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.

Palaeotectonic and palaeogeographic evolution of the western Tethys and PeriTethyan domain (IGCP Project 369)

Project No. 369 (''Comparative Evolution of PeriTethyan Rift Basins'') of the International Geological Correlation Program produced a new palaeotectonic-palaeo-geographic atlas of the western PeriTethyan domain. The atlas contains more than two hundred new maps and documents grouped in nine regional sets (Iberia, Polish Trough, Eastern European and Scythian Platforms, Moesian Platform, Levant, Arabian Platform,, Northern Africa, NE Africa-NW Arabia, Libya-Pelagian Shelf plus a set of reconstructions for the whole western Tethys. The area, considered in the atlas stretches. from west to east, from the eastern Atlantic shores to the Urals and, from north to south, from the Baltic shield to equatorial Africa; the time span covered extends from the Late. Carboniferous to the Present. The dataset, resulting from an extensive cooperation between industrial and academic sources, is accessible interactively on a CD-ROM (Stampfli et al., 2001a) and includes legend, timetable, short explanatory notes, full references and additional supporting data. This dataset provides information on the development of the Tethyan realm in space and time. In particular, the relation between the Variscan and Cimmerian cycles in the Mediterranean realm is illustrated by numerous palaeogeographic and palaeotectonic maps.

Comparison of central macular thickness (CMT) measurement between time domain and spectral domain (SD) optical coherence tomography (OCT) and reproducibility of SD OCT in active neovascular AMD

Purpose: To evaluate the reproducibility of Cirrus-SD OCT measurements and to compare central macular thickness (CMT) measurements between TD-Stratus and SD-Cirrus OCT in patients with active exudative AMD. Methods: Consecutive case series of patients with active exudative AMD seen in the Medical Retina Department. Patients underwent 1 scan with Stratus (macular thickness map protocol) and 5 scans with Cirrus (Macular Cube protocol) at the same visit by the same experienced examiner. To be included, patients best-corrected visual acuity (BCVA) had to be >20/200 while all scans had to be of sufficient quality, well-centered and at least one Cirrus scan with CMT >300 microns. The repeatability of the SD Cirrus was estimated by using all 5 CMT measurements and the mean of the Cirrus measurements was compared with the CMT obtained by TD Stratus. Results: Cirrus OCT demonstrated high intraobserver repeatability at the central foveal region (ICC 96%). The mean of the CMT measurements was 321microns for Stratus and 387 microns for Cirrus. The average difference was 65m (SD=30). The coefficient of concordance between Stratus and Cirrus CMT measurements was rho=0,749 with a high precision and a moderate accuracy. The equation of the line of regression between Stratus and meanCirrus is given by the following: M_stratus = 0,848 x m_cirrus - 4,496 (1).Conclusions: The Cirrus macular cube protocol allows reproducible CMT measurements in patients with active exudative AMD. In cases of upgrading from TD to SD use and vice versa, there is the possibility to predict the measurements by using the equation (1). These real life data and conclusions can help in improving our clinical management of patients with neovascular AMD.

Insights into the biogeographical history of the Lower Guinea Forest Domain: evidence for the role of refugia in the intraspecific differentiation of Aucoumea klaineana.

Determining the biogeographical histories of rainforests is central to our understanding of the present distribution of tropical biodiversity. Ice age fragmentation of central African rainforests strongly influenced species distributions. Elevated areas characterized by higher species richness and endemism have been postulated to be Pleistocene forest refugia. However, it is often difficult to separate the effects of history and of present-day ecological conditions on diversity patterns at the interspecific level. Intraspecific genetic variation could yield new insights into history, because refugia hypotheses predict patterns not expected on the basis of contemporary environmental dynamics. Here, we test geographically explicit hypotheses of vicariance associated with the presence of putative refugia and provide clues about their location. We intensively sampled populations of Aucoumea klaineana, a forest tree sensitive to forest fragmentation, throughout its geographical range. Characterizing variation at 10 nuclear microsatellite loci, we were able to obtain phylogeographic data of unprecedented detail for this region. Using Bayesian clustering approaches, we demonstrated the presence of four differentiated genetic units. Their distribution matched that of forest refugia postulated from patterns of species richness and endemism. Our data also show differences in diversity dynamics at leading and trailing edges of the species' shifting distribution. Our results confirm predictions based on refugia hypotheses and cannot be explained on the basis of present-day ecological conditions.

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA-binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

A case of acute retinal pigment epithelitis: spectral domain optical coherence tomography time course and physiopathologic hypothesis.

PURPOSE: To report the time course of retinal morphologic changes in a patient with acute retinal pigment epithelitis (ARPE) using spectral domain optical coherence tomography (SD-OCT). METHODS: A 30-year old man was referred for blurred vision of his right eye after five days that appeared suddenly 15 days after recovery from a flu-like syndrome. SD-OCT was performed immediately, followed by fluorescein and infracyanine angiography at eight days and then at three weeks. RESULTS: At presentation, a bubble of sub-macular deposit was observed on the right macula with central golden micronodules in a honeycomb pattern. SD-OCT showed an "anterior dislocation" of all the retinal layers up to the inner/outer segment (IS/OS) line and irregular deposits at the OS level together with thickening of the retinal pigment epithelial (RPE) layer. As visual acuity increased, eight days later, the OCT showed reduction of the sub-retinal deposits and an abnormal hyperflectivity of the sub-retinal and RPE layers was observed. The patient showed a positive serology for picornavirus. DISCUSSION: The acute SD-OCT sections of this patient with ARPE were compared with histological sections of a 35 day old Royal College of Surgeons rat. Similar findings could be observed, with preservation of the IS/OS line and accumulation of debris at the OS level, suggesting that ARPE symptoms could result from a transient phagocytic dysfunction of the RPE at the fovea, inducing reversible accumulation of undigested OS. Picornaviruses comprising enterovirus and coxsachievirus described as being associated with acute chorioretinitis. In this case, it was responsible for ARPE. CONCLUSION: We hypothesize that ARPE syndrome results from a transient dysfunction of RPE, which can occur as a post viral reaction.

Tectonic and metamorphic evolution of the Central Himalayan Domain in Southeast Zanskar (Kashmir, India)

La région du Zanskar, étudiée dans le cadre de ce travail, se situe au passage entre deux domaines himalayens fortement contrastés, la Séquence Cristalline du Haut Himalaya (HHCS), composée de roches métamorphiques et l'Himalaya Tethysien (TH), composé de séries sédimentaires. La transition entre ces deux domaines est marquée par une structure tectonique majeure, la Zone de Cisaillement du Zanskar (ZSZ), au sein de laquelle on observe une augmentation extrêmement rapide, mais néanmoins graduelle, du degré du métamorphisme entre le TH et le HHCS. Il a été établi que le HHCS n'est autre que l'équivalent métamorphique des séries sédimentaires de la base du TH. C'est principalement lors d'un épisode de mise en place de nappes à vergence sudouest, entre l'Eocène moyen et l'Oligocène, que les séries sédimentaires de la base du TH ont été entraînées en profondeur où elles ont subi un métamorphisme de type barrovien. Au début du Miocène, le HHCS à été exhumé en direction du sud-ouest sous forme d'une grande nappe, délimitée a sa base par le MCT (principal chevauchement central) et à son sommet par la Zone de Cisaillement du Zanskar. L'ensemble des zones barroviennes, de la zone à biotite jusqu'à la zone à disthène, a été cisaillée par les mouvements en faille normale au sommet du HHCS et se retrouve actuellement sur une épaisseur d'environ 1 kilomètre au sein de la ZSZ. La décompression associée à l'exhumation du HHCS a provoqué la fusion partielle d'une partie du HHCS et a donné naissance à des magmas de composition leucogranitiques. Grâce à la géothermobarometrie, et connaissant la géométrie de la ZSZ, il nous a été possible de déterminer que le rejet le long de cette structure d'extension est d'au moins 35?9 kilomètres. Une série d'arguments nous permet cependant de suggérer que ce rejet aurait pu être encore bien plus important (~100km). Les données géochronologiques nous permettent de contraindre la durée des mouvements d'extension le long de la ZSZ à 2.4?0.2 Ma entre 22.2?0.2 Ma et 19.8?0.1 Ma. Ce travail apporte de nouvelles données sur les processus métamorphiques, magmatiques et tectoniques liés aux phénomènes d'extension syn-orogeniques.<br/><br/>The southeastern part of Zanskar is located at the transition between two major Himalayan domains of contrasting metamorphic grade, the High Himalayan Crystalline Sequence (HHCS) and the Tethyan Himalaya (TH). The transition between the TH and the HHCS is marked by a very rapid, although perfectly gradual, decrease in metamorphic grade, which coincides with a major tectonic structure, the Zanskar Shear Zone (ZSZ). It is now an established fact that the relation between the HHCS and the TH is not one of basement-cover type, but that the metasedimentary series of the HHCS represent the metamorphic equivalent of the lowermost sedimentary series of the TH. This transformation of sedimentary series into metamorphic rocks, and hence the differentiation between the TH and the HHCS, is the consequence of crustal thickening associated to the formation of large scale southwest vergent nappes within the Tethyan Himalaya sedimentary series. This, Middle Eocene to Oligocene, episode of crustal thickening and associated Barrovian metamorphism is followed, shortly after, by the exhumation of the HHCS as a, large scale, south-west vergent, nappe. Foreword The exhumation of the HHCS nappe is marked by the activation of two contemporaneous structures, the Main Central Thrust at its base and the Zanskar Shear Zone at its top. Extensional movements along the ZSZ, caused the Barrovian biotite to the kyanite zones to be sheared and constricted within the ~1 km thick shear zone. Decompression associated with the exhumation of the HHCS induced the formation of leucogranitic magmas through vapour-absent partial melting of the highest-grade rocks. The combination of geothermobarometric data with a geometric model of the ZSZ allowed us to constrain the net slip at the top of the HHCS to be at least 35?9 kilometres. A set of arguments however suggests that these movements might have been much more important (~ 100 km). Geochronological data coupled with structural observations constrain the duration of ductile shearing along the ZSZ to 2.4?0.2 Ma between 22.2?0.2 Ma and 19.8?0.1 Ma. This study also addresses the consequences of synorogenic extension on the metamorphic, tectonic and magmatic evolution of the upper parts of the High Himalayan Crystalline Sequence.

Between Immunology And Tolerance: Controlling Immune Responses Employing Tolerogenic Dendritic Cells

Dendritic cells (DCs) are the most efficient antigen presenting cells, they provide co-stimulation, are able to secrete various proinflammatory cytokines and therefore play a pivotal role in shaping adaptive immune responses. Moreover, they are important for the promotion and maintenance of central and peripheral tolerance through several mechanisms like the induction of anergy or apoptosis in effector T cells or by promoting regulatory T cells. The murine CD8α+ (MuTu) dendritic cell line was previously derived and described in our laboratory. The MuTu cell line has been shown to maintain phenotypical and functional characteristics of endogenous CD8α+ DCs. They are able to cross-present exogenous antigens to CD8+ T cells and produce interleukin (IL-) 12 upon engagement of Toll like receptors. The cell line constitutes an infinite source of homogenous, phenotypically well-defined dendritic cells. This allows us to investigate the role and potential of specific molecules in the induction as well as regulation of immune responses by DCs in a rational and standardized way. In a first project the MuTu dendritic cell line was transduced in order to stably express the immunosuppressive molecules IL-10, IL-35 or the active form of TGF-β (termed IL-10+DC, IL-35+DC or actTGFβ+DC). We investigated the capability of these potentially suppressive or tolerogenic dendritic cell lines to induce immune tolerance and explore the mechanisms behind tolerance induction. The expression of TGF-β by the DC line did not affect the phenotype of the DCs itself. In contrast, IL-10+ and IL-35+DCs were found to exhibit lower expression of co-stimulatory molecules and MHC class I and II, as well as reduced secretion of pro-inflammatory cytokines upon activation. In vitro co-culture with IL-35+, IL10+ or active TGFβ+ DCs interfered with function and proliferation of CD4+ and CD8+ T cells. Furthermore, IL-35 and active TGF-β expressing DC lines induced regulatory phenotype on CD4+ T cells in vitro without or with expression of Foxp3, respectively. In different murine cancer models, vaccination with IL-35 or active TGF-β expressing DCs resulted in faster tumor growth. Interestingly, accelerated tumor growth could be observed when IL-35-expressing DCs were injected into T cell-deficient RAG-/- mice. IL-10expressing DCs however, were found to rather delay tumor growth. Besides the mentioned autocrine effects of IL-35 expression on the DC line itself, we surprisingly observed that the expression of IL-35 or the addition of IL-35 containing medium enhances neutrophil survival and induces proliferation of endothelial cells. Our findings indicate that the cytokine IL-35 might not only be a potent regulator of adaptive immune responses, but it also implies IL-35 to mediate diverse effects on an array of cellular targets. This abilities make IL-35 a promising target molecule not only for the treatment of auto-inflammatory disease but also to improve anti-cancer immunotherapies. Indeed, by applying active TGFβ+ in murine autoimmune encephalitis we were able to completely inhibit the development of the disease, whereas IL-35+DCs reduced disease incidence and severity. Furthermore, the preventive transfer of IL-35+DCs delayed rejection of transplanted skin to the same extend as the combination of IL-10/actTGF-β expressing DCs. Thus, the expression of a single tolerogenic molecule can be sufficient to interfere with the adequate activation and function of dendritic cells and of co-cultured T lymphocytes. The respective mechanisms of tolerance induction seem to be different for each of the investigated molecule. The application of a combination of multiple tolerogenic molecules might therefore evoke synergistic effects in order to overcome (auto-) immunity. In a second project we tried to improve the immunogenicity of dendritic cell-based cancer vaccines using two different approaches. First, the C57BL/6 derived MuTu dendritic cell line was genetically modified in order to express the MHC class I molecule H-2Kd. We hypothesized that the expression of BALB/c specific MHC class I haplotype (H-2Kd) should allow the priming of tumor-specific CD8+ T cells by the otherwise allogeneic dendritic cells. At the same time, the transfer of these H-2Kd+ DCs into BALB/c mice was thought to evoke a strong inflammatory environment that might act as an "adjuvant", helping to overcome tumor induced immune suppression. Using this so called "semi-allogeneic" vaccination approach, we could demonstrate that the delivery of tumor lysate pulsed H-2Kd+ DCs significantly delayed tumor growth when compared to autologous or allogeneic vaccination. However, we were not able to coherently elucidate the cellular mechanisms underlying the observed effect. Second, we generated MuTu DC lines which stably express the pro-inflammatory cytokines IL-2, IL-12 or IL-15. We investigated whether the combination of DC vaccination and local delivery of pro-inflammatory cytokines might enhance tumor specific T cell responses. Indeed, we observed an enhanced T cell proliferation and activation when they were cocultured in vitro with IL-12 or IL-2-expressing DCs. But unfortunately we could not observe a beneficial or even synergistic impact on tumor development when cytokine delivery was combined with semi-allogeneic DC vaccination.