a way of working adapted to neonatology. The adoption of the regulation of 25 April 2000, at the Regional Teaching Hospital of Lille, France, has been jointly implemented in three other hospital complexes of different size and with different structures and means. This global approach led to the elaboration of a common guide of neonatology, including steps and actions to be taken in order to meet the requirements. Besides audit, physical inventory, purchases according to the new rule of public market, quality control and maintenance, this study puts the patient at the centre of the collaboration between the medical services, paramedical, industrials and biomedical engineers. Apart from means to mobilize, this close collaboration should rely on the experience of all the participants in order to optimise the conception and maintenance prevention of medical instruments.     

Organisation, caractérisation et analyse multidimensionnelle des données de la marche: aspects méthodologiquesOrganisation, characterisation and multidimensional analysis ofgait data: methodological aspects

This first article states the problem of the biomechanical data multivariate analysis of the gait. The data base is organised according to a parallelepiped PO where the three directions indicate the subject, the examination number and the trial number. Each non empty cell of PO contains the signals, as angles, moments or EGM activities, recorded during the corresponding empirical situation. Aiming at analysing globally these data, the signals are characterized through average membership values to space-time windows. These values are organised according a data table where the rows correspond to the triplet (cell of PO, time window, limb), and the columns to the space windows of the time variables. The table is investigated through the multiple correspondence analysis (MCA) which yields the more discriminant variables, their relationships and the intra and interindividual differences. The MCA potentialities vs. those of the principal component analysis (PCA) are shown using a didactic example based on simulated data. A second article will illustrate our approach on gait data.