Interleukin-4 (IL-4) enhances and soluble interleukin-4 receptor (sIL-4R) inhibits histamine release from peripheral blood basophils and mast cells in vitro and in vivo

The aim of the study was to analyse the effect of interleukin-4 (IL-4) on allergen and anti-IgE mediated histamine release from basophils and human skin mast cells and to assess whether soluble recombinant interleukin-4 receptor (sIL4R) can inhibit these effects. Anti-IgE stimulated histamine release from peripheral blood basophils and mast cells of atopic donors was enhanced after preincubation with IL-4, whereas after preincubation with sIL-4R it was inhibited. These effects were even more pronounced when samples were stimulated with a clinically relevant allergen. In IL-4 preincubated skin mast cells, there was a similar enhancement of anti-IgE stimulated histamine release, which could again be inhibited by sIL-4R. The effects of IL-4 and sIL4R were dose- and time-dependent. Mice sensitized to ovalbumin and treated with soluble recombinant murine sIL-4R showed significantly reduced immediate-type cutaneous hypersensitivity responses compared with untreated mice. These in vivo effects were IgE independent, since there were no significant differences in total and allergen specific IgE/IgG1 antibody titres between treated and untreated mice. This indicates that IL4 exerts priming effects on histamine release by effector cells of the allergic response and that these effects are potently antagonized by soluble IL-4R both in vitro and in vivo

Interactions of Host Proteins with the Murine Leukemia Virus Integrase

Retroviral infections cause a variety of cancers in animals and a number of diverse diseases in humans such as leukemia and acquired immune deficiency syndrome. Productive and efficient proviral integration is critical for retroviral function and is the key step in establishing a stable and productive infection, as well as the mechanism by which host genes are activated in leukemogenesis. Host factors are widely anticipated to be involved in all stages of the retroviral life cycle, and the identification of integrase interacting factors has the potential to increase our understanding of mechanisms by which the incoming virus might appropriate cellular proteins to target and capture host DNA sequences. Identification of MoMLV integrase interacting host factors may be key to designing efficient and benign retroviral-based gene therapy vectors; key to understanding the basic mechanism of integration; and key in designing efficient integrase inhibitors. In this review, we discuss current progress in the field of MoMLV integrase interacting proteins and possible roles for these proteins in integration

Foamy Virus Biology and Its Application for Vector Development

Spuma- or foamy viruses (FV), endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system

Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors

Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies

The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway

Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. NMD has been shown to also regulate normal gene expression and thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Recently, NMD efficiency has been shown to vary between cell types and individuals thus implicating NMD as a modulator of genetic disease severity. We have now specifically analysed the molecular mechanism of variable NMD efficiency and first established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains. In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model. We conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that cell type specific co-factor availability represents a novel principle that controls NMD

Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting

Suppression of BCL6 Function by HDAC Inhibitor Mediated Acetylation and Chromatin Modification Enhances BET Inhibitor Effects in B-cell Lymphoma Cells

Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occur in clinically aggressive B-cell lymphomas and lead to higher expression levels and activity of this transcriptional repressor. BCL6 is, therefore, an attractive target for therapy in aggressive lymphomas. In this study romidepsin, a potent histone deacetylase inhibitor (HDACi), induced apoptosis and cell cycle arrest in Burkitt and diffuse large B-cell lymphoma cell lines, which are model cells for studying the mechanism of action of BCL6. Romidepsin caused BCL6 acetylation at early timepoints inhibiting its function, while at later timepoints BCL6 expression was reduced and target gene expression increased due to chromatin modification. MYC contributes to poor prognosis in aggressive lymphoma. MYC function is reduced by inhibition of chromatin readers of the bromodomain and extra-terminal repeat (BET) family, which includes BRD4. The novel combination of romidepsin and JQ1, a BRD4 inhibitor was investigated and showed synergy. Collectively we suggest that the combination of HDACi and BRD4i should be pursued in further pre-clinical testing.Funding: The work was supported by grants SAF2014-53526-R and SAF2017-88026-R from MINECO, Spanish Government, to M.D.D. and J.L. (partially funded by FEDER program from European Union). M.G.C. was recipient of a “Marcos Fernández” fellowship from Leukemia and Lymphoma foundation. L.G.G. was recipient of a FPI fellowship from Spanish Government

A bottom-up and citizen-centred approach to transitional justice: public opinion towards reparations in post-Franco Spain

[eng] Forty-five years after the death of the Spanish dictator Francisco Franco dealing with the legacies of the country's repressive past and addressing the needs of the regime's victims of human rights violations continues to be a highly current and controversial topic in contemporary Spain. The exhumation of the former dictator from the mausoleum 'Valley of the Fallen', carried out by state authorities in October 2019, was praised as a long overdue step by relatives of victims, while right-wing conservative circles criticized it as an unnecessary resurgence of old supposedly overcome conflicts. However, what does the broader Spanish population think about repairing victims of Francoism? An open question in the research field of transitional justice remains the opinion of the public towards state- sponsored victim reparations including different measures ranging from material compensation payments and recognition of individual pension rights to symbolic and collective measures such as renaming of streets or the establishment of public memorial sites and places of remembrance. Using a qualitative bottom-up research approach applying a combination of online-survey and focus group methodology various forms of reparations are identified, which seem to meet with positive feedback from both the victims and the general public. Based on theoretical considerations of the academic literature on transitional justice such publicly supported reparations measures could possibly contribute to reconcile divided societies resurging from a conflicting past.[spa] Cuarenta y cinco años después de la muerte del dictador español Francisco Franco, abordar los legados del pasado represivo del país y atender las necesidades de las víctimas de violaciones de los derechos humanos del régimen sigue siendo un tema muy actual y controvertido en la España contemporánea. La exhumación del ex dictador del mausoleo "Valle de los Caídos", llevada a cabo por las autoridades estatales en octubre de 2019, fue elogiada como un paso largamente esperado por los familiares de las víctimas, mientras que los círculos conservadores de derecha la criticaron como un resurgimiento innecesario de viejos conflictos supuestamente superados. Sin embargo, ¿qué piensa la población española en general sobre la reparación de las víctimas del franquismo? Una cuestión abierta en el campo de la investigación de la justicia de transición sigue siendo la opinión del público sobre las reparaciones de las víctimas patrocinadas por el Estado, que incluyen diferentes medidas que van desde el pago de compensaciones materiales y el reconocimiento de los derechos individuales de pensión hasta medidas simbólicas y colectivas como el cambio de nombre de las calles o el establecimiento de lugares de conmemoración pública y lugares de recuerdo. Utilizando un enfoque cualitativo de investigación ascendente que aplica una combinación de encuestas en línea y la metodología de grupos de discusión, se identifican diversas formas de reparación que parecen recibir una respuesta positiva tanto de las víctimas como del público en general. Sobre la base de consideraciones teóricas de la literatura académica sobre la justicia de transición, esas medidas de reparación apoyadas públicamente podrían contribuir a reconciliar a las sociedades divididas que resurgen de un pasado conflictivo.[cat] Quaranta-cinc anys després de la mort del dictador espanyol Francisco Franco, abordar els llegats del passat repressiu del país i abordar les necessitats de les víctimes de violacions dels drets humans del règim continua sent una qüestió molt actual i controvertida a l'Espanya contemporània. L'exhumació de l'exdictador del mausoleu "Vall dels Caiguts", duta a terme per les autoritats estatals a l'octubre de 2019, va ser elogiada com un pas esperat pels familiars de les víctimes, mentre que els cercles conservadors de dretes la van criticar com un ressorgiment innecessari dels vells conflictes suposadament superats. Però, què pensa la població espanyola en general sobre la reparació de les víctimes del franquisme? Una qüestió oberta en l'àmbit de la investigació de la justícia transicional segueix sent l'opinió pública sobre les reparacions de les víctimes patrocinades per l'Estat, incloent diverses mesures que van des del pagament de la indemnització material i el reconeixement dels drets individuals de pensions a mesures simbòliques i col·lectives, com canviar els noms dels carrers o establir memorials públics i llocs de record. Mitjançant un enfocament qualitatiu de recerca a l'alça que aplica una combinació d'enquestes en línia i metodologia de grup de discussió, s'identifiquen diverses formes de reparació que semblen rebre una resposta positiva tant de les víctimes com del públic en general. Basant-se en consideracions teòriques en la literatura acadèmica sobre justícia transicional, aquestes mesures de reparació recolzades públicament podrien ajudar a conciliar societats dividides que ressorgeixen d'un passat conflicti