Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>The PFD1235w <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and <it>Escherichia coli </it>based system was used to express single and double domains encoded by the <it>pfd1235w var </it>gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7_PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed.</p> <p>Methods</p> <p>The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and <it>E. coli</it>-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7_PFD1235w-IE.</p> <p>Results</p> <p>All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the <it>E. coli </it>system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the <it>E. coli </it>produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7_PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay.</p> <p>Conclusions</p> <p>The baculovirus based insect cell system was distinctly superior to the <it>E. coli </it>expression system in producing a larger number of different recombinant PFD1235w protein domains and these were significantly easier to purify at a useful yield. However, proteins produced in both systems were able to induce antibodies in rats, which can recognize the native PFD1235w on the surface of IE.</p

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of <it>Plasmodium falciparum </it>adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.</p> <p>Methods</p> <p>A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy</p> <p>Results</p> <p>Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.</p> <p>Conclusion</p> <p>A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.</p

Expression of a type B RIFIN in Plasmodium falciparum merozoites and gametes

BACKGROUND: The ability of Plasmodium falciparum to undergo antigenic variation, by switching expression among protein variants encoded by multigene families, such as var, rif and stevor, is key to the survival of this parasite in the human host. The RIFIN protein family can be divided into A and B types based on the presence or absence of a 25 amino acid motif in the semi-conserved domain. A particular type B RIFIN, PF13_0006, has previously been shown to be strongly transcribed in the asexual and sexual stages of P. falciparum in vitro. METHODS: Antibodies to recombinant PF13_0006 RIFIN were used in immunofluorescence and confocal imaging of 3D7 parasites throughout the asexual reproduction and sexual development to examine the expression of PF13_0006. Furthermore, reactivity to recombinant PF13_0006 was measured in plasma samples collected from individuals from both East and West African endemic areas. RESULTS: The PF13_0006 RIFIN variant appeared expressed by both released merozoites and gametes after emergence. 7.4% and 12.1% of individuals from East and West African endemic areas, respectively, carry plasma antibodies that recognize recombinant PF13_0006, where the antibody responses were more common among older children. CONCLUSIONS: The stage specificity of PF13_0006 suggests that the diversity of RIFIN variants has evolved to provide multiple specialized functions in different stages of the parasite life cycle. These data also suggest that RIFIN variants antigenically similar to PF13_0006 occur in African parasite populations

Ionization via Chaos Assisted Tunneling

A simple example of quantum transport in a classically chaotic system is studied. It consists in a single state lying on a regular island (a stable primary resonance island) which may tunnel into a chaotic sea and further escape to infinity via chaotic diffusion. The specific system is realistic : it is the hydrogen atom exposed to either linearly or circularly polarized microwaves. We show that the combination of tunneling followed by chaotic diffusion leads to peculiar statistical fluctuation properties of the energy and the ionization rate, especially to enhanced fluctuations compared to the purely chaotic case. An appropriate random matrix model, whose predictions are analytically derived, describes accurately these statistical properties.Comment: 30 pages, 11 figures, RevTeX and postscript, Physical Review E in pres

Corticotroph Aggressive Pituitary Tumors and Carcinomas Frequently Harbor ATRX Mutations

Context: Aggressive pituitary tumors (APTs) are characterized by unusually rapid growth and lack of response to standard treatment. About 1% to 2% develop metastases being classified as pituitary carcinomas (PCs). For unknown reasons, the corticotroph tumors are overrepresented among APTs and PCs. Mutations in the alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene, regulating chromatin remodeling and telomere maintenance, have been implicated in the development of several cancer types, including neuroendocrine tumors. Objective: To study ATRX protein expression and mutational status of the ATRX gene in APTs and PCs. Design: We investigated ATRX protein expression by using immunohistochemistry in 30 APTs and 18 PCs, mostly of Pit-1 and T-Pit cell lineage. In tumors lacking ATRX immunolabeling, mutational status of the ATRX gene was explored. Results: Nine of the 48 tumors (19%) demonstrated lack of ATRX immunolabelling with a higher proportion in patients with PCs (5/18; 28%) than in those with APTs (4/30;13%). Lack of ATRX was most common in the corticotroph tumors, 7/22 (32%), versus tumors of the Pit-1 lineage, 2/24 (8%). Loss-of-function ATRX mutations were found in all 9 ATRX immunonegative cases: nonsense mutations (n = 4), frameshift deletions (n = 4), and large deletions affecting 22-28 of the 36 exons (n = 3). More than 1 ATRX gene defect was identified in 2 PCs. Conclusion: ATRX mutations occur in a subset of APTs and are more common in corticotroph tumors. The findings provide a rationale for performing ATRX immunohistochemistry to identify patients at risk of developing aggressive and potentially metastatic pituitary tumors.Peer reviewe

Surface Co-Expression of Two Different PfEMP1 Antigens on Single Plasmodium falciparum-Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required

Le Moyen Âge par le Moyen Âge, même: Réception, relectures et réécritures des textes médiévaux dans la littérature française des XIVe et XVe siècles

Actes d'un colloque tenu à l'Université de Stockholm et au château de Skokloster (Suède) du 2 au 4 juillet 2009. - Textes en français. - Autre tirage : 2015International audienceLes contributions rassemblées dans le présent volume s’intéressent toutes, à travers des œuvres différentes appartenant à des genres divers – romans, arthuriens ou non, chansons de geste, poésie lyrique, théâtre, mais aussi compilation historique ou traduction –, à la même question, à savoir la façon dont la littérature française de la fin du Moyen Âge a reçu, relu, réécrit les textes médiévaux antérieurs, et spécialement ceux du Moyen Âge central. Se dévoilent ainsi des postérités plus (Chrétien de Troyes, toujours lui) ou moins attendues (qui se souvient de la mise en prose de Guillaume de Palerne par Pierre Durand ?) ; des phénomènes subtils ou massifs de rajeunissement linguistique, deréécriture – entre le vers et la prose, la « prose en dévers » – ou d’échos, évidemment lyriques ; d’autres de croisement et d’hybridation des matières, des genres, théâtre et allégorie, compilation historique et roman arthurien – sans oublier la légende de Barlaam et Josaphat, qui traverse plus d’un genre. De tous ces textes nouveaux greffés sur d’anciens textes, il peut ainsi ressortir l’image d’une littérature qui ne se construit pas tant dans le recul ou la distanciation vis-à-vis d’elle-même que dans «un toujours jeune recommencement »