Early diagnostic of B cell chronic leukemia by novel biomarkers

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Functional proteomics approaches for high throuput determination of CKIT and small molecules

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Translational biomarker: identification of biomarkers related to capecitabine response in solid tumors by nucleic acids programmable protein microarrays (NAPPA), antibody arrays, IFISH and SNPS approaches

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Longevity of the adaptive immune cell compartment in the human small intestine

The intestinal immune system must rapidly respond to pathogens while maintaining the tolerance against commensals and dietary antigens. To accomplish this task, the gut contains the largest number of adaptive immune cells in the body, including antibody-producing plasma cells (PCs) and antigen-experienced T cells. The focus of this thesis is to study the homeostatic turnover, phenotype and function of different populations of memory T cells, including CD8+ and CD4+ resident memory T cells (Trm), regulatory T cells (Treg) as well as PCs in the human small intestine. During the last decade, it has been shown that T cell immunosurveillance in peripheral tissues is accomplished for the most part by resident populations (Trm cells) that do not recirculate. In the first study of this thesis we evaluated the turnover of CD8+ T cell subsets in transplanted small intestine and we found that the majority of the CD103+ CD8+ T cells persisted in the graft for one year after transplantation, suggesting that they constitute bona fide Trm cells. In addition, they displayed a TCR repertoire dominated by expanded clones that were conserved over time. In contrast, CD103- CD8+ T cells presented different phenotype, functional capabilities and immune repertoire compared to CD103+ CD8+ T cells. Moreover, CD103- CD8+ T cells were rapidly exchanged after one year, indicating that they constitute recently recruited cells. In the second study we demonstrated that intestinal CD4+ T cell compartment is mainly composed by CD103- and CD103+ resident populations that survive for at least one year in transplanted duodenum and present a polyfunctional Th1 profile. Furthermore, in the third study we revealed that the human small intestine contains two functionally distinct populations of CD4+ Treg cells: a resident subset of Helios- Treg cells and a subset of Helios+ Treg cells that is dynamically exchanged. Finally, we demonstrated that most of the intestinal PCs constitute long-lived cells with a lifespan of several decades and these cells are enriched for specificities against previous infections. Taken together, these results show that the human small intestine harbors very persistent PCs and T cells, which has profound implications for the development of mucosal vaccines and new immunotherapies

Ensayos : revista de la Escuela Universitaria de Formación del Profesorado de Albacete

Resumen basado en el de la publicaciónLa conquista de la autonomía adolescente puede llevar consigo la ruptura familiar, pero también es posible alcanzar la autonomía sin que se produzca el conflicto intergeneracional. La autonomía y la continuación de relaciones estrechas con los padres no son excluyentes. Frente al mito del conflicto intergeneracional, la literatura científica confirma las buenas relaciones entre los adolescentes y sus padres. Se ha comprobado que el estilo educativo democrático de los padres, donde hay un equilibrio entre control y autonomía, muestra su eficacia en el desarrollo óptimo del adolescente y en la menor probabilidad de comportamientos problemáticos. Por ello, se estudian las variables del contexto familiar que actúan como factores de riesgo o de protección ante las conductas problemáticas de los adolescentes y que cobran una gran relevancia a la hora de plantear programas de prevención en este campo. No obstante, la falta de resultados consistentes hace pensar que son necesarios más estudios longitudinales al respecto.ES

Functional Proteomics: Beads-based array system for Biomarkers Discovery

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High-throughgput phage-display screening in array format

et al.Emerging technologies for the design and generation of human antibodies require improved approaches enabling their screening, characterization and validation. Currently, strategies based on ELISA or western blot are used to that aim. However, the ever increasing number of novel antibodies generated would benefit from the development of new high-throughput (HT) platforms facilitating rapid antibody identification and characterization. Herein, we describe a protein chip bearing recombinant phage particles and based on a large phage antibody library. In this paper we have set forth a novel implementation which provides a powerful and simple methodology enabling the identification of single-chain variable fragments (scFv). As a proof-of-principle of this method, we tested it with recombinant antigen (human recombinant interleukin 8). Additionally, we developed a novel bioinformatics tool that serves to compare this novel strategy with traditional methods. The method described here, together with associated informatics tools, is robust, relatively fast and represents a step-forward in protocols including phage library screenings.We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI11/02114)-Fondos FEDER and Junta Castilla-León SA198A12-2. The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. Paula Díez and Noelia Dasilva are supported by a JCYLEDU/346/2013 Ph.D., scholarship. María González-González and Raquel Bartolomé are supported by ISCIII FIS08/00721 Ph.D., scholarship and MEC 2009 scholarship, respectively. J. Casado-Vela is a JAE-DOC (CSIC) holder supported by Ministerio de Economía y Competitividad, Spain, co-funded by the European Social Fund (FEDER).Peer Reviewe

Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays

et al.Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.This work was supported by grants FIS PI081884 and PI1102114 from the Instituto de Salud Carlos III (ISCIII; Ministerio de Ciencia e Innovación, Madrid, Spain) and grant JCYL-SA198A12-2 from the Consejería de Educación de la Junta de Castilla y León (Spain). María González-González is supported by a PhD scholarship from the ISCIII (FI08/00721), Raquel Bartolome is supported by a PhD scholarship from the Ministerio de Educación (FPU, AP2009-2633), and Noelia Dasilva is supported by a PhD scholarship from JCyL (Orden EDU 346/2013). Juan Casado-Vela is a JAE–DOC (CSIC) holder supported by the Ministerio de Economía y Competitividad (Spain), cofunded by the European Social Fund.Peer Reviewe

Immune microniches shape intestinal Treg function.

Acknowledgements: The authors thank the Kennedy Institute of Rheumatology (KIR) Flow Cytometry Facility and the manager, J. Webber, for help with flow cytometry and FACS; the KIR Biomedical Services Unit, especially L. Barker for help with animal care and husbandry; the KIR microscopy facility and manager C. Lagerholm; I. Parisi, B. Stott and R. Cook for tissue processing and staining; A. Lee and M. Attar (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the generation and initial processing of sequencing data; S. van Dongen and P. V. Mazin and the Teichmann laboratory for discussion and support with scripts. We acknowledge the generous support of the Kennedy Trust for Rheumatology Research, IDRM and Carl Zeiss GMBH for the microscopy facilities (Zeiss 980) used in this research. We acknowledge the generous support of the Kennedy Trust for Rheumatology Research and a Wellcome Trust Multi-User Equipment Grant 202911/Z/16/Z for the microscope purchase (Zeiss 880 multiphoton) and facilities used in this research. Experimental design and summary diagrams were created with BioRender.com. Y.G. was funded by a Wellcome Trust Clinical Research Fellowship (CRTF), grant reference 201224/Z/16/Z. RB-C Grant 315307, Forskerprosjekt 2020, Researcher Project/International Mobility Grant from the Research Council of Norway and travel grant from the Per Brandtzæg’s Fund for Research in Mucosal Immunology. E.E.T. was supported by Wellcome Trust (095688/Z/11/Z and 212240/Z/18/Z, awarded to F.P.), Nuffield Department of Medicine, and MRC core grant reference MC_UU_00008. F.P. was supported by Wellcome Trust (095688/Z/11/Z and 212240/Z/18/Z). This research was funded in whole, or in part, by the Wellcome Trust 212240/Z/18/Z. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identified3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103+SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies