Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4

Reporte de Mercados Financieros - primer trimestre de 2019

El Banco de la República (BR) genera información para la toma de decisiones, la rendición de cuentas y la difusión al público. En particular, el Reporte de Mercados Financieros está enmarcado dentro del principio de difusión al público y contribuye a cumplir con el servicio que presta el Banco de ofrecer información e investigación económica de calidad

Reporte de Mercados Financieros - cuarto trimestre de 2018

El Banco de la República (BR) genera información para la toma de decisiones, la rendición de cuentas y la difusión al público. En particular, el Reporte de Mercados Financieros está enmarcado dentro del principio de difusión al público y contribuye a cumplir con el servicio que presta el Banco de ofrecer información e investigación económica de calidad

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Biological and molecular characterization of lifeguard : an inhibitor of the Fas apoptotic pathway

Apoptosis or programmed cell death participates in several biological processes, including embryonic development, tissue remodeling, tumor surveillance and immune system regulation. The Fas pathway is among the most studied apoptotic pathways. Its misregulation can contribute to many diseases including autoimmunity and cancer. Lifeguard (LFG) is a membrane protein isolated as an inhibitor of the Fas apoptotic pathway. LFG has wide tissue distribution with highest expression in the brain, predominantly in neurons of hippocampus and cerebellum. In order to study the biological role of LFG, we used two different models; mice with reduced LFG expression by siRNA lentiviral transgenesis and LFG null mice. Phenotypic characterization of these mice indicated that loss of LFG affected several organs, such as lungs, kidneys, spleen, thymus and brain. The work presented here reports the investigation of LFG's role in the CNS (1), in the immune system (2) as well as the molecular and biochemical characterization of LFG (3). 1. We focused in the study of LFG's biological role in the brain, especially in cerebellum, where the phenotype was most severe. LFG affected cerebellar size in early development, internal granular layer size, and Purkinje cell differentiation, morphology and susceptibility to apoptosis. In the last decade there has been increasing evidence for an involvement of the Fas apoptotic pathway in cerebral ischemia. Therefore, we have started to test the hypothesis that LFG could have a neuroprotective role against stroke, by inhibiting Fas induced apoptosis. 2. We also investigated the role of LFG in the immune system, especially in T cell development. Based on LFG's expression pattern and on the phenotypical characterization of the T cell subpopulations in the thymus of siLFG mice we hypothesized that LFG is a survival factor whose expression is upregulated after the major selection checkpoints in T cell development. 3. We demonstrated that LFG blocks the Fas apoptotic cascade at the level of caspase 8 activation. We also showed that LFG is ubiquitinated and it interacts with the E3 Ubiquitin ligase AIP4. Finally, deletion mutant studies revealed that the last intracellular loop and transmembrane domain of LFG are essential for its antiapoptotic functio

Antiapoptotic Role for Lifeguard in T Cell Mediated Immune Response

<div><p>Anti-apoptotic protein Lifeguard (LFG) is upregulated on T cells upon <i>in vitro</i> activation. To investigate its role in T cell immunity we infected wild type and LFG knockout bone marrow chimaeras mice with LCMV. We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells. WT and KO T cells proliferated at the same rate, however, LFG KO CD44^hi T cells showed increased cell death during the initial phase of the immune response. LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in <i>ex vivo</i> cultures, and blocking extrinsic pathways of cell death <i>in vivo</i> with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells. Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.</p></div

Apoptosis induced by Jo2.

<p>This table represents the percentage of AnnexinV⁺ WT and KO CD44^hiCD8 and CD44^hiCD4 cells after culturing the splenocytes of naïve (day 0) or day 2 p.i. chimaeric mice in the presence of either IgG control or the Fas agonist Jo2 for 16 hours. (n = 4 mice per timepoint, ± = SEM).</p><p>*p ≤ 0.05 between IgG and Jo2 conditions</p><p>**p ≤ 0.05 between WT and KO under the IgG or Jo2 condition</p><p>Apoptosis induced by Jo2.</p

Decreased immune response of LFG KO T cells after LCMV infection.

<p>(A) The graphs represent the percentage of WT (diamonds) and KO (squares) CD44^hiCD8 T cells (top panel) or CD44^hiCD4 T cells (bottom panel) over the time course of the immune response to LCMV infection. We evaluated the pre-infection phase (day 0), expansion phase (days 6 and 8), contraction phase (day 15) and memory phase (day 35). (B) Decreased LFGKO antigen specific CD8 response to LCMV infection. The graph represent the combined absolute number of WT (diamonds) and KO (squares) CD8 T cells that are specific to NP396 and GP33 over the time course of the immune response to LCMV infection. Splenocytes from the infected chimera mice were stimulated with the two peptides for 6 hours in the presence of BFA and intracellularly stained for Interferon gamma. (n = 4 mice per timepoint, *p ≤ 0.05, **p ≤ 0.01, ± = SEM).</p

LFG is expressed in T cells and upregulated upon TCR stimulation.

<p>(A) Histograms showing LFG staining in blue compared to IgG control in red. The left panel is gated on CD8 T cells and the right panel on CD4 T cells. (B) FACS plots representing LFG and CD44 expression before (red) or after TCR stimulation by adding anti-CD3/CD28 (blue) in CD8 (left panel) and CD4 (right panel) T cells. The CD44^hi/lo gate was created according to the unstimulated condition. The bottom panel represents the values of LFG mean fluorescence intensity for CD8 or CD4 T cells in the no stimulation versus the CD3/CD28 condition. (C) Histograms representing LFG expression in CD4 (blue), CD8 (green) and non-T cells (red) before and after CD3/CD28 stimulation. (n = 4 mice, *p ≤ 0.05, **p ≤ 0.01, ± = SEM).</p

Phosphorylation of p53 by IκB kinase 2 promotes its degradation by β-TrCP

Functional inactivation of p53 and constitutive activation of the NF-κB pathway has been associated with several human cancers. In this study, we show that IκB kinase 2 (IKK2/IKKβ), which is critical for NF-κB activation, also phosphorylates p53. Phosphorylation of p53 at serines 362 and 366 by IKK2 leads to its recruitment to and ubiquitination by β-TrCP1. Degradation of ubiquitinated p53 is independent of Mdm2, because it occurs in both wild-type and Mdm2−/− cells. SiRNA-mediated reduction in the levels of β-TrCP1 and other members of the SCFβ−TrCP1E3 ubiquitin ligase complex or overexpression of a dominant negative form of β-TrCP1 enhances p53 stability. Substitutions at Ser-362 and 366 of p53 by alanines (p53 AA) result in reduced phosphorylation of p53 by IKK2, decreased association with β-TrCP1, and thus increased stability of p53 and expression of p53 target genes such as p21, altering the G1 phase of the cell cycle. Our results identify IKK2 and β-TrCP1 as novel regulators of the p53 pathway and suggest that blocking of IKK2 and β-TrCP1 could be a means of regulating p53 stability and thereby modulating its biological activity