CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Television before TV : a transnational history of an experimental medium on display, 1928 - 1939

Dès le milieu des années 1920 et le développement des premiers prototypes fonctionnels, la télévision se fait objet d'exposition. La nouvelle technologie est montrée à des foires industrielles et dans des grands magasins, à des expositions universelles et nationales : exposée, la télévision devient un mass media avant même qu'elle ne diffuse des émissions régulières. En étudiant les présentations publiques de la télévision en Allemagne, en Grande-Bretagne et aux Etats-Unis entre 1928 - date de l'ouverture des premières expositions annuelles dans les trois pays - et 1939 - moment de l'interruption de celles-ci suite à l'éclatement de la Deuxième Guerre mondiale - Television before TV montre que les lieux où le médium rencontre son premier public constituent les mêmes espaces qui, rétrospectivement, permettent de comprendre son avènement. Cette recherche propose ainsi de nouvelles pistes pour l'histoire des médias en interrogeant la définition de la télévision en tant que médium du privé. Elle rappelle l'importance historique de l'espace public pour la « vision à distance » et, dépassant la perspective nationale, reconstitue les différences et similarités techniques, médiatiques et institutionnelles de la télévision allemande, américaine et britannique dans l'entre-deux-guerres. Développée dans un contexte qui est simultanément nationaliste et nourri d'échanges et de compétitions internationales, la télévision sert de comparatif dans les rivalités entre pays, mais stimule également la circulation de savoir-faire et de personnes. Sa forte valeur symbolique comme emblème du progrès scientifique et, dans le cas allemand, comme preuve de la modernité du régime national-socialiste se traduit dans les salles d'expositions par des scénographies souvent innovatrices, dont les photographies et descriptions se propagent au-delà de la presse locale. Au centre d'un réseau discursif et représentationnel, les expositions jouent alors un rôle essentiel pour la construction sociale, politique et culturelle de la télévision. Remplaçant l'analyse de textes audiovisuels par l'étude de la machine exposée, ma recherche reformule la question bazinienne de qu'est-ce que la télévision ? pour demander où le médium se situe-t-il ? Cette prémisse méthodologique permet de contourner une évaluation qualitative de la technologie qui déplorerait ses multiples imperfections, et éclaire d'un nouveau jour des dispositifs télévisuels qui, même sans programme, intègrent un univers voué à la culture de consommation et du loisir.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Inner-membrane transporters for the siderophores pyochelin in Pseudomonas aeruginosa and enantio-pyochelin in Pseudomonas fluorescens display different enantioselectivities.

Iron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch (ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two AraC-type regulators PchR, which are activated by their cognate siderophore. Here, stereospecific siderophore recognition is shown to occur at the inner membrane also. In P. aeruginosa, translocation of ferriPch across the inner membrane is carried out by the single-subunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational fetA-gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination with either of the two siderophore receptors FetA and FptA in a siderophore-negative P. aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific siderophore recognition occurs at the inner membrane by the FetCDE transporter.

Mice with chronic GSH deficit display phenotypical anomalies that resemble key aspects of schizophrenia

ABSTRACTSchizophrenia is a major psychiatric disorder occurring with a prevalence of 1% in the worldwide population. It develops progressively with psychosis onset in late adolescence or earlyadulthood. The disorder can take many different facets and has a highly diffuse anddistributed neuropathology including deficits in major neurotransmitter systems,myelination, stress regulation, and metabolism. The delayed onset and the heterogeneouspathology suggest that schizophrenia is a developmental disease that arises from interplayof genetic and environmental factors during sensitive periods. Redox dysregulation due to animbalance between pro-oxidants and antioxidant defence mechanisms is among the riskfactors for schizophrenia. Glutathione (GSH) is the major cellular redox regulator andantioxidant. Levels of GSH are decreased in cerebrospinal fluid, prefrontal cortex and postmortemstriatum of schizophrenia patients. Moreover, polymorphisms of the key GSHsynthesizingenzyme, glutamate-cysteine ligase, modifier (GCLM) subunit, are associatedwith the disease, suggesting that GSH deficit is of genetic origin. Here we used miceknockout (KO) for the GCLM gene, which display chronic GSH deficit (~70 to 80% decrease)to investigate the direct link between redox dysregulation and schizophrenia. Accordingly,we evaluated whether GCLM KO compared to normal wildtype mice display behavioralchanges that relate to schizophrenia symptoms and whether their brains showmorphological, functional or metabolic alterations that resemble those in patients.Moreover, we exposed pubertal GCLM mice to repeated mild stress and measured theirhormonal and behavioral stress reactivity. Our data show that chronic GSH deficit isassociated with altered emotion- and stress-related behaviors, deficient prepulse inhibition,pronounced amphetamine-induced hyperlocomotion but normal spatial learning andworking memory. These changes represent important schizophrenia endophenotypes.Moreover, this particular pattern of change indicates impairment of the ventralhippocampus (VH) and related circuitry as opposed to the dorsal hippocampus (DH), which isimplicated in spatial information processing. This is consistent with a selective deficit ofparvalbumin positive interneurons and gamma oscillation in the VH but not DH. Increasedlevels of circulating stress hormones in KO mice following pubertal stress corroborate VHdysfunction as it is involved in negative feedback control of the stress response. VHstructural and functional deficits are frequently found in the schizophrenic brain. Metabolicevaluation of the developing GCLM KO anterior cortex using in vivo magnetic resonancespectroscopy revealed elevated glutamine (Gln), glutamate (Glu), Gln/Glu and N-acetylaspartate(NAA) during the pre-pubertal period. Similar changes are reported in earlyschizophrenia. Overall, we observe phenotypic anomalies in GSH deficient GCLM KO micethat correspond to major schizophrenia endophenotypes. This supports an important rolefor redox dysregulation in schizophrenia and validates the GCLM KO mouse as model for thedisease. Moreover, our results indicate that puberty may be a sensitive period for redoxsensitivechanges highliting the importance of early intervention. Gln, Gln/Glu, Glu and NAAmay qualify as early metabolic biomarkers to identify young at-risk individuals. Since chronictreatment with NAC normalized most metabolic changes in GCLM KO mice, NAC may be oneadjunct treatment of choice for early intervention in patients.RESUMELa schizophrénie est une maladie psychiatrique majeure avec une prévalence de 1% dans lapopulation. Son développement est progressif, les premières psychoses apparaissant àl'adolescence ou au début de l'âge adulte. La maladie a plusieurs présentations et uneneuropathologie étendue, qui inclut des déficits neurochimiques, métaboliques, de lamyélination et de la régulation du stress. L'émergence tardive et l'hétérogénéité de lapathologie suggèrent que la schizophrénie est une maladie développementale, favorisée pardes facteurs génétiques et environnementaux durant des périodes sensibles. La dérégulationrédox, due à un déséquilibre entre facteurs pro-oxidantes et défenses anti-oxidantes,constitue un facteur de risque. Le glutathion (GSH) est le principal régulateur rédox et antioxidantdes cellules, ses taux sont diminués dans le liquide céphalorachidien, le cortexpréfrontal et le striatum de patients. De plus, des variations du gène codant la sous-unitémodulatrice (GCLM) de la glutamate-cystéine ligase, enzyme de synthèse du GSH, sontassociés la maladie, suggérant que le déficit observé chez les patients est d'originegénétique. Nous avons donc utilisé des souris ayant une délétion du gène GCLM (KO), quiont un déficit chronique en GSH (70-80%), afin d'étudier le lien entre une dérégulation rédoxet la schizophrénie. Nous avons évalué si ces souris présentent des altérationscomportementales analogues aux symptômes de la maladie, et des modificationsstructurelles, fonctionnelles et métaboliques au niveau du cerveau, ressemblant à celles despatients. De plus, nous avons soumis les souris à des stresses modérés durant la puberté,puis mesuré les réponses hormonales et comportementales. Les animaux présentent undéficit pré-attentionnel du traitement des informations moto-sensorielles, un déficit pourcertains apprentissages, une réponse accrue à l'amphétamine, mais leurs mémoires spatialeet de travail sont préservées. Ces atteintes comportementales sont analogues à certainsendophénotypes de la schizophrénie. De plus, ces changements comportementaux sontlargement expliqués par une perturbation morphologique et fonctionnelle de l'hippocampeventral (HV). Ainsi, nous avons observé un déficit sélectif des interneurones immunoréactifsà la parvalbumine et une désynchronisation neuronale dans l'HV. L'hippocampe dorsal,impliqué dans l'orientation spatiale, demeure en revanche intact. L'augmentationd'hormones de stress dans le sang des souris KO suite à un stress prépubertal soutien aussil'hypothèse d'une dysfonction de l'HV, connu pour moduler ce type de réponse. Des déficitsstructurels et fonctionnels dans l'hippocampe antérieur (ventral) ont d'ailleurs été rapportéschez des patients schizophrènes. Par de résonance magnétique, nous avons également suivile profil métabolique du le cortex antérieur au cours du développement postnatal des sourisKO. Ces mesures ont révélé des taux élevés de glutamine (Gln), glutamate (Glu), du ratioGln/Glu, et de N-acétyl-aspartate (NAA) durant la période prépubertale. Des altérationssimilaires sont décrites chez les patients durant la phase précoce. Nous avons donc révélédes anomalies phénotypiques chez les souris GCLM KO qui reflètent certainsendophénotypes de la schizophrénie. Nos résultats appuient donc le rôle d'une dérégulationrédox dans l'émergence de la maladie et le potentiel des souris KO comme modèle. De plus,cette étude met en évidence la puberté comme période particulièrement sensible à unedérégulation rédox, renforçant l'importance d'une intervention thérapeutique précoce. Dansce cadre, Gln, Gln/Glu, Glu and NAA seraient des biomarqueurs clés pour identifier de jeunesindividus à risque. De part son efficacité dans notre modèle, NAC pourrait être unesubstance de choix dans le traitement précoce des patients.

Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin.

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.

Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology.

The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.

Serious games for rehabilitation using head-mounted display and haptic devices

In the health domain, the field of rehabilitation suffers from a lack specialized staff while hospital costs only increase. Worse, almost no tools are dedicated to motivate patients or help the personnel to carry out monitoring of therapeutic exercises. This paper demonstrates the high potential that can bring the virtual reality with a platform of serious games for the rehabilitation of the legs involving a head-mounted display and haptic robot devices. We first introduce SG principles and the current context regarding rehabilitation interventions followed by the description of an original haptic device called Lambda Health System. The architecture of the model is then detailed, including communication specifications showing that lag is imperceptible for user (60Hz). Finally, four serious games for rehabilitation using haptic robots and/or HMD were tested by 33 health specialists.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

Sexual dimorphism in resource acquisition and deployment: both size and timing matter.

Background and Aims The males and females of many dioecious plant species differ from one another in important life-history traits, such as their size. If male and female reproductive functions draw on different resources, for example, one should expect males and females to display different allocation strategies as they grow. Importantly, these strategies may differ not only between the two sexes, but also between plants of different age and therefore size. Results are presented from an experiment that asks whether males and females of Mercurialis annua, an annual plant with indeterminate growth, differ over time in their allocation of two potentially limiting resources (carbon and nitrogen) to vegetative (below-and above-ground) and reproductive tissues.Methods Comparisons were made of the temporal patterns of biomass allocation to shoots, roots and reproduction and the nitrogen content in the leaves between the sexes of M. annua by harvesting plants of each sex after growth over different periods of time.Key Results and Conclusions Males and females differed in their temporal patterns of allocation. Males allocated more to reproduction than females at early stages, but this trend was reversed at later stages. Importantly, males allocated proportionally more of their biomass towards roots at later stages, but the roots of females were larger in absolute terms. The study points to the important role played by both the timing of resource deployment and the relative versus absolute sizes of the sinks and sources in sexual dimorphism of an annual plant.

Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR-669a downregulation has been linked to the severe DCM progression displayed by Sgcb-null dystrophic mice. However, the impact of long-term overexpression of miR-669a on muscle structure and functionality of the dystrophic heart is yet unknown. METHODS AND RESULTS: Here, we demonstrate that intraventricular delivery of adeno-associated viral (AAV) vectors induces long-term (18 months) miR-669a overexpression and improves survival of Sgcb-null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR-669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long-term miR-669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. CONCLUSIONS: Our findings provide the first evidence of long-term beneficial impact of AAV-mediated miRNA therapy in a transgenic model of severe, chronic MD-associated DCM.