Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis

Culturing of the first 37:4 predominant lacustrine haptophyte : geochemical, biochemical, and genetic implications

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 78 (2012): 51–64, doi:10.1016/j.gca.2011.11.024.Long chain alkenones (LCAs) are potential biomarkers for quantitative paleotemperature reconstructions from lacustrine environments. However, progress in this area has been severely hindered by the lack of culture studies of haptophytes responsible for alkenone distributions in lake sediments: the predominance of C37:4 LCA. Here we report the first enrichment culturing of a novel haptophyte phylotype (Hap-A) from Lake George, ND that produces predominantly C37:4-LCA. Hap-A was enriched from its resting phase collected from deep sediments rather than from water column samples. In contrast, enrichments from near surface water yielded a different haptophyte phylotype (Hap-B), closely related to Chrysotila lamellosa and Pseudoisochrysis paradoxa, which does not display C37:4-LCA predominance (similar enrichments have been reported previously). The LCA profile in sediments resembles that of Hap-A enrichments, suggesting that Hap-A is the dominant alkenone producer of the sedimentary LCAs. In enrichments, excess lighting appeared to be crucial for triggering blooms of Hap-A. Both and indices show a linear relationship with temperature for Hap-A in enrichments, but the relationship appears to be dependent on the growth stage. Based on 18S rRNA gene analyses, several lakes from the Northern Great Plains, as well as Pyramid Lake, NV and Tso Ur, Tibetan Plateau, China contain the same two haptophyte phylotypes. The Great Plains lakes show the Hap-A-type LCA distribution, whereas Pyramid and Tso Ur show the Hap-B type distribution. Waters of the Great Plain lakes are dominated by sulfate, whereas those Pyramid and Tso Ur are dominated by carbonate, suggesting that the sulfate to carbonate ratio may be a determining factor for the competitiveness of the Hap-A and Hap-B phylotypes in natural settings.This work was supported by a grant from the National Science Foundation to Y. Huang (EAR06-02325) and a Brown University Graduate School Dissertation Fellowship to J. L. Toney

A Single-Domain Antibody Targeting Complement Component C5 Acts as a Selective Inhibitor of the Terminal Pathway of the Complement System and Thus Functionally Mimicks the C-Terminal Domain of the Staphylococcus aureus SSL7 Protein

The complement system is an efficient anti-microbial effector mechanism. On the other hand abnormal complement activation is involved in the pathogenesis of multiple inflammatory and hemolytic diseases. As general inhibition of the complement system may jeopardize patient health due to increased susceptibility to infections, the development of pathway-specific complement therapeutics has been a long-lasting goal over the last decades. In particular, pathogen mimicry has been considered as a promising approach for the design of selective anti-complement drugs. The C-terminal domain of staphylococcal superantigen-like protein 7 (SSL7), a protein secreted by Staphylococcus aureus, was recently found to be a specific inhibitor of the terminal pathway of the complement system, providing selective inhibition of cell lysis mediated by the membrane attack complex (MAC). We describe here the selection by phage display of a humanized single-domain antibody (sdAb) mimicking the C-terminal domain of SSL7. The antibody, called sdAb_E4, binds complement C5 with an affinity in the low micromolar range. Furthermore, sdAb_E4 induces selective inhibition of MAC-mediated lysis, allowing inhibition of red blood cell hemolysis and inhibition of complement deposition on apopto-necrotic cells, while maintaining efficient bactericidal activity of the complement terminal pathway. Finally, we present preliminary results indicating that sdAb_E4 may also be efficient in inhibiting hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. Our data provide a proof of concept for the design of a selective MAC inhibitor capable of retaining complement bacteriolytic activity and this study opens up promising perspectives for the development of an sdAb_E4-derived therapeutics with application in the treatment of complement-mediated hemolytic disorders

Expression of the T Cell Receptor αβ on a CD123+ BDCA2+ HLA-DR+ Subpopulation in Head and Neck Squamous Cell Carcinoma

Human Plasmacytoid Dendritic Cells (PDCs) infiltrating solid tumor tissues and draining lymph nodes of Head and Neck Squamous Cell Carcinoma (HNSCC) show an impaired immune response. In addition to an attenuated secretion of IFN-α little is known about other HNSCC-induced functional alterations in PDCs. Particular objectives in this project were to gain new insights regarding tumor-induced phenotypical and functional alterations in the PDC population. We showed by FACS analysis and RT-PCR that HNSCC orchestrates an as yet unknown subpopulation exhibiting functional autonomy in-vitro and in-vivo besides bearing phenotypical resemblance to PDCs and T cells. A subset, positive for the PDC markers CD123, BDCA-2, HLA-DR and the T cell receptor αβ (TCR-αβ) was significantly induced subsequent to stimulation with HNSCC in-vitro (p = 0.009) and also present in metastatic lymph nodes in-vivo. This subgroup could be functionally distinguished due to an enhanced production of IL-2 (p = 0.02), IL-6 (p = 0.0007) and TGF-β (not significant). Furthermore, after exposure to HNSCC cells, mRNA levels revealed a D-J-beta rearrangement of the TCR-beta chain besides a strong enhancement of the CD3ε chain in the PDC population. Our data indicate an interface between the PDC and T cell lineage. These findings will improve our understanding of phenotypical and functional intricacies concerning the very heterogeneous PDC population in-vivo

A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5′ nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/IIIHC) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/IIIHC genome (within the 3′NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5′NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5′NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5′NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3′NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/IIIHC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5′NTR, 3′NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses

ARTEFACTS: How do we want to deal with the future of our one and only planet?

The European Commission’s Science and Knowledge Service, the Joint Research Centre (JRC), decided to try working hand-in-hand with leading European science centres and museums. Behind this decision was the idea that the JRC could better support EU Institutions in engaging with the European public. The fact that European Union policies are firmly based on scientific evidence is a strong message which the JRC is uniquely able to illustrate. Such a collaboration would not only provide a platform to explain the benefits of EU policies to our daily lives but also provide an opportunity for European citizens to engage by taking a more active part in the EU policy making process for the future. A PILOT PROGRAMME To test the idea, the JRC launched an experimental programme to work with science museums: a perfect partner for three compelling reasons. Firstly, they attract a large and growing number of visitors. Leading science museums in Europe have typically 500 000 visitors per year. Furthermore, they are based in large European cities and attract local visitors as well as tourists from across Europe and beyond. The second reason for working with museums is that they have mastered the art of how to communicate key elements of sophisticated arguments across to the public and making complex topics of public interest readily accessible. That is a high-value added skill and a crucial part of the valorisation of public-funded research, never to be underestimated. Finally museums are, at present, undergoing something of a renaissance. Museums today are vibrant environments offering new techniques and technologies to both inform and entertain, and attract visitors of all demographics.JRC.H.2-Knowledge Management Methodologies, Communities and Disseminatio

Evidenzbasierte Entwicklung von Bildungserträgen durch Assessments. Überlegungen zu einem Länder- und kultursensiblen Monitoring in Ländern der Entwicklungszusammenarbeit

Im Kontext des Diskurses zu internationalen Vergleichsstudien werden im folgenden Beitrag Möglichkeiten ausgeleuchtet, eine entsprechend empirische Datenbasis anhand von Assessments in Ländern der Entwicklungszusammenarbeit aufzubauen, die den Ertrag von Bildungsprozessen für die Steuerung von Bildungssystemen zur Verfügung stellt. Hierzu wird der Zusammenhang zwischen der Durchführung von Bildungsassessments und Bildungsqualität in den Blick genommen und ein ‚länder- und kultursensibles Bildungsmonitoring‘ beschrieben. Zudem werden Qualitätsindikatoren für Assessments beschrieben, die potenziell zu einer Verbesserung von Bildungsqualität beitragen können. Abschließend werden Überlegungen für den deutschen Beitrag zur internationalen Entwicklungszusammenarbeit ausgeführt. (DIPF/Orig.)In the context of the discourse on international large-scale assessments the following contribution explores opportunities for the development of a respective empirical data base for assessments in development cooperation countries, which provide the outcome of educational processes for the controlling of education systems. For this purpose the relation between the implementation of educational assessments and educational quality is taken into account to describe a country- and culture- sensitive educational monitoring. Furthermore, quality indicators for assessments are described, which potentially can contribute to the improvement of educational quality. In conclusion, observations are made in regard to the German contribution to international development cooperation. (DIPF/Orig.

Impact of pollen on human health: more than allergen carriers?

The transfer of pollen from floral anther to recipient stigma is the critical reproductive event among higher plants--this is the botanical view of pollen. Proteins and glycoproteins from pollen can function as allergens, environmental molecules interacting with the human immune system to elicit an allergic response in susceptible individuals--this is how allergists and immunologists see pollen grains. Between 10 and 25% of the population now have symptoms of hay fever or allergic asthma and the incidence has more than doubled in the past three decades while the reason(s) for this increment are only hypothetical, but there is a multitude of them. Despite our natural focus on this impact of pollen on human health, pollen have to be considered in a larger context. First of all, to evaluate the bioavailability of allergens from pollen, we have to understand their function and their influence factors. Furthermore, pollen grains are not only releasing proteins eliciting specific immune responses, but they also liberate bioactive lipid mediators and this much more rapidly. And last but not least, recent observations indicate, that pollen do not only induce allergy and thus have a much broader impact on human health. This review is an attempt to favour this holistic view of pollen and their impact on human health