1,720,993 research outputs found
Biochemical and functional characterization of the binding of Neisseria Adhesin A (NadA) with Siglec-5 and 14
Full text linkNeisseria adhesin A (NadA) is a proteic recombinant antigen included in Bexsero, a vaccine against serogroup B Neisseria meningitidis (N. meningitidis). NadA belongs to the trimeric autotransporters family and is composed of a long coiled-coil with three protruding wing-like structures that create an unusual N-terminal head domain. Even though NadA has been extensively described as promoter of both adhesion to and invasion into human epithelial cells, its receptors at the interface with human cells have never been found. To identify the targeted human receptors, we used recombinant NadA (rNadA) as probe on a protein microarray including a expanded collection of surface and secreted human proteins. The screening disclosed three putative interactors, Siglec-5, Siglec-14 and FcγRIIA, expressed on cells of myeloid lineage. We first validated these interactions addressing the biochemical features of the binding, confirming Siglec-5 and -14 as high affinity NadA binding proteins, while FcγRIIA was not. Next, we assessed the membrane properties of NadA binding to Siglecs by using Escherichia coli as an heterologous system. Full-lenght Siglecs, expressed on CHO-K1 cells showed an increase of adhesion of N. meningitidis, whereas the isogenic strain knock-out NadA did not. Although recombinant Siglec-5 ectodomain was shown to bind to NadA on both capsulated and unencapsulated N. meningitidis, we could not find any relevant evidence of NadA/Siglecs interaction on primary and immortalized monocytic cell lines.
In recent years, soluble forms of Siglecs were detected in human serum. In this study, the soluble Siglecs presence was confirmed in human pooled sera and in culture media of monocytes and macrophages. Interestingly, we found that during infection assays N. meningitids was able to bind monocytes-released Siglecs in NadA dependent manner. The exogenous addition of the soluble species increased the attachment of bacteria on monocytic cells surface but reduced internalization.
We also demonstrated a novel interaction between Siglecs and complement component 1 (C1q), whose in vitro interaction was inhibited by recombinant NadA in a concentration dependent manner. Further, we observed a survival advantage for N. meningitidis in presence of bactericidial antibodies anti-NadA by testing sera, as source of complement, depleted of Siglec-5 and -14.
In summary, this work revealed two new human interactors for the neisserial adhesin that could be encountered not only on phagocytes surfaces but also in the bloodstream. On the host side, we characterized a new proteic target for Siglecs on N. meningitidis bacterial surface. Moreover, the C1q binding to Siglec-5 and -14 was revealed for the first time, opening questions on Siglecs biology.
In conclusion we found that NadA may not contribute exclusively to the crossing of the epithelial nasopharyngeal barrier but also it may help the bacterium to survive into extracellular host milieu
Development of a modular and multiplexed assay to predict the coverage of BexseroTM against Neisseria meningitidis type B
Full text linkDevelopment of a modular and multiplex assay on Luminex platform to predict the coverage of BexseroTM against Neisseria meningitidis type B
a,b Sara Favale, b Brunella Brunelli, b Bruno Galletti, b Francois Legay, b Marzia Giuliani,
a Developmental and Cellular Biology Department, La Sapienza, University of Rome, 00185 Roma, Piazzale Aldo Moro 5, Italy
bGlaxoSmithKline, 53100 Siena, Via Fiorentina 1, Italy
e-mail: [email protected]
Keywords: N. meningitidis, meningitis, BexseroTM, MATS, Luminex.
BexseroTM vaccine against Neisseria meningitidis type B has been approved in several countries. It is composed by three sub-capsular antigens, selected by Reverse Vaccinology approach: factor H binding protein (fHbp) variant 1.1, Neisserial Heparin Binding Antigen (NHBA) peptide 2 and Neisserial Adesin A (NadA) variant 3. Bexsero vaccine includes also Outer Membrane Vescicles (OMVs) derived from the New Zealand strain NZ 98/254, expressing PorA serosubtype P1.4. Evaluation of antigen expression by circulating N. meningitidis strains is a very critical step in order to predict the vaccine coverage and, a specific test has been set up for this purpose (Ref. 1).
The Meningococcal Antigen Typing System (MATS) was designed to measure immunologic cross-reactivity and quantify antigen expression by target strains of N. meningitidis B. MATS results from a combination of three sandwich Enzyme-Linked Immunosorbent Assay (ELISA) assays, one for each vaccine antigen, plus sequencing of PorA P1.4 (Ref.2). Although working, conventional ELISA makes immunogenicity evaluation of a multi-component vaccine difficult, laborious, time-consuming and expensive, since only one immunogen per assay run can be tested.
xMap Luminex Technology allows the development of multiplex immunoassays where, multiple antibody types can be determined simultaneously in one assay run.
Taking the case of MATS-ELISA assay and BexseroTM vaccine as reference, this PhD project aim to: (i) develop a flexible multiplexed and quantitative sandwich assay (on Luminex platform) allowing the simultaneous measurement of all vaccine antigens expressed by bacterial strains, in order to predict the coverage of BexseroTM; (ii) qualify the multiplex assay performance (reproducibility, sensitivity, accuracy, precision, intra/inter assay variability); (iii) evaluate comparability of the new assay with the currently accepted ones, taking also into account results of the Serum Bactericidal Assay (SBA), the only accepted reference test for functional antibodies directed to meningococcus.
We developed and qualified a multiplex 4-plex assay based on Luminex technology able to simultaneously quantify BexseroTM vaccine antigens in Men B isolates. The assay was also designed to measure the OMVs content, avoiding PorA, sequencing in order to speculate on a possible role of the entire OMVs in the coverage of BexseroTM, avoiding MATS underestimation (Ref.3).
The new multiplex assay RPs shows a good correlation with MATS RPs for each BexseroTM antigen. It represents a promising assay to obtain information about BexseroTM coverage in easy, fast, cheap and more reproducible way. Due to the high flexibility of this technology in the future it will be possible to increase the antigens panel (from 4 up to 100 microspheres in the single well) and detect other vaccine antigens expressed on bacterial strain to predict the coverage of a new generation multi-component vaccine.
Ref.1 G. Boccadifuoco, B. Brunelli, M. G. Pizza, and M. M. Giuliani, “A combined approach to assess the potential coverage of a multicomponent protein-based vaccine,” Journal of Preventive Medicine and Hygiene, vol. 53, no. 2, pp. 56–60, 2012
Ref.2 Serruto, D., M. J. Bottomley, et al. (2012). "The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens." Vaccine 30 Suppl 2: B87-97.
Ref.3 Frosi, G. et al. Bactericidal antibody against a representative epidemiological meningococcal serogroup B panel confirms that MATS underestimates 4CMenB vaccine strain coverage. (2013). Vaccine 31:4968– 497
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
p38 MAPK is a critical regulator of the constitutive and the beta4 integrin-regulated expression of IL-6 in human normal thymic epithelial cells.
No full textCytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor alpha6beta4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-kappaB and NF-IL6 transactivators. Here we show that alpha6beta4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, beta4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and beta4 integrin-induced production of IL-6 preventing NF-kappaB and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-kappaB activation. Overall, our results indicate that p38 MAPK and alpha6beta4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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