RESUMEN
Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Supervivencia Celular/fisiología , Células Cultivadas , Clonación Molecular , Femenino , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Proteínas/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Alport syndrome (AS) is a genetic disease characterized by haematuric glomerulopathy variably associated with hearing loss and anterior lenticonus. It is caused by mutations in the COL4A3, COL4A4 or COL4A5 genes encoding the α3α4α5(IV) collagen heterotrimer. AS is rare, but it accounts for >1% of patients receiving renal replacement therapy. Angiotensin-converting enzyme inhibition slows, but does not stop, the progression to renal failure; therefore, there is an urgent requirement to expand and intensify research towards discovering new therapeutic targets and new therapies. The 2015 International Workshop on Alport Syndrome targeted unmet needs in basic science, genetics and diagnosis, clinical research and current clinical care. In three intensive days, more than 100 international experts including physicians, geneticists, researchers from academia and industry, and patient representatives from all over the world participated in panel discussions and breakout groups. This report summarizes the most important priority areas including (i) understanding the crucial role of podocyte protection and regeneration, (ii) targeting mutations by new molecular techniques for new animal models and potential gene therapy, (iii) creating optimal interaction between nephrologists and geneticists for early diagnosis, (iv) establishing standards for mutation screening and databases, (v) improving widespread accessibility to current standards of clinical care, (vi) improving collaboration with the pharmaceutical/biotech industry to investigate new therapies, (vii) research in hearing loss as a huge unmet need in Alport patients and (viii) the need to evaluate the risk and benefit of novel (including 'repurposing') therapies on an international basis.
Asunto(s)
Nefritis Hereditaria/genética , Animales , Colágeno Tipo IV/genética , Terapia Genética , Humanos , Mutación , Evaluación de Necesidades , Nefritis Hereditaria/terapia , Podocitos , Mejoramiento de la CalidadRESUMEN
COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.
Asunto(s)
COVID-19 , Animales , Humanos , SARS-CoV-2 , Factor D del Complemento , Células Endoteliales , HaplorrinosRESUMEN
IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein. Removal of cellular kappaB-Ras enhances whereas excess kappaB-Ras blocks induced IkappaBbeta degradation. Remarkably, kappaB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of kappaB-Ras abrogate its ability to block degradation of IkappaBbeta. kappaB-Ras also directly blocks the in vitro phosphorylation of IkappaBbeta by IKKbeta. These observations suggest that IkappaBbeta in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IkappaBbeta.
Asunto(s)
Proteínas I-kappa B/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas ras/metabolismo , Animales , Línea Celular , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Quinasa I-kappa B , Sustancias Macromoleculares , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Proteínas ras/genéticaRESUMEN
Currently, products containing interferon beta (IFNß) are injected either intramuscularly or subcutaneously. To avoid the necessity of injection, we developed a novel monomeric Fc fusion protein of IFNß (IFNßFc) that is absorbed via an immunoglobulin transport system present in the upper and central airways upon administration of the drug as an inhaled aerosol. The systemic absorption of IFNßFc through the lung in non-human primates, at deposited doses of 1, 3, and 10 µg/kg, was compared to the absorption of a single 3 µg/kg dose of IFNß-1a (Avonex®) subcutaneously administered. IFNßFc was well absorbed through the lung, displaying dose proportional increases in serum concentrations, and was biologically active, as shown by increases in plasma neopterin levels. The circulating half-life of IFNßFc was â¼3 times longer (â¼30 h) than that of IFNß-1a, (8-9 h). At approximately equimolar doses of IFNßFc (10 µg/kg) and IFNß-1a (3 µg/kg), the stimulation of neopterin over background levels was approximately equivalent, demonstrating that the longer half-life of IFNßFc compensated for the lower relative specific antiviral activity of IFNßFc measured in vitro. In conclusion, IFNßFc was efficiently absorbed after pulmonary delivery in non-human primates, retained its biological activity, and may offer a convenient alternative to injectable IFNß.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulinas/metabolismo , Interferón beta/administración & dosificación , Interferón beta/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacología , Administración por Inhalación , Animales , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Inyecciones Subcutáneas , Interferón beta-1a , Interferón beta/biosíntesis , Interferón beta/farmacocinética , Macaca fascicularis/sangre , Masculino , Neopterin/sangre , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.
Asunto(s)
Polaridad Celular/fisiología , Citocinas/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/genética , Polaridad Celular/efectos de los fármacos , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Citocinas/farmacología , Tolerancia a Medicamentos/genética , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1/metabolismo , Interleucina-1/farmacología , Interleucina-8/genética , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Microvellosidades/efectos de los fármacos , Microvellosidades/enzimología , Microvellosidades/genética , FN-kappa B/genética , Proteína Quinasa C-delta , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Pretreatment of HT29-D4 epithelial adenocarcinoma colic cells with des-IGF-1 upregulated TNF alpha-mediated activation of IL-8 expression at different levels (protein, mRNA, and hnRNA). RNA transcription but not RNA stabilization was found to be involved. In this cell line, cooperation of NF-kappa B with other factors appeared essential for IL-8 expression. Indeed, TNF alpha-induced NF-kappa B translocation was not sufficient to support enhancement of the transcription and des-IGF-1 did not promote but partly inhibited both the TNF alpha-induced NF-kappa B activation and I kappa B alpha degradation through a PI-3K-dependent pathway. A CCAAT/enhancer binding protein (C/EBP) site located on the IL-8 gene enhancer cooperated with a NF-kappa B binding site and led to the upregulation of IL-8 expression. Binding of C/EBP alpha to this sequence disappeared in IGF-1 treated cells. This event may be important for the cross-talk between IGF-1- and TNF alpha-mediated pathways leading to the control of inflammatory processes and the decision concerning apoptosis or cell survival.