Development of a High-Throughput Calcium Mobilization Assay for CCR6 Receptor Coupled to Hydrolase Activity Readout

CCR6 is a chemokine receptor highly implicated in inflammatory diseases and could be a potential therapeutic target; however, no therapeutic agents targeting CCR6 have progressed into clinical evaluation. Development of a high-throughput screening assay for CCR6 should facilitate the identification of novel compounds against CCR6. To develop a cell-based assay, RBL-2H3 cells were transfected with plasmids encoding β-hexosaminidase and CCR6. Intracellular calcium mobilization of transfected cells was measured with a fluorescent substrate using the activity of released hexosaminidase as readout of the assay. This stable, transfected cell showed a specific signal to the background ratio of 19.1 with low variability of the signal along the time. The assay was validated and optimized for high-throughput screening. The cell-based calcium mobilization assay responded to the specific CCR6 ligand, CCL20, in a dose-dependent manner with an EC50 value of 10.72 nM. Furthermore, the assay was deemed robust and reproducible with a Z’ factor of 0.63 and a signal window of 7.75. We have established a cell-based high-throughput calcium mobilization assay for CCR6 receptor. This assay monitors calcium mobilization, due to CCR6h activation by CCL20, using hexosaminidase activity as readout. This assay was proved to be robust, easy to automate and could be used as method for screening of CCR6 modulators

Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity

AIMS: Obesity is characterized by a low-grade systemic inflammatory state and adipose tissue (AT) dysfunction, which predispose individuals to the development of insulin resistance (IR) and metabolic disease. However, a subset of obese individuals, referred to as metabolically healthy obese (MHO) individuals, are protected from obesity-associated metabolic abnormalities. Here, we aim at identifying molecular factors and pathways in adipocytes that are responsible for the progression from the insulin-sensitive to the insulin-resistant, metabolically unhealthy obese (MUHO) phenotype. RESULTS: Proteomic analysis of paired samples of adipocytes from subcutaneous (SC) and omental (OM) human AT revealed that both types of cells are altered in the MUHO state. Specifically, the glutathione redox cycle and other antioxidant defense systems as well as the protein-folding machinery were dysregulated and endoplasmic reticulum stress was increased in adipocytes from IR subjects. Moreover, proteasome activity was also compromised in adipocytes of MUHO individuals, which was associated with enhanced accumulation of oxidized and ubiquitinated proteins in these cells. Proteasome activity was also impaired in adipocytes of diet-induced obese mice and in 3T3-L1 adipocytes exposed to palmitate. In line with these data, proteasome inhibition significantly impaired insulin signaling in 3T3-L1 adipocytes. INNOVATION: This study provides the first evidence of the occurrence of protein homeostasis deregulation in adipocytes in human obesity, which, together with oxidative damage, interferes with insulin signaling in these cells. CONCLUSION: Our results suggest that proteasomal dysfunction and impaired proteostasis in adipocytes, resulting from protein oxidation and/or misfolding, constitute major pathogenic mechanisms in the development of IR in obesity.IMIBIC/Universidad de Córdoba-SCAI (ProteoRed, PRB2-ISCIII)MINECO/FEDERJunta de Andalucía/FEDERCIBERobn(Instituto de Salud Carlos III

From vasospasm to early brain injury: New frontiers in subarachnoid haemorrhage research

Introduction: Delayed vasospasm has traditionally been considered the most important determinant of poor outcome after subarachnoid haemorrhage (SAH). Consequently, most of the research and therapies are directed towards reducing the incidence of vasospasm (VSP). To date, however, clinical trials based on this strategy have not delivered a definitive treatment for preventing or reducing brain injury after SAH. This fact has caused a paradigm shift in research, which now focuses on early brain injury (EBI) occurring in the first 72 hours after SAH. It has also changed the idea of VSP's role in brain damage, and suggests the need for re-evaluating the pathophysiological process of SAH. Development: This review examines the current state of knowledge on the pathophysiological mechanisms associated with EBI and summarises the diagnostic options currently available. Conclusion: It seems that the research approach needs to be changed, so that investigators will focus on prevention of EBI, reduction of secondary brain complications and ultimately, the optimisation neurological outcome. Resumen: Introducción: El vasoespasmo (VSP) ha sido tradicionalmente considerado como el principal determinante de mal pronóstico tras sufrir una hemorragia subaracnoidea (HSA). Como consecuencia, la mayoría de las líneas de investigación y los tratamientos están dirigidos hacia la reducción de la incidencia de dicho VSP. Hasta la fecha, sin embargo, los resultados de los ensayos clínicos basados en esta estrategia no se han traducido en un tratamiento definitivo capaz de prevenir o mejorar la lesión cerebral tras una HSA.Este hecho ha provocado un cambio de paradigma en el interés investigativo, focalizándolo hacia la lesión cerebral precoz (LCP), que se produce en las primeras 72 horas tras la HSA. Así mismo, ha modificado la visión que se tenía de la responsabilidad del VSP sobre el daño cerebral y sugiere la necesidad de una re-evaluación del proceso fisiopatológico de la HSA. Desarrollo: Esta revisión examina el estado actual del conocimiento de los mecanismos fisiopatológicos relacionados con la LCP y resume las opciones diagnósticas disponibles en la actualidad. Conclusión: Parece necesario cambiar la dirección en la investigación de esta enfermedad, centrándose en la prevención de la LCP, la reducción de las complicaciones cerebrales secundarias y en última instancia, la optimización de los resultados neurológicos. Keywords: Biomarkers, CT perfusion, Early brain injury, Subarachnoid haemorrhage, Vasospasm, Palabras clave: Hemorragia subaracnoidea, Lesión cerebral precoz, Marcadores biológicos, Tomografía computarizada de perfusión, Vasoespasm

Del vasoespasmo a la lesión cerebral precoz: una nueva frontera en la investigación de la hemorragia subaracnoidea

Resumen: Introducción: El vasoespasmo (VSP) ha sido tradicionalmente considerado como el principal determinante de mal pronóstico tras sufrir una hemorragia subaracnoidea (HSA). Como consecuencia, la mayoría de las líneas de investigación y los tratamientos están dirigidos hacia la reducción de la incidencia de dicho VSP. Hasta la fecha, sin embargo, los resultados de los ensayos clínicos basados en esta estrategia no se han traducido en un tratamiento definitivo capaz de prevenir o mejorar la lesión cerebral tras una HSA. Este hecho ha provocado un cambio de paradigma en el interés investigativo, focalizándolo hacia la lesión cerebral precoz (LCP), que se produce en las primeras 72 h tras la HSA. Así mismo, ha modificado la visión que se tenía de la responsabilidad del VSP sobre el daño cerebral y sugiere la necesidad de una re-evaluación del proceso fisiopatológico de la HSA. Desarrollo: Esta revisión examina el estado actual del conocimiento de los mecanismos fisiopatológicos relacionados con la LCP y resume las opciones diagnósticas disponibles en la actualidad. Conclusión: Parece necesario cambiar la dirección en la investigación de esta enfermedad, centrándose en la prevención de la LCP, la reducción de las complicaciones cerebrales secundarias y en última instancia, la optimización de los resultados neurológicos. Abstract: Introduction: Delayed vasospasm has traditionally been considered the most important determinant of poor outcome after subarachnoid haemorrhage (SAH). Consequently, most of the research and therapies are directed towards reducing the incidence of vasospasm (VSP). To date, however, clinical trials based on this strategy have not delivered a definitive treatment for preventing or reducing brain injury after SAH. This fact has caused a paradigm shift in research, which now focuses on early brain injury (EBI) occurring in the first 72 hours after SAH. It has also changed the idea of VSP's role in brain damage, and suggests the need for re-evaluating the pathophysiological process of SAH. Development: This review examines the current state of knowledge on the pathophysiological mechanisms associated with EBI and summarises the diagnostic options currently available. Conclusion: It seems that the research approach needs to be changed so that investigators will focus on prevention of EBI, reduction of secondary brain complications and ultimately, the optimisation neurological outcome. Palabras clave: Hemorragia subaracnoidea, Lesión cerebral precoz, Marcadores biológicos, Tomografía computarizada de perfusión, Vasoespasmo, Keywords: Biomarkers, CT perfusion, Early brain injury, Subarachnoid haemorrhage, Vasospas

Effect of melatonin on myocardial oxidative stress induced by experimental obstructive jaundice Efecto de la melatonina en el estrés oxidativo del miocardio en un modelo experimental de obstrucción biliar

Objective: melatonin has been demonstrated to have active antioxidant properties in different tissues during experimental cholestasis. The aim of this research was to study myocardial oxidative stress on obstructive jaundice, and to analyze the effect of melatonin on myocardial oxidative lesions. Material and methods: we achieved cholestasis by ligature and sectioning of the main bile duct. Melatonin was administered intraperitoneally (500 µg/kg/day). We measured malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxydase (GPx) antioxidant enzyme levels in the heart tissue. Results: obstructive cholestasis increased MDA and decreased GSH as well as all antioxidant enzymes. Melatonin administration significantly decreased MDA values, and increased GSH and antioxidant enzymes on the icteric animal myocardium. Conclusions: melatonin treatment prevents oxidative stress in the cardiac tissue as induced by experimental cholestasis

Clinical and Neurochemical Effects of Transcranial Magnetic Stimulation (TMS) in Multiple Sclerosis: A Study Protocol for a Randomized Clinical Trial

Background: Transcranial Magnetic Stimulation (TMS) is a technique based on the principles of electromagnetic induction. It applies pulses of magnetic radiation that penetrate the brain tissue, and it is a non-invasive, painless, and practically innocuous procedure. Previous studies advocate the therapeutic capacity of TMS in several neurodegenerative and psychiatric processes, both in animal models and in human studies. Its uses in Parkinson's disease, Alzheimer's disease and in Huntington's chorea have shown improvement in the symptomatology and in the molecular profile, and even in the cellular density of the brain. Consequently, the extrapolation of these TMS results in the aforementioned neurodegenerative disease to other entities with etiopathogenic and clinical analogy would raise the relevance and feasibility of its use in multiple sclerosis (MS). The overall objective will be to demonstrate the effectiveness of the TMS in terms of safety and clinical improvement, as well as to observe the molecular changes in relation to the treatment. Methods and Design: Phase II clinical trial, unicentric, controlled, randomized, single blind. A total of 90 patients diagnosed with relapsing-remitting multiple sclerosis (RRMS) who meet all the inclusion criteria and do not present any of the exclusion criteria that are established and from which clinically evaluable results can be obtained. The patients included will be assigned under the 1:1:1 randomization formula, constituting three groups for the present study: 30 patients treated with natalizumab + white (placebo) + 30 patients treated with natalizumab + TMS (1 Hz) + 30 patients treated with natalizumab + TMS (5 Hz). Discussion: Results of this study will inform on the efficiency of the TMS for the treatment of MS. The expected results are that TMS is a useful therapeutic resource to improve clinical status (main parameters) and neurochemical profile (surrogate parameters); both types of parameters will be checked. Ethics and Dissemination: The study is approved by the Local Ethics Committee and registered in https://clinicaltrials.gov (NCT04062331). Dissemination will include submission to a peer-reviewed journal, patients, associations of sick people and family members, healthcare magazines and congress presentations. Trial Registration: ClinicalTrials.gov ID: NCT04062331 (registration date: 19th/ August/2019). Version Identifier: EMTr-EMRR, ver-3, 21/11/2017.Ye