The differential regulation of Lck kinase phosphorylation sites by CD45 is critical for T cell receptor signaling responses

SummaryThe molecular mechanisms whereby the CD45 tyrosine phosphatase (PTPase) regulates T cell receptor (TCR) signaling responses remain to be elucidated. To investigate this question, we have reconstituted CD45 (encoded by Ptprc)-deficient mice, which display severe defects in thymic development, with five different expression levels of transgenic CD45RO, or with mutant PTPase null or PTPase-low CD45R0. Whereas CD45 PTPase activity was absolutely required for the reconstitution of thymic development, only 3% of wild-type CD45 activity restored T cell numbers and normal cytotoxic T cell responses. Lowering the CD45 expression increased CD4 lineage commitment. Peripheral T cells with very low activity of CD45 phosphatase displayed reduced TCR signaling, whereas intermediate activity caused hyperactivation of CD4+ and CD8+ T cells. These results are explained by a rheostat mechanism whereby CD45 differentially regulates the negatively acting pTyr-505 and positively acting pTyr-394 p56lck tyrosine kinase phosphorylation sites. We propose that high wild-type CD45 expression is necessary to dephosphorylate p56lck pTyr-394, suppressing CD4 T+ cell lineage commitment and hyperactivity

The Mitochondrial SDHD Gene Is Required for Early Embryogenesis, and Its Partial Deficiency Results in Persistent Carotid Body Glomus Cell Activation with Full Responsiveness to Hypoxia

The SDHD gene encodes one of the two membrane-anchoring proteins of the succinate dehydrogenase (complex II) of the mitochondrial electron transport chain. This gene has recently been proposed to be involved in oxygen sensing because mutations that cause loss of its function produce hereditary familiar paraganglioma, a tumor of the carotid body (CB), the main arterial chemoreceptor that senses oxygen levels in the blood. Here, we report the generation of a SDHD knockout mouse, which to our knowledge is the first mammalian model lacking a protein of the electron transport chain. Homozygous SDHD_/_ animals die at early embryonic stages. Heterozygous SDHD_/_ mice show a general, noncompensated deficiency of succinate dehydrogenase activity without alterations in body weight or major physiological dysfunction. The responsiveness to hypoxia of CBs from SDHD_/_ mice remains intact, although the loss of an SDHD allele results in abnormal enhancement of resting CB activity due to a decrease of K_ conductance and persistent Ca2_ influx into glomus cells. This CB overactivity is linked to a subtle glomus cell hypertrophy and hyperplasia. These observations indicate that constitutive activation of SDHD_/_ glomus cells precedes CB tumor transformation. They also suggest that, contrary to previous beliefs, mitochondrial complex II is not directly involved in CB oxygen sensing

MYH10 activation rescues contractile defects in arrhythmogenic cardiomyopathy (ACM).

The most prevalent genetic form of inherited arrhythmogenic cardiomyopathy (ACM) is caused by mutations in desmosomal plakophilin-2 (PKP2). By studying pathogenic deletion mutations in the desmosomal protein PKP2, here we identify a general mechanism by which PKP2 delocalization restricts actomyosin network organization and cardiac sarcomeric contraction in this untreatable disease. Computational modeling of PKP2 variants reveals that the carboxy-terminal (CT) domain is required for N-terminal domain stabilization, which determines PKP2 cortical localization and function. In mutant PKP2 cells the expression of the interacting protein MYH10 rescues actomyosin disorganization. Conversely, dominant-negative MYH10 mutant expression mimics the pathogenic CT-deletion PKP2 mutant causing actin network abnormalities and right ventricle systolic dysfunction. A chemical activator of non-muscle myosins, 4-hydroxyacetophenone (4-HAP), also restores normal contractility. Our findings demonstrate that activation of MYH10 corrects the deleterious effect of PKP2 mutant over systolic cardiac contraction, with potential implications for ACM therapy.This study was supported by MCIU grant BFU2016-75144-R and PID2020- 116935RB-I00, and by a “la Caixa” Banking Foundation grant under the project code HR18-00304” to J.A.B.; The study was also supported by the “Ayudas a la Investigación Cátedra Real Madrid-Universidad Europea” (2017/RM01). C.M.-L. and S.S. hold MCIU predoctoral contracts BES-2017-079715, and BES-2017-079707 respectively. R.G. acknowledges funding from the European Research Council under grant ERCAG-340177 (3DNanoMech) and from the MCIU under grant MAT2016- 76507-R. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence, grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033. The microscopy experiments were carried out at the Dynamic Microscopy and Image Unit, CNIC, ICTS-ReDib, co-financed by MCIN/AEI /10.13039/ 501100011033 and FEDER “A way of making Europe” (#ICTS-2018-04- CNIC-16). Imaris full analysis were carried out at the Microscopy & Dynamic Imaging, CNIC, ICTS-ReDib, co-funded by MCIN/AEI /10.13039/501100011033. Biomedical Imaging has been conducted at the Advanced Imaging Unit of the CNIC (Centro Nacional de Investigaciones Cardiovasculares Carlos III), Madrid, Spain. This project used the ReDIB ICTS infrastructure TRIMA@CNIC, Ministerio de Ciencia e Innovación (MCIN).S

The pharmaceutical solvent N-methyl-2-pyrollidone (NMP) attenuates inflammation through Krüppel-like factor 2 activation to reduce atherogenesis.

N-methyl-2-pyrrolidone (NMP) is a versatile water-miscible polar aprotic solvent. It is used as a drug solubilizer and penetration enhancer in human and animal, yet its bioactivity properties remain elusive. Here, we report that NMP is a bioactive anti-inflammatory compound well tolerated in vivo, that shows efficacy in reducing disease in a mouse model of atherosclerosis. Mechanistically, NMP increases the expression of the transcription factor Kruppel-like factor 2 (KLF2). Monocytes and endothelial cells treated with NMP express increased levels of KLF2, produce less pro-inflammatory cytokines and adhesion molecules. We found that NMP attenuates monocyte adhesion to endothelial cells inflamed with tumor necrosis factor alpha (TNF-α) by reducing expression of adhesion molecules. We further show using KLF2 shRNA that the inhibitory effect of NMP on endothelial inflammation and subsequent monocyte adhesion is KLF2 dependent. Enhancing KLF2 expression and activity improves endothelial function, controls multiple genes critical for inflammation, and prevents atherosclerosis. Our findings demonstrate a consistent effect of NMP upon KLF2 activation and inflammation, biological processes central to atherogenesis. Our data suggest that inclusion of bioactive solvent NMP in pharmaceutical compositions to treat inflammatory disorders might be beneficial and safe, in particular to treat diseases of the vascular system, such as atherosclerosis

p38γ is essential for cell cycle progression and liver tumorigenesis

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)–cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease

Natural History of MYH7-Related Dilated Cardiomyopathy

BACKGROUND Variants in myosin heavy chain 7 (MYH7) are responsible for disease in 1% to 5% of patients with dilated cardiomyopathy (DCM); however, the clinical characteristics and natural history of MYH7-related DCM are poorly described. OBJECTIVES We sought to determine the phenotype and prognosis of MYH7-related DCM. We also evaluated the influence of variant location on phenotypic expression. METHODS We studied clinical data from 147 individuals with DCM-causing MYH7 variants (47.6% female; 35.6 +/- 19.2 years) recruited from 29 international centers. RESULTS At initial evaluation, 106 (72.1%) patients had DCM (left ventricular ejection fraction: 34.5% +/- 11.7%). Median follow-up was 4.5 years (IQR: 1.7-8.0 years), and 23.7% of carriers who were initially phenotype-negative developed DCM. Phenotypic expression by 40 and 60 years was 46% and 88%, respectively, with 18 patients (16%) first diagnosed at <18 years of age. Thirty-six percent of patients with DCM met imaging criteria for LV noncompaction. During follow-up, 28% showed left ventricular reverse remodeling. Incidence of adverse cardiac events among patients with DCM at 5 years was 11.6%, with 5 (4.6%) deaths caused by end-stage heart failure (ESHF) and 5 patients (4.6%) requiring heart transplantation. The major ventricular arrhythmia rate was low (1.0% and 2.1% at 5 years in patients with DCM and in those with LVEF of <= 35%, respectively). ESHF and major ventricular arrhythmia were significantly lower compared with LMNA-related DCM and similar to DCM caused by TTN truncating variants. CONCLUSIONS MYH7-related DCM is characterized by early age of onset, high phenotypic expression, low left ventricular reverse remodeling, and frequent progression to ESHF. Heart failure complications predominate over ventricular arrhythmias, which are rare. (C) 2022 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Nuevos modelos animales de hipercolesterolemia familiar y tratamientos de la aterosclerosis

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 08-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 08-09-2019La aterosclerosis es una enfermedad crónica y una de las principales causas de mortalidad en todo el mundo por lo que nuevos tratamientos son una necesidad urgente. Los modelos animales de aterosclerosis han ayudado enormemente a entender la enfermedad, identificar dianas terapéuticas, y a desarrollar potenciales tratamientos. Estudios recientes en animales demuestran que la proproteina convertasa subtilisin/kexin tipo 9 (PCSK9) reduce la absorción de LDL hepática incrementando la degradación endosomal y lisosomal del receptor de LDL (LDLR). Pacientes con PCSK9 mutante desarrollan hipercolesterolemia familiar y tienen un riesgo exagerado de desarrollar afecciones cardiovasculares. Expresar, por tanto, el mutante D374Y de ganancia de función de PCSK9 (PCSK9DY) en animales silvestres debería inducir aterosclerosis. Hemos generado vectores basados en virus adenoasociados (AAV) para transferir y expresar PCSK9DYen hígado. Una inyección intravenosa de AAV-PCSK9DY es una manera fácil, rápida y económica para obtener animales que desarrollen hipercolesterolemia y aterosclerosis, sin la necesidad de generar colonias transgénicas. Esta nueva estrategia permite, además, estudiar interacciones genéticas entre distintos mutantes (y diferentes fondos genéticos) sin la necesidad que realizar cruces. En definitiva, esta manera de generar “transgénicos” permitirá explorar más aún las interacciones funcionales de PCSK9 y como contribuye al desarrollo de la aterosclerosis. La discontinuidad en el flujo circulatorio activa una respuesta transcripcional pro-inflamatoria en el endotelio vascular que contribuye al desarrollo de la aterosclerosis. El factor de transcripción Krüppel-Like factor 2 (Klf2 ) es crítico en la activación de la respuesta antiinflamatoria en células endoteliales por lo que incrementar la expresión de Klf2 puede ser una opción en el tratamiento clínico de la aterosclerosis. Usando un sistema de activación transcripcional de luciferasa con el promotor del gen de Klf2 in vitro , encontramos que una molécula que se utiliza habitualmente por la industria farmacéutica como cosolvente, N-metil-2-pirolidona (NMP), que activa transcripcionalmente Klf2 . El tratamiento con NMP, se tolera bien in vivo , y reduce el tamaño de la placa en un modelo de hipercolesterolemia y aterosclerosis con alimentación rica en grasas. En ensayos in vitro de adhesión en condiciones de flujo continuo, NMP inhibe la capacidad de los linfocitos a unirse al endotelio activado, lo que demuestra que NMP inhibe la adhesión intercelular que son esenciales para la extravasación de células linfoides a la vasculatura. Estos hallazgos demuestran que NMP tiene un efecto sobre procesos básicos en la iniciación y desarrollo de la aterosclerosis. Proponemos que la administración de NMP pudiera ser un compuesto que se utilizase de manera segura

Exercise triggers ARVC phenotype in mice expressing a disease-causing mutated version of human plakophilin-2.

BACKGROUND Exercise has been proposed as a trigger for arrhythmogenic right ventricular cardiomyopathy (ARVC) phenotype manifestation; however, research is hampered by the limited availability of animal models in which disease-associated mutations can be tested. OBJECTIVES This study evaluated the impact of exercise on ARVC cardiac manifestations in mice after adeno-associated virus (AAV)-mediated gene delivery of mutant human PKP2, which encodes the desmosomal protein plakophilin-2. METHODS We developed a new model of cardiac tissue-specific transgenic-like mice on the basis of AAV gene transfer to test the potential of a combination of a human PKP2 mutation and endurance training to trigger an ARVC-like phenotype. RESULTS Stable cardiac expression of mutant PKP2 (c.2203C>T), encoding the R735X mutant protein, was achieved 4 weeks after a single AAV9-R735X intravenous injection. High-field cardiac magnetic resonance over a 10-month postinfection follow-up did not detect an overt right ventricular (RV) phenotype in nonexercised (sedentary) mice. In contrast, endurance exercise training (initiated 2 weeks after AAV9-R735X injection) resulted in clear RV dysfunction that resembled the ARVC phenotype (impaired global RV systolic function and RV regional wall motion abnormalities on cardiac magnetic resonance). At the histological level, RV samples from endurance-trained R735X-infected mice displayed connexin 43 delocalization at intercardiomyocyte gap junctions, a change not observed in sedentary mice. CONCLUSIONS The introduction of the PKP2 R735X mutation into mice resulted in an exercise-dependent ARVC phenotype. The R735X mutation appears to function as a dominant-negative variant. This novel system for AAV-mediated introduction of a mutation into wild-type mice has broad potential for study of the implication of diverse mutations in complex cardiomyopathies.S