Realive (2016): from cellular to human cryopreservation. Myth and reality for teacher training in the area of health sciences

There is a growing thought of delaying the progression of a terminal illness by cryopreservation of the bodies of patients. Faced with the fear of dying from diseases, they are confident that in the future they will be able to be revived and treated with novel therapies that will lead to the cure and elimination of the pathology. Although it seems science fiction, the reality is that it is already being carried out with cellular organisms for the conservation of species and against diseases that alter reproduction. However, when it comes to embryos, the ethical committees do not yet have a defined strategy. For this reason, this work tries to focus on an interrelation between teachers and students for the discussion and reflection of the cryopreservation of patients, showing the latest advances and reports on this aspect.Existe un creciente pensamiento de postergar el avance de una enfermedad terminal mediante la criopreservación de los cuerpos de los pacientes. Ante el miedo a morir de enfermedades se confían que en el futuro podrán ser reanimados y tratados con novedosas terapias que le supondrán la cura y eliminación de la patología. Aunque parezca ciencia ficción la realidad es que ya se está realizando con organismos celulares para la conservación de especies y ante enfermedades que alteran la reproducción. Sin embargo, cuando se trata de embriones los comités éticos no tienen todavía una estrategia definida. Por esa razón, este trabajo intenta enfocar una interrelación entre profesorado y alumnado para la discusión y reflexión de la criopreservación de pacientes, mostrando los últimos avances e informes sobre este aspecto

Increased Protein Stability and Interleukin-2 Production of a LAT(G131D)Variant With Possible Implications for T Cell Anergy

The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-gamma activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LAT(G131D)) increases phosphorylation of Tyr132, PLC-gamma activation and calcium influx generation. Interestingly, the LAT(G131D)mutant has a slower protein turnover while being equally sensitive to Fas-mediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LAT(G131D)secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LAT(G131D)mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance

A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.This research was funded by Consejeria de Salud de Andalucia, Junta de Andalucia (grant PI-0055-2017 to E.A.), and Fundacion Biomedica Cadiz Proyectos INIBICA 2019 (grant LI19/I14NCO15 to E.A. and M.M.A.-E.)

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT-Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT-Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.Consejería de Salud de Andalucía, Junta de Andalucía (grants PI-0365-2013 and PI-0055-2017); Instituto de Salud Carlos III (grant PI16-00784 from the “Plan Estatal de I+D+I 2013–2016/FEDER”

Taller “Aproximaciones experimentales en terapia génica: Las células sanguíneas y su reprogramación”

BREVE RESEÑA DE LA MENTORÍA (principales contenidos que abordará): Como todo tejido, la sangre se compone de células y componentes extracelulares (su matriz extracelular). Estas dos fracciones tisulares vienen representadas por los elementos formes y un medio isotónico denominado plasma sanguíneo. Los elementos formes de la sangre son variados en tamaño, estructura y función, y se agrupan en las células sanguíneas, que son los glóbulos blancos o leucocitos, los glóbulos rojos (también llamados hematíes o eritrocitos), y los derivados celulares, que no son células estrictamente sino fragmentos celulares, están representados por las plaquetas. El primer taller consistirá en la determinación de la presencia de antígenos A, B y D en las membranas de los hematíes. Esto puede llevarse a cabo muy fácilmente si disponemos de los correspondientes anticuerpos antiA, antiB o antiD, respectivamente. La interacción de estos anticuerpos con sus correspondientes antígenos desencadena la aglutinación de hematíes que, al agregarse entre sí, se separan del plasma. Este fenómeno puede visualizarse fácilmente mezclando una gota de sangre y una gota del antisuero correspondiente sobre un portaobjetos. En función del resultado obtenido en las tres mezclas, el alumnado deberá deducir el grupo sanguíneo (A, B, O, AB; Rh+, Rh-). Emplearemos la herramienta de trabajo grupal “lluvia de ideas”, para facilitar el surgimiento de nuevas ideas que ayuden a abordar la prevención de la enfermedad hemolítica del recién nacido (eritroblastosis fetal). El segundo taller consistirá en la visualización de linfocitos por microscopía óptica. Conoceremos la morfología de estas células, las diferencias con otros tipos celulares. Contaje de células y visualización de células muertas. Realizaremos una charla acerca de cómo podemos realizar modificaciones génicas a las células con el fin de curar enfermedades. El tercer taller consistirá en el uso de proteínas fluorescentes para la detección de genes que se han introducido en las células (Linfocitos). Los estudiantes conocerán cómo se pueden detectar estas proteínas usando la técnica de Citometría de flujo. El objetivo del primer taller tiene como finalidad el manejo y el aprendizaje reflexivo del funcionamiento de los distintos tipos de anticuerpos del sistema ABO y Rh sobre la superficie de los eritrocitos. El objetivo de los dos últimos talleres es que los estudiantes conozcan las técnicas que se utilizan actualmente para realizar terapia génica. -TEMPORALIZACIÓN (Mínimo 3 sesiones en horario de tarde): La realización de este taller comprenderá de tres sesiones de laboratorio con una duración aproximada de 2 horas por sesión, donde el primer objetivo será abordado el primer día, el segundo objetivo el segundo día, finalizando con el tercer objetivo en la última sesión.TALLER DE MENTORIA PARA EL ALUMNADO DE ALTAS CAPACIDADES INTELECTUALES: Aproximaciones experimentales en terapia génica: Las células sanguíneas y su reprogramación.Elaboración y creacción de nuevo material docente para impartir docencia a alumnado de altas capacidades en la Universidad de CádizÁrea de Fisiología y Área de Inmunología de la Facultad de Medicina de la Universidad de Cádiz.Este taller está orientado para ser llevado a cabo con alumnos denominados "de altas capacidades". Incluye material innovador desarollado por profesores investigadores que desarrollan su trabajo en la Universidad de Cádiz. La mentoría está formado por tres sesiones prácticas de una duración de dos horas cada uno. Todos los talleres contienen de su propio guión, elaborado con materiales de uso libre y/o creado por los autores de esta mentoría a raíz de su actividad docente y resultados investigadores. La práctica 1 se denomina GRUPOS SANGUÍNEOS. El guión consta de 8 páginas, con detalles de introducción, historia de los grupos sanguíneos, explicaciones, ejercicios prácticos de determinación de grupos sanguíneos en laboratorio y ejercicios de razonamiento y debate, metodología de aprendizaje y marcadores de realidad aumentada para proyectar figuras en tres dimensiones con el uso de una webcam de un ordenador portátil. Adjunto un video creado por el autor para la explicación visual de la metodología de la práctica a través de un kit de sangre simulada para el estudio de los grupos sanguíneos en los laboratorios de los Servicios Centrales de la Universidad de Cádiz. El objetivo de esta práctica es seguir el procedimiento utilizado por el personal médico en bancos de sangre y laboratorios médicos para la determinación de la sangre en transfusiones y como evidencias forenses en un procedimiento penal o en disputas de paternidad. La práctica 2 se denomina MICROSCOPÍA DE LINFOCITOS. El guión consta de 5 páginas, con detalles de introducción, explicaciones teóricas y un ejercicio de visualización de linfocitos y cuantificación clásica en el laboratorio de cultivo celular. Sus objetivos son: 1. Visualizar células por microscopía óptica, células que crecen adheridas sobre un soporte sólido y células que crecen en suspensión. 2. Conocer cómo se puede estimar la viabilidad de una suspensión celular (colorantes vitales). 3. Saber calcular el número de células que hay en un volumen determinado conociendo su concentración celular. La práctica 3 se denomina TRANSDUCCIÓN DE CÉLULAS– PROTEÍNAS ETIQUETADORAS. El guión consta de 3 páginas, con detalles de introducción, explicaciones teóricas y un ejercicio práctico de visualización de cuantificación de linfocitos a través de Citometría de Flujo en los Servicios Centrales de la Universidad de Cádiz. Sus objetivos son: 1. Conocer los métodos que se usan actualmente para introducir genes en las células. 2. Familiarizarse con proteínas fluorescentes que sirven para la detección de células que expresan los genes que se han introducido. 3. Analizar células a las que se les han realizado modificaciones genéticas, por la técnica de citometría de flujo

Mutation of the glycine residue preceding the sixth tyrosine of the LAT adaptor severely alters T cell development and activation

International audienceThe LAT transmembrane adaptor is essential to transduce intracellular signals triggered by the TCR. Phosphorylation of its four C-terminal tyrosine residues (136, 175, 195, and 235 in mouse LAT) recruits several proteins resulting in the assembly of the LAT signalosome. Among those tyrosine residues, the one found at position 136 of mouse LAT plays a critical role for T cell development and activation. The kinetics of phosphorylation of this residue is delayed as compared to the three other C-terminal tyrosines due to a conserved glycine residue found at position 135. Mutation of this glycine into an aspartate residue (denoted LAT G135D ) increased TCR signaling and altered antigen recognition in human Jurkat T cells and ex vivo mouse T cells. Here, using a strain of LAT G135D knockin mice, we showed that the LAT G135D mutation modifies thymic development, causing an increase in the percentage of CD4+CD8+ double-positive cells, and a reduction in the percentage of CD4+ and CD8+ single-positive cells. Interestingly, the LAT G135D mutation alters thymic development even in a heterozygous state. In the periphery, the LAT G135D mutation reduces the percentage of CD8+ T cells and results in a small increment of γδ T cells. Remarkably, the LAT G135D mutation dramatically increases the percentage of central memory CD8+ T cells. Finally, analysis of the proliferation and activation of T lymphocytes shows increased responses of T cells from mutant mice. Altogether, our results reinforce the view that the residue preceding Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation

Ultrastructural Localization and Molecular Associations of HCV Capsid Protein in Jurkat T Cells

Hepatitis C virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. When expressed in CD4+ T lymphocytes, it can induce modifications in several essential cellular and biological networks. To shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed HCV-core subcellular localization and its associations with host proteins in Jurkat T cells. In order to investigate the intracellular localization of Hepatitis C virus core protein, we have used a lentiviral system to transduce Jurkat T cells and subsequently localize the protein using immunoelectron microscopy techniques. We found that in Jurkat T cells, Hepatitis C virus core protein mostly localizes in the nucleus and specifically in the nucleolus. In addition, we performed pull-down assays combined with Mass Spectrometry Analysis, to identify proteins that associate with Hepatitis C virus core in Jurkat T cells. We found proteins such as NOLC1, PP1γ, ILF3, and C1QBP implicated in localization and/or traffic to the nucleolus. HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4+ T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. Thus, in the current work, we show the ultrastructural localization of Hepatitis C virus core and the first profile of HCV core associated proteins in T cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of Hepatitis C virus core protein. Thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying HCV core mediated alterations that had been described in relevant biological processes in CD4+ T cells

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT–Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT–Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex