Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells

AbstractOX40 is a member of the TNF/NGF-receptor family expressed on activated T cells, whose ligand is found on activated T and B cells. In the present study, we show that cross-linking of OX40L on CD40L-stimulated B cells, αlgD dextran-stimulated B cells, or both results in a significantly enhanced proliferative response with no change In the cell survival rate. Furthermore, OX40 stimulation increases Immunoglobulin heavy chain mRNA levels and Immunoglobulin secretion, which could not be blocked by anti-cytokine antibodies. In additional molecular studies, we show that OX40L cross-linking results In the down-regulation of the transcription factor BSAP. This, In turn, leads to a change In the In vivo binding pattern of the imunoglobulin heavy chain gene 3′ α enhancer, suggesting its activation. This effect may thus be one mechanism for OX40-induced Increase In Immunoglobulin secretion. In conclusion, our data suggest that the OX40-OX40L interaction is a novel pathway in T cell-dependent B cell proliferation and differentiation

Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

<p>Abstract</p> <p>Background</p> <p>RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.</p> <p>Results</p> <p>Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.</p> <p>Conclusion</p> <p>The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types.</p

The Immunosuppressive Properties of the HIV Vpr Protein Are Linked to a Single Highly Conserved Residue, R90

BACKGROUND: A hallmark of AIDS progression is a switch of cytokines from Th1 to Th2 in the plasma of patients. IL-12, a critical Th1 cytokine secreted by antigen presenting cells (APCs) is suppressed by Vpr, implicating it as an important virulence factor. We hypothesize that Vpr protein packaged in the virion may be required for disabling APCs of the first infected mucosal tissues. Consistent with this idea are reports that defects in the C-terminus of Vpr are associated with long-term non-progression. PRINCIPAL FINDINGS: Vpr RNA amplified from various sources was electroporated into monocyte-derived DC and IL-12 levels in supernatants were analyzed. The analysis of previously reported C-terminal Vpr mutations demonstrate that they do not alleviate the block of IL-12 secretion. However, a novel single conservative amino acid substitution, R90K, reverses the IL-12 suppression. Analysis of 1226 Vpr protein sequences demonstrated arginine (R) present at position 90 in 98.8%, with other substitutions at low frequency. Furthermore, none of sequences report lysine (K) in position 90. Vpr clones harboring the reported substitutions in position 90 were studied for their ability to suppress IL-12. Our data demonstrates that none of tested substitutions other than K relieve IL-12 suppression. This suggests a natural selection for sequences which suppress IL-12 secretion by DC and against mutations which relieve such suppression. Further analyses demonstrated that the R90K, as well as deletion of the C-terminus, directs the Vpr protein for rapid degradation. CONCLUSION: This study supports Vpr as an HIV virulence factor during HIV infection and for the first time provides a link between evolutionary conservation of Vpr and its ability to suppress IL-12 secretion by DC. DC activated in the presence of Vpr would be defective in the production of IL-12, thus contributing to the prevailing Th2 cytokine profile associated with progressive HIV disease. These findings should be considered in the design of future immunotherapies that incorporate Vpr as an antigen

Ten Simple Rules for Effective Computational Research

<p>Ten Simple Rules for Effective Computational Research</p

Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

BACKGROUND: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS: The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE: This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral loads in the absence of conventional antiretroviral drug therapy

Diez reglas sencillas para una exitosa colaboración transdisciplinar

El presente artículo es la versión en castellano de la publicación: KNAPP, B.; BARDENET, R.; BERNABEU, M.O.; BORDAS, R.; BRUNA, M.; CALDERHEAD, B. ET AL. (2015) “Ten Simple Rules for a Successful Cross-Disciplinary Collaboration”. PLoS Comput Biol 11(4): e1004214, disponible en: https://doi.org/10.1371/journal.pcbi.1004214. La traducción, autorizada por la entidad editora, ha sido llevada a cabo por Ona Lorda Roure y Leila Adim, colaboradoras del Instituto de Investigación TransJus y supervisada por el Dr. Juli Ponce Solé, Director del TransJus. En la misma se han incluido algunas notas aclaratorias para el lector en español, así como bibliografía complementaria en español.[spa] En el auge de las colaboraciones interdisciplinarias entre los distintos campos científicos, la transdisciplinariedad se presenta como la clave para encontrar soluciones a una variedad de problemas globales. Este trabajo, situado en el marco de la biología informática, se centra en exponer una lista extensa de reglas y consejos útiles para lograr una exitosa sinergia entre los varios colaboradores de un proyecto transdisciplinar. Se trata, de hecho, de una guía que pretende dirigirse tanto a investigadores noveles como a aquellos investigadores consolidados que se adentran en un espacio transdisciplinar por primera vez. En particular, este trabajo expone los beneficios principales de establecer una colaboración transdisciplinar, así como los problemas que de ella puedan surgir.[cat] En l'auge de les col·laboracions interdisciplinàries entre els diferents camps científics, la transdisciplinarietat es presenta com la clau per trobar solucions a una varietat de problemes globals. Aquest treball, situat en el marc de la biologia informàtica, es centra en exposar una llista extensa de regles i consells útils per aconseguir una reeixida sinergia entre els varis col·laboradors d'un projecte transdisciplinar. Es tracta, de fet, d'una guia que pretén dirigir-se tant a recercadors novells com a aquells recercadors consolidats que s'endinsen en un espai transdisciplinar per primera vegada. En particular, aquest treball exposa els beneficis principals d'establir una col·laboració transdisciplinar, així com els problemes que d'ella puguin sorgir.[eng] At a time of increasing interdisciplinary collaboration between different scientific fields, cross-disciplinarity represents a key for finding solutions to a variety of global problems. This work, located within the framework of computer biology, focuses on exposing an extensive list of rules and useful tips to achieve a successful synergy among the various collaborators of a transdisciplinary project. It is, in fact, a guide aimed at addressing both first-time researchers and consolidated researchers who enter a transdisciplinary space for the first time. In particular, this work exposes the main benefits of establishing a cross-disciplinary collaboration, as well as the problems that may arise from it

Ten Simple Rules for a Successful Cross-Disciplinary Collaboration

Cross-disciplinary collaborations have become an increasingly important part of science. They are seen as key if we are to find solutions to pressing, global-scale societal challenges, including green technologies, sustainable food production, and drug development. Regulators and policy- makers have realized the power of such collaborations, for example, in the 80 billion Euro "Horizon 2020" EU Framework Programme for Research and Innovation. This programme puts special emphasis on “breaking down barriers to create a genuine single market for knowledge, research and innovation

A Nondegenerate Code of Deleterious Variants in Mendelian Loci Contributes to Complex Disease Risk

SummaryAlthough countless highly penetrant variants have been associated with Mendelian disorders, the genetic etiologies underlying complex diseases remain largely unresolved. By mining the medical records of over 110 million patients, we examine the extent to which Mendelian variation contributes to complex disease risk. We detect thousands of associations between Mendelian and complex diseases, revealing a nondegenerate, phenotypic code that links each complex disorder to a unique collection of Mendelian loci. Using genome-wide association results, we demonstrate that common variants associated with complex diseases are enriched in the genes indicated by this “Mendelian code.” Finally, we detect hundreds of comorbidity associations among Mendelian disorders, and we use probabilistic genetic modeling to demonstrate that Mendelian variants likely contribute nonadditively to the risk for a subset of complex diseases. Overall, this study illustrates a complementary approach for mapping complex disease loci and provides unique predictions concerning the etiologies of specific diseases