Transcription, Editing, and Switching of Antibody Genes

B lymphocytes are the antibody producing cells of the immune system. During B cell lymphopoiesis in the bone marrow, immunoglobulin molecules are assembled from V, D, and J gene segments through a process known as V(D)J recombination. Because of its stochastic nature, V(D)J recombination is expected to create self-reactive specificities. In targeted animals carrying autoantibodies, these specificities were shown to be silenced by deletion, anergy, and receptor editing. While anergy and deletion physically eliminate the autoreactive clone, receptor editing salvages it by replacing its immunoglobulin receptor through ongoing V(D)J recombination. Once a functional non-self -reactive receptor is generated, B lymphocytes migrate to the periphery. Upon antigen encounter, B cells undergo another recombination process, class switching, by replacing their heavy chain p constant region with downstream isotypes, such as y, a, or e. The isotype choice is dictated by a variety of mitogens and cytokines produced during the immune response. In the first part of this thesis, I describe experiments that elucidate the role of DNA double-stranded break repair in class switch recombination. In the second part, I determine how frequently B cell receptors are successfully replaced in vivo by editing. Finally, I provide evidence that secondary gene recombination, as well as allelic exclusion of light chain genes is under transcriptional regulation

RAGs and Regulation of Autoantibodies

Autoreactive antibodies are etiologic agents in a number of autoimmune diseases. Like all other antibodies these antibodies are produced in developing B cells by V(D)J recombination in the bone marrow. Three mechanisms regulate autoreactive B cells: deletion, receptor editing, and anergy. Here we review the prevalence of autoantibodies in the initial antibody repertoire, their regulation by receptor editing, and the role of the recombinase proteins (RAG l and RAG2) in this process

Turismo, tecnología y narrativas: reflexiones para el diseño de espacios turísticos

Caracteritzem el turisme essencialment com un fenomen comunicatiu entre una arquitectura, un territori, un espai i els seus visitants. El disseny d'espais turístics ha evolucionat al llarg del temps segons les tecnolgies emprades per construir-los. L'aparició de noves tecnologies permet noves interaccions amb el nostre entorn i una connexió constant amb fonts d'informació, a més de geoposició i realitat augmentada. Es tracta d'unes tecnologies que estan reconfigurant l'espai turístic i, per tant, obrint les possibilitats. Per donar-nos noves capacitats d'experiència i significat per al coneixement, la interpretació, el descobriment i la relació amb l'entorn. Aquestes eines permeten dissenyar espais inèdits (suma de lloc i comunicació) d'experiències culturals (i de lleure) articulant-se a l'entorn de noves narratives, la qual cosa permet posar en valor patrimonis emergents i obrir-se a un camp que cal explorar i construir.We characterize tourism essentially as a phenomenon of communication between architecture, a territory, a space and its visitors. The design of tourist spaces has evolved over time, according to the technologies used to build them. The emergence of new technologies enables new interactions with our environment and a constant connection with sources of information, in addition to geo-location and augmented reality. These are technologies that are re-configuring the tourist space and, therefore, opening up possibilities. This gives us new capacities for experience and meening for knowledge, interpretation, discovery and the relationship with the environment. These tools allow the design of hitherto unknown spaces (the combination of location and communication) of cultural experiences (and leisure) which are articulated around new narratives, placing value on emerging assets and opening up a field that must be explored and constricted.Caracterizamos el turismo esencialmente como una fenómeno comunicativo entre una arquitectura, un territorio, un espacio y sus visitantes. El diseño de espacios turísticos ha evolucionado a lo largo del tiempo según las tecnologías empleadas para construirlos. La aparición de tecnologías nuevas permite interacciones nuevas con nuestro entorno y una conexión constante con fuentes de información, además de geoposición y realidad aumentada. Se trata de unas tecnologías que están volviendo a configurar el espacio turístico y, por tanto, abriendo las posibilidades. Para darnos nuevas capacidades de experiencia y significado para el conocimiento, la interpretación, el descubrimiento y la relación con el entorno. Estas herramientas permiten diseñar espacios inéditos (suma de lugar y comunicación) de experiencias culturales (y de ocio) que se articulan en torno a narrativas nuevas, lo que permite poner en valor patrimonios emergentes y abrirse a un campo que se debe explorar y construir

Multidisciplinary models for pregnancy care in patients with rheumatic diseases: Clinical experiences and experts opinion

Pregnancy care; Rheumatic diseasesCuidado del embarazo; Enfermedades reumáticasCura de l'embaràs; Malalties reumàtiquesObjectives: To describe different models of multidisciplinary pregnancy care for patients with inflammatory and autoimmune rheumatic diseases, and the steps to follow concerning their implementation. Methods: A qualitative study was conducted including: (1) a comprehensive literature search in PUBMED focused on multidisciplinary care models; (2) structured interviews with seven rheumatologists from multidisciplinary pregnancy clinics for patients with inflammatory and autoimmune rheumatic diseases. Data were collected related to the hospitals, medical departments, populations cared for, and multidisciplinary care models (type, material, and human resources, professional requirements, objectives, referral criteria, agendas, protocols, responsibilities, decision-making, research and educational activities, multidisciplinary clinical sessions, initiation/start, planning, advantages/disadvantages, and barriers/facilitators for implementation); (3) a nominal meeting group in which the results of searches and interviews were analyzed and the recommendations for the implementation of the multidisciplinary care models defined. Results: We analyzed seven models of multidisciplinary care in pregnancy, implemented 3-10 years ago, which can all be summarized by two different subtypes: parallel (patients are assessed the same day in the involved medical services) and preferential (patients are assessed on different days in the involved medical services) circuits. The implementation of a specific model results rather from an adaptation to the hospital’s and professionals’ circumstances. Correct planning and good harmony among professionals are key points to implementing a model. Conclusion: Different multidisciplinary care models have been implemented for patients with inflammatory and autoimmune rheumatic diseases during pregnancy. They pretend to improve care, system efficiency, and collaboration among specialists and should be carefully implemented.The project was supported by UCB Pharma

Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization

Diabetic retinopathy; Endostatin; Intravitreal vesselsRetinopatia diabètica; Endostatina; Vasos intravítresRetinopatía diabética; Endostatina; Vasos intravítreosEndostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels.This work was supported by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain (grant number PI16/00719); the Fundação para a Ciência e a Tecnologia, Ministerio da Educação e Ciência, Portugal (grant numbers SFRH/BD/95330/2013 and SFRH/BPD/102573/2014); and Fondo Europeo de Desarrollo Regional (FEDER)

Igκ allelic inclusion is a consequence of receptor editing

The discovery of lymphocytes bearing two light chains in mice carrying self-reactive antibody transgenes has challenged the “one lymphocyte–one antibody” rule. However, the extent and nature of allelically included cells in normal mice is unknown. We show that 10% of mature B cells coexpress both Igκ alleles. These cells are not the result of failure in allelic exclusion per se, but arise through receptor editing. We find that under physiological conditions, editing occurs both by deletion and by inclusion with equal probability. In addition, we demonstrate that B lymphocytes carrying two B-cell receptors are recruited to germinal center reactions, and thus fully participate in humoral immune responses. Our data measure the scope of allelic inclusion and provide a mechanism whereby autoreactive B cells might “escape” central tolerance

Alterations in the Chromatin Environment Following the Introduction of DNA Breaks

The presence of DNA breaks has extensive biochemical implications for the integrity of the genome. It is well established that distinct DNA damage response proteins are recruited to, and accumulate at, sites of genomic lesions, including kinases that initiate multiple DNA damage signaling cascades. The repair of DNA breaks is facilitated by the phosphorylation of H2AX, which organizes DNA damage response factors in the vicinity of the lesion. Metabolism of the DNA breaks occurs in a chromatin environment and modulating chromatin structure is necessary for the fidelity of the DNA damage response. We set out to determine in living cells both how chromatin is remodeled in the presence of DNA breaks and whether the establishment of large sub-cellular DNA damage response domains influences other DNA metabolic processes, such as transcription. Using a photoactivatable histone H2B, we examined the mobility and structure of chromatin immediately after the introduction of DNA breaks. We find that chromatin-containing damaged DNA exhibits limited mobility but undergoes an initial energy-dependent local expansion that occurs independently of H2AX and ATM. We also took advantage of the large copy number, tandem gene arrangement, and spatial organization of ribosomal transcription units as a model system to measure the kinetics of transcription in real time in the presence of DNA breaks. We find that RNA polI inhibition is not the direct result of the physical DNA break but mediated by ATM kinase activity and surrogate DNA repair proteins. We propose that the localized opening of chromatin at DNA breaks establishes an accessible biochemically unique sub-nuclear environment that facilitates DNA damage signaling and repair

Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization

Research Areas: OphthalmologyABSTRACT - Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels.info:eu-repo/semantics/publishedVersio

c-Myc Is a Universal Amplifier of Expressed Genes in Lymphocytes and Embryonic Stem Cells

SummaryThe c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and differentiation at the cellular, tissue, or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Myc’s targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature