RESUMEN
Limited T cell persistence restrains chimeric antigen receptor (CAR)-T cell therapy in solid tumors. To improve persistence, T cells have been engineered to secrete proinflammatory cytokines, but other possible methods have been understudied. Runx3 has been considered a master regulator of T cell development, cytotoxic T lymphocyte differentiation, and tissue-resident memory T (Trm)-cell formation. A study using a transgenic mouse model revealed that overexpression of Runx3 promoted T cell persistence in solid tumors. Here, we generated CAR-T cells overexpressing Runx3 (Run-CAR-T cells) and found that Run-CAR-T cells had long-lasting antitumor activities and achieved better tumor control than conventional CAR-T cells. We observed that more Run-CAR-T cells circulated in the peripheral blood and accumulated in tumor tissue, indicating that Runx3 coexpression improved CAR-T cell persistence in vivo. Tumor-infiltrating Run-CAR-T cells showed less cell death with enhanced proliferative and effector activities. Consistently, in vitro studies indicated that AICD was also decreased in Run-CAR-T cells via downregulation of tumor necrosis factor (TNF) secretion. Further studies revealed that Runx3 could bind to the TNF promoter and suppress its gene transcription after T cell activation. In conclusion, Runx3-armored CAR-T cells showed increased antitumor activities and could be a new modality for the treatment of solid tumors.
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Neoplasias , Linfocitos T , Animales , Ratones , Neoplasias/genética , Neoplasias/terapia , Inmunoterapia Adoptiva/métodos , Citocinas/metabolismo , Muerte Celular/genética , Línea Celular Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Killer-cell immunoglobulin-like receptors (KIRs) are mainly expressed on natural killer (NK) cells and are key regulators of innate immune responses. NK cells are the first responders in the face of infection and help promote placentation during pregnancy; the importance of KIRs in these NK-mediated processes is well-established. However, mounting evidence suggests that KIRs also have a prominent and long-lasting effect on the adaptive immune system. Here, we review the evidence for the impact of KIRs on T cell responses with a focus on the clinical significance of this interaction.
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Linfocitos T , Virosis , Embarazo , Femenino , Humanos , Receptores KIR , Inmunidad Innata , Células Asesinas NaturalesRESUMEN
BACKGROUND: Leukocyte phytohemagglutinin (PHA-L), derived from the L4 tetramer of PHA, has been frequently employed as a mitogen to induce T lymphocyte proliferation in vitro. The biological application of PHA-L in cancer diagnosis and treatment has gained traction in recent years. However, it has been noted that PHA-L obtained using traditional procedures has a massive amount of impurities or toxic components, which interfere with the activity of PHA-L. Preparation of a monoclonal antibody against active PHA-L is a significant tool for studying PHA-L's function and therapeutic potential. RESULTS: We successfully prepared monoclonal antibodies against the active components of PHA-L based on the whole PHA-L protein as an antigen, and found that monoclonal antibody 3C1C6G11 can be employed in western blot, immunofluorescence, and immunohistochemistry detection. Importantly, preliminary result shows that the mAb 3C1C6G11 may prevent PHA-L-induced cell aggregation and AICD (activation-induced cell death). CONCLUSIONS: The monoclonal antibody mAb 3C1C6G11 prepared in this study can be used as an effective tool for detecting PHA-L active components, investigating PHA-L's function and antineoplastic application.
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Anticuerpos Monoclonales , Phaseolus , Fitohemaglutininas/farmacología , Activación de LinfocitosRESUMEN
Amyloid-ß precursor protein (APP) is sequentially cleaved by secretases and generates amyloid-ß, the major components in senile plaques in Alzheimer's disease. APP is upregulated in human Alzheimer's disease brains. However, the molecular mechanism of how APP contributes to Alzheimer's disease pathogenesis remains incompletely understood. Here we show that truncated APP C586-695 fragment generated by δ-secretase directly binds to CCAAT/enhancer-binding protein beta (CEBPB), an inflammatory transcription factor, and enhances its transcriptional activity, escalating Alzheimer's disease-related gene expression and pathogenesis. The APP C586-695 fragment, but not full-length APP, strongly associates with CEBPB and elicits its nuclear translocation and augments the transcriptional activities on APP itself, MAPT (microtubule-associated protein tau), δ-secretase and inflammatory cytokine mRNA expression, finally triggering Alzheimer's disease pathology and cognitive disorder in a viral overexpression mouse model. Blockade of δ-secretase cleavage of APP by mutating the cleavage sites reduces its stimulatory effect on CEBPB, alleviating amyloid pathology and cognitive dysfunctions. Clearance of APP C586-695 from 5xFAD mice by antibody administration mitigates Alzheimer's disease pathologies and restores cognitive functions. Thus, in addition to the sequestration of amyloid-ß, APP implicates in Alzheimer's disease pathology by activating CEBPB upon δ-secretase cleavage.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica/fisiología , Anciano , Animales , Cisteína Endopeptidasas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana EdadRESUMEN
Activation of self-reactive T cells is a major driver to autoimmunity and is suppressed by mechanisms of regulation. In a humanized model of autoimmune thyroiditis, we investigated the mechanism underlying break of tolerance. Here, we found that a human TCR specific for the self-antigen thyroid peroxidase (TPO) is positively selected in the thymus of RAG KO mice on both T effector (Teff) and T regulatory (Treg) CD4+Foxp3+ cells. In vivo Teff are present in all immune organs, whereas the TPO-specific Treg are present in all lymphoid organs with the exception of the thyroid-draining lymph nodes. We suggest that the presence of TPO in the thyroid draining lymph nodes induces the activation of Teff and the depletion of Treg via activation-induced cell death (AICD). Our findings provide insights on the failure of the mechanisms of immune tolerance, with potential implications in designing immunotherapeutic strategies.
RESUMEN
Recognising the need for a national approach for the recommended best practice for the follow-up of implanted cardiac rhythm devices to ensure patient safety, this document has been produced by the Cardiac Society of Australia and New Zealand (CSANZ). It draws on accepted practice standards and guidelines of international electrophysiology bodies. It lays out methodology, frequency, and content of follow-up, including remote monitoring; personnel, including physician, allied health, nursing and industry; paediatric and adult congenital heart patients; and special considerations including magnetic resonance imaging scanning, perioperative management, and hazard alerts.
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Desfibriladores Implantables , Marcapaso Artificial , Adulto , Australia , Niño , Electrónica , Estudios de Seguimiento , Humanos , Nueva ZelandaRESUMEN
OBJECTIVE: To study the protective effect of oleanolic acid liposomes (OA-Lips) on cisplatin-induced oligoasthenospermia (COAS) in mice. METHODS: Sixty ICR mice were randomly divided into a normal control, a COAS model control, a positive control and a low-, a medium- and a high-dose OA-Lips group. The animals in the low-, medium- and high-dose OA-Lips and positive control groups were given intragastrically OA-Lips solution at 25, 50, and 100 mg/kg/d and vitamin E at 50 mg/kg/d, respectively. On the 28th day, the mice in the COAS model control, positive control and OA-Lips groups were injected intraperitoneally with cisplatin solution at 10 mg/kg, while those in the normal control group with the same dose of normal saline. Three days after administration, all the mice were sacrificed and their testis tissues collected for detection of the semen parameters and observation of the testicular morphology. RESULTS: Both the percentage of motile sperm and sperm concentration were significantly increased in the high-dose OA-Lips group (P < 0.05). HE staining showed that OA-Lips remarkably improved the damaged testis tissue (P < 0.05) and protected the seminiferous tubules and interstitial cells. The percentage of progressively motile sperm (PMS) and the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF) of sperm were gradually increased in a dose-dependent manner in the OA-Lips groups. The serum T level was significantly higher (P < 0.05) in the OA-Lips-treated mice than in the COAS model controls while the percentage of morphologically abnormal sperm (MAS) markedly lower in the high-dose OA-Lips group than in the model control, positive control and low-dose OA-Lips groups (P < 0.05). CONCLUSION: OA-Lips can relieve oligoaspermia and improve the productive ability of mice.
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Cisplatino , Ácido Oleanólico , Animales , Masculino , Ratones , Cisplatino/toxicidad , Liposomas , Ratones Endogámicos ICR , Ácido Oleanólico/farmacología , Semen , Motilidad Espermática , EspermatozoidesRESUMEN
Lung cancer remains the leading cause of cancer-related deaths and despite extensive research, the survival rate of lung cancer patients remains significantly low. Recent data reveal that aberrant Kras signaling drives regulatory T cells (Tregs) present in lung tumor microenvironment to establish immune deregulation and immunosuppression but the exact pathogenic mechanism is still unknown. In this study, we investigate the role of oncogenic Kras in Treg-related immunosuppression and its involvement in tumor-associated metabolic reprogramming. Findings reveal Tregs to prompt GATA3/NOS2-related immunosuppression via STING inhibition which triggers a decline in CD4+ T infiltration, and a subsequent increase in lung metastatic burden. Enhanced Treg expression was also associated with low T/MDSC ratio through restriction of CD8+CD44+CD62L- T effector cells, contributing to a tumor-promoting status. Specifically, TIM3+/LAG3+ Tregs prompted Kras-related immunosuppressive chemoresistance and were associated with T cell dysfunction. This Treg-dependent immunosuppression correlated with CD8 T cell exhaustion phenotype and ILC2 augmentation in mice. Moreover, enhanced Treg expression promoted activation-induced cell death (AICD) of T lymphocytes and guided lymph node metastasis in vivo. Overall, these findings demonstrate the multifaceted roles of Tregs in sustaining lung immunosuppressive neoplasia through tumor microenvironment remodeling and provide new opportunities for effective metastasis inhibition, especially in chemoresistant tumors.
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Linfocitos T CD8-positivos/inmunología , Neoplasias Pulmonares/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Células A549 , Animales , Línea Celular Tumoral , Factor de Transcripción GATA3/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Inmunidad Innata/inmunología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Trasplante de Neoplasias , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T Reguladores/patología , Trasplante HeterólogoRESUMEN
CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Antígenos CD28/inmunología , Antígenos CD5/inmunología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos , Antígenos CD28/antagonistas & inhibidores , Antígenos CD28/genética , Antígenos CD5/antagonistas & inhibidores , Antígenos CD5/genética , Extractos Celulares/farmacología , Línea Celular Tumoral , Proliferación Celular , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Femenino , Regulación de la Expresión Génica , Interferón gamma/genética , Interferón gamma/inmunología , Activación de Linfocitos/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Noqueados , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
Proteolytic processing of the amyloid precursor protein (APP) releases the APP intracellular domain (AICD) from the membrane. Bound to the APP adaptor protein Fe65 and the lysine acetyltransferase (KAT) Tip60, AICD translocates to the nucleus. Here, the complex forms spherical condensates at sites of endogenous target genes, termed AFT spots (AICD-Fe65-Tip60). We show that loss of Tip60 KAT activity prevents autoacetylation, reduces binding of Fe65 and abolishes Fe65-mediated stabilization of Tip60. Autoacetylation is a prerequisite for AFT spot formation, with KAT-deficient Tip60 retained together with Fe65 in speckles. We identify lysine residues 204 and 701 of Fe65 as acetylation targets of Tip60. We do not detect acetylation of AICD. Mutation of Fe65 K204 and K701 to glutamine, mimicking acetylation-induced charge neutralization, increases the transcriptional activity of Fe65 whereas Tip60 inhibition reduces it. The lysine deacetylase (KDAC) class III Sirt1 deacetylates Fe65 and pharmacological modulation of Sirt1 activity regulates Fe65 transcriptional activity. A second acetylation/deacetylation cycle, conducted by CBP and class I/II KDACs at different lysine residues, regulates stability of Fe65. This is the first report describing a role for acetylation in the regulation of Fe65 transcriptional activity, with Tip60 being the only KAT tested that supports AFT spot formation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lisina Acetiltransferasas/metabolismo , Acetilación , Células Cultivadas , Humanos , Activación TranscripcionalRESUMEN
BACKGROUND: Cardiac pacing can be challenging after a Fontan operation, and limited data exist regarding strategies to plan these epimyocardial systems while minimizing the number of surgical procedures. METHODS: A retrospective review of all our 47 patients (mean age 18 years, standard deviation 9 years) with a Fontan palliation who received an epimyocardial cardiac implantable electronic device (CIED) between 2002 and 2020 with regard to the stability of the epimyocardial lead parameters and the incidence of system revisions. RESULTS: Over the last 18 years, 84 implantations or revisions of the epimyocardial CIED in 47 Fontan patients were performed. Mean age at time of the first implantation was 9.4 (range 0.28-29.3) years. Follow-up period ranges from 0.11 to 18.2 (mean 7.7, standard deviation 4.2) years. A total of 123 pacing leads were implanted of which 99 are still active. From 2010 triple lead cardiac resynchronization devices were used in 17 patients to better cope with lead problems. The initial pacing threshold of the leads inactivated during this study period proved significantly higher (mean 1.66 V) than in the "all leads" group (mean 1.27 V, p = .0005) or the group of the still active leads (mean 1.17 V, p = .00004). CONCLUSIONS: When implanted with a low pacing threshold, the bipolar epimyocardial electrodes show stable and good long-term results in young patients with a Fontan circulation. Resynchronization pacing systems and the prospective implantation of reserve leads may help to reduce the rate of resternotomies and provide a flexible concept to deal with lead failure.
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Estimulación Cardíaca Artificial/métodos , Electrodos Implantados , Procedimiento de Fontan , Marcapaso Artificial , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Estudios RetrospectivosRESUMEN
Mitochondrial dynamics and functionality are linked to the autophagic degradative pathway under several stress conditions. However, the interplay between mitochondria and autophagy upon cell death signalling remains unclear. The T-cell receptor pathway signals the so-called activation-induced cell death (AICD) essential for immune tolerance regulation. Here, we show that this apoptotic pathway requires the inhibition of macroautophagy. Protein kinase-A activation downstream of T-cell receptor signalling inhibits macroautophagy upon AICD induction. This leads to the accumulation of damaged mitochondria, which are fragmented, display remodelled cristae and release cytochrome c, thereby driving apoptosis. Autophagy-forced reactivation that clears the Parkin-decorated mitochondria is as effective in inhibiting apoptosis as genetic interference with cristae remodelling and cytochrome c release. Thus, upon AICD induction regulation of macroautophagy, rather than selective mitophagy, ensures apoptotic progression.
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Apoptosis , Autofagia , Mitocondrias/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/fisiología , Animales , Células Cultivadas , Citocromos c/metabolismo , Humanos , Ratones Endogámicos C57BL , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Transducción de SeñalRESUMEN
BACKGROUND: Implantable cardioverter defibrillator (ICD) is recommended for patients with ventricular tachycardia (VT) due to cardiac sarcoidosis (CS). Programming supraventricular tachycardia (SVT) discriminators (onset, stability, and morphology/template match) is generally recommended to minimize inappropriate therapies. However, VT in patients with CS is known to show cycle length variability (CLV) and pleomorphism. OBJECTIVE: To determine whether the stability criterion, designed to prevent inappropriate therapy during atrial fibrillation with rapid ventricular rates, could potentially lead to incorrect classification of VT as SVT and inappropriately delay or inhibit ICD therapy. METHODS: Cases of biopsy-proven CS with VT were analyzed. For patients with implanted devices, all recorded electrograms of tachycardia episodes and ICD therapies were analyzed at last follow up. RESULTS: A total of 142 patients were included (mean age 38 years, 87 males). One hundred and three of 142 patients had implanted devices (ICD or CRT-D). Thirty eight of 103 (36.9%) patients received appropriate ICD therapies over 3 ± 2.2 years follow up. Four of 38 (10.5%) of patients experienced delayed-detection or underdetection of VT related to CLV, resulting in VT counters being repeatedly "reset" (classified as "unstable" rhythms). Retrospective analysis of other VT episodes in 70 of 103 (68%) patients revealed that 25 of 80 (31.3%) episodes had > 50 ms cycle length oscillations. CONCLUSION: Among CS patients with VT, CLV is a common occurrence seen in two-thirds of VT episodes. Routine programming of the stability criterion may result in underdetection of VT in a subset of such patients. We recommend that the stability criterion should be programmed "OFF" for patients with CS, unless the patient has documented atrial fibrillation.
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Cardiomiopatías/fisiopatología , Desfibriladores Implantables , Corazón/fisiopatología , Sarcoidosis/fisiopatología , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/fisiopatología , Adulto , Cardiomiopatías/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Sarcoidosis/complicaciones , Taquicardia Ventricular/complicacionesRESUMEN
The enzyme Cypridina luciferase (CLase) enables Cypridina luciferin to emit light efficiently through an oxidation reaction. The catalytic mechanism on the substrate of CLase has been studied, but the details remain to be clarified. Here, we examined the luminescence of Cypridina luciferin in the presence of several proteins with drug-binding ability. Luminescence measurements showed that the mixture of human plasma alpha 1-acid glycoprotein (hAGP) and Cypridina luciferin produced light. The total value of the luminescence intensity over 60 s was over 12.6-fold higher than those in the presence of ovalbumin, human serum albumin, or bovine serum albumin. In the presence of heat-treated hAGP, the luminescence intensity of Cypridina luciferin was lower than in the presence of intact hAGP. Chlorpromazine, which binds to hAGP, showed an inhibitory effect on the luminescence of Cypridina luciferin, both in the presence of hAGP and a recombinant CLase. Furthermore, BlastP analysis showed that hAGP had partial amino acid sequence similarity to known CLases in the region including amino acid residues involved in the drug-binding ability of hAGP. These findings indicate enzymological similarity between hAGP and CLase and provide insights into both the enzymological understanding of CLase and development of a luminescence detection method for hAGP.
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Luciferasas/metabolismo , Luminiscencia , Mediciones Luminiscentes , Orosomucoide/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Luciferasas/química , Luciferasas/genética , Mediciones Luminiscentes/métodos , Estructura Molecular , TemperaturaRESUMEN
Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cell-based drug screening methods for nuclear translocation.
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Proteínas Nucleares/metabolismo , Transcripción Genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Fluorescencia , Vectores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Mutación , Transporte de Proteínas , Transducción de SeñalRESUMEN
Activation-induced cytidine deaminase (AID) is required for the immunoglobulin diversification processes of somatic hypermutation, gene conversion and class-switch recombination. The targeting of AID's deamination activity is thought to be a combination of cis- and trans-acting elements, but has not been fully elucidated. Deletion analysis of putative proximal cis-regulatory motifs, while helpful, fails to identify additive versus cumulative effects, redundancy, and may create new motifs where none previously existed. In contrast, gain-of-function analysis can be more insightful with fewer of the same drawbacks and the output is a positive result. Here, we show five defined DNA regions of the avian Igλ locus that are sufficient to confer events of hypermutation to a target gene. In our analysis, the essential cis-targeting elements fully reconstituted diversification of a transgene under heterologous promotion in the avian B-cell line DT40. Furthermore, to the best of our knowledge two of the five regions we report on here have not previously been described as individually having an influence on somatic hypermutation.
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Linfocitos B/inmunología , Citidina Desaminasa/genética , Mutación con Ganancia de Función , Cambio de Clase de Inmunoglobulina , Animales , Línea Celular , Pollos , Conversión Génica , Hipermutación Somática de InmunoglobulinaRESUMEN
CSP-1103 (formerly CHF5074) has been shown to reverse memory impairment and reduce amyloid plaque as well as inflammatory microglia activation in preclinical models of Alzheimer's disease. Moreover, it was found to improve cognition and reduce brain inflammation in patients with mild cognitive impairment. Recent evidence suggests that CSP-1103 acts through a single molecular target, the amyloid precursor protein intracellular domain (AICD), a transcriptional regulator implicated in inflammation and apoptosis. We here tested the possible anti-apoptotic and neuroprotective activity of CSP-1103 in a cell-based model of post-ischemic injury, wherein the primary mouse cortical neurons were exposed to oxygen-glucose deprivation (OGD). When added after OGD, CSP-1103 prevented the apoptosis cascade by reducing cytochrome c release and caspase-3 activation and the secondary necrosis. Additionally, CSP-1103 limited earlier activation of p38 and nuclear factor κB (NF-κB) pathways. These results demonstrate that CSP-1103 is neuroprotective in a model of post-ischemic brain injury and provide further mechanistic insights as regards its ability to reduce apoptosis and potential production of pro-inflammatory cytokines. In conclusion, these findings suggest a potential use of CSP-1103 for the treatment of brain ischemia.
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Apoptosis/efectos de los fármacos , Ciclopropanos/farmacología , Flurbiprofeno/análogos & derivados , Glucosa/deficiencia , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Oxígeno/farmacología , Animales , Caspasa 3/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Corteza Cerebral/patología , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Flurbiprofeno/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ibuprofeno/farmacología , Ratones Endogámicos C57BL , Necrosis , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Currently, deficit of amyloid ß-peptide (Aß) clearance from the brain is considered as one of the possible causes of amyloid accumulation and neuronal death in the sporadic form of Alzheimer's disease (AD). Aß clearance can involve either specific proteases present in the brain or Aß-binding/transport proteins. Among amyloid-degrading enzymes the most intensively studied are neprilysin (NEP) and insulin-degrading enzyme (IDE). Since ageing and development of brain pathologies is often accompanied by a deficit in the levels of expression and activity of these enzymes in the brain, there is an urgent need to understand the mechanisms involved in their regulation. We have recently reported that NEP and also an Aß-transport protein, transthyretin are epigenetically co-regulated by the APP intracellular domain (AICD) and this regulation depends on the cell type and APP695 isoform expression in a process that can be regulated by the tyrosine kinase inhibitor, Gleevec. We have now extended our work and shown that, unlike NEP, another amyloid-degrading enzyme, IDE, is not related to over-expression of APP695 in neuroblastoma SH-SY5Y cells but is up-regulated by APP751 and APP770 isoforms independently of AICD but correlating with reduced HDAC1 binding to its promoter. Studying the effect of the nuclear retinoid X receptor agonist, bexarotene, on NEP and IDE expression, we have found that both enzymes can be up-regulated by this compound but this mechanism is not APP-isoform specific and does not involve AICD but, on the contrary, affects HDAC1 occupancy on the NEP gene promoter. These new insights into the mechanisms of NEP and IDE regulation suggest possible pharmacological targets in developing AD therapies.
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Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Epigénesis Genética , Insulisina/metabolismo , Neprilisina/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Bexaroteno , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Humanos , Insulisina/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Receptores X Retinoide/antagonistas & inhibidores , Tetrahidronaftalenos/farmacologíaRESUMEN
Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3-P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower ßamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Síndrome de Down/tratamiento farmacológico , Proteínas Hedgehog/metabolismo , Neurogénesis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Pirazoles/farmacología , Quinolinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Síndrome de Down/patología , Síndrome de Down/fisiopatología , Inhibidores Enzimáticos/farmacología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipocampo/fisiopatología , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurogénesis/fisiología , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Nicho de Células Madre/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/patología , Sinapsis/fisiologíaRESUMEN
Several data indicate that neuronal infection with herpes simplex virus type 1 (HSV-1) causes biochemical alterations reminiscent of Alzheimer's disease (AD) phenotype. They include accumulation of amyloid-ß (Aß), which originates from the cleavage of amyloid precursor protein (APP), and hyperphosphorylation of tau protein, which leads to neurofibrillary tangle deposition. HSV-1 infection triggers APP processing and drives the production of several fragments including APP intracellular domain (AICD) that exerts transactivating properties. Herein, we analyzed the production and intracellular localization of AICD following HSV-1 infection in neurons. We also checked whether AICD induced the transcription of two target genes, neprilysin (nep) and glycogen synthase kinase 3ß (gsk3ß), whose products play a role in Aß clearance and tau phosphorylation, respectively. Our data indicate that HSV-1 led to the accumulation and nuclear translocation of AICD in neurons. Moreover, results from chromatin immunoprecipitation assay showed that AICD binds the promoter region of both nep and gsk3ß. Time course analysis of NEP and GSK3ß expression at both mRNA and protein levels demonstrated that they are differently modulated during infection. NEP expression and enzymatic activity were initially stimulated but, with the progression of infection, they were down-regulated. In contrast, GSK3ß expression remained nearly unchanged, but the analysis of its phosphorylation suggests that it was inactivated only at later stages of HSV-1 infection. Thus, our data demonstrate that HSV-1 infection induces early upstream events in the cell that may eventually lead to Aß deposition and tau hyperphosphorylation and further suggest HSV-1 as a possible risk factor for AD.