Aspectos docentes sobre enfermedades raras en las universidades españolas

Trabajo presentado al VI Congreso Internacional de Medicamentos Huérfanos y Enfermedades Raras: IV Mesa Redonda y Coloquio: Acciones institucionales enrelación con lasEnfermedades Raras, celebrado en Sevilla (España) del 14 al 16 de febrero de 2013 y organizado por el Real e Ilustre Colegio Oficial de Farmacéuticos de Sevilla, la Federación Española de Enfermedades Raras (FEDER), la Fundación MEHUER y la Fundación FEDER.Mesa patrocinada por AMGEN.Peer Reviewe

Characterization and Drugs Screening in Human Dermal Fibroblasts Derived From Patient With Amiotrophic Lateral Sclerosis

Motivation: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the loss of motor neurons. Though currently we unknow its etiology, there are several alterations related to its physiopathology, such as mutation in Superoxide dismutase-1 (SOD-1), an enzyme which prevents free radical production, and intracellular non-avaliable iron accumulation, both alterations have been observed in the patient. The mutated form of this enzyme tends to form fibrillar aggregates on the cytoplasm. In this way, the research line have two parts: the molecular characterization of the disease, and the elimination or reduction of  intracellular iron accumulation and the reestablishment of modified protein levels. Treatments screening allows to increase patient's survival due to we use commercialized compounds, with this approximation we can skip long proccess of drugs commercialization.Methods: Human Dermal Fibroblast (HDF) primary cultures with and without pathological background are used. Iron accumulation in this cultures is observed by Prussian Blue technique. Expression protein levels are measured by Western Blotting, TransferBlot, InmmunoBlot and ChemicDoc developing. Quantifications were calculated with ImageJ software.Preliminar Results: We observed differences in the expression protein levels involved in autophagy proccess (P62, LC3B), antioxidative activity (GPX, SOD1), lipid peroxidation (PLA2G6) and lisosomal dynamic (LAMP1). Drugs screening allowed to select several drugs which reduced intracellular iron levels. With this technique we did another screening combining that drugs to select the best combination

Búsqueda de nuevos tratatamientos en las enfermedades mitocondriales

Trabajo presentado al I Encuentro Nacional de Familias con Niños Enfermos Mitocondriales organizado por AEPMI junto con la Fundación Ana Carolina Díez Maho y celebrado en Burgos del 13 al 16 de noviembre de 2013.Peer Reviewe

PLA2G6-associated neurodegeneration (PLAN): Characterization of patients and drug screening

Neurodegeneration with brain iron accumulation (NBIA) envolve a group of rare neurodegenerative disorders characterized by brain iron accumulation, progressive extrapyramidal dysfunction (dystonia, stiffness, choreoathetosis), and presence of axonal spheroids, usually limited by the central nervous system.   Within the differents subtypes of NBIA, in this study we focussed in PLA2G6-associated neurodegeneration (PLAN), diseases caused by a mutation in the phospholipase A2 group VI (PLA2G6). PLA2G6 encodes the enzyme iPLA2b, a calcium-independent phospholipase A2 which is involved in lipid metabolism. The loss of iPLA2b’s function result in mitochondrial abnormalities and synaptic transmission impairment in neurons among other alterations.   In the current work we studied the pathophysiology of three confirmed cases of PLAN using fibroblasts derived from the patients: PLAN 10 (heterozygous mutation), PLAN 11(heterozygous mutation) and PLAN 8 (homozygous mutation). The aim of this study is to characterize, in patient-derived fibroblasts, the pathological alterations produced by PLA2G6 mutations. Main methods used were Western blot and Prussian Blue Staining. Our results confirm iron accumulation in patients-derived fibroblasts, impaired autophagy and ferritinophagy, especially in the patient that suffers the homozygous mutation (Plan 8). The purpose is to carry out a drug screening able to reverse the pathophysiology observed

Importance of food sensitivities and personalized diet in patients with fibromyalgia

Fibromyalgia (FM) is a disease characterized by generalized chronic pain located in the locomotor system. Patients usually present obesity, metabolic syndrome, alterations in the microbiota and food sensitivities. The prevalence is 2.1% in the world population, affecting 2.3% of Europeans and 2.4% of Spaniards. By sex, the prevalence among men is estimated at 0.2%, compared to 4.2% in women. FM is one of the rheumatic diseases with more impact on the quality of life. Nutrition has been suggested as a relevant factor in the treatment and improvement of FM symptoms. The aim of this study was to measure mitochondrial homeostasis using several biomarkers for the diagnosis and monitoring of FM before and after the application of a personalized nutritional intervention with a rich antioxidant diet. Furthermore, we  evaluated the role of personalized nutrition as a potential treatment for patients with FM and identified new approaches for a better understanding of the disease.Motivation: Currently, a significant percentage of patients who suffer FM are not well diagnosed and face inadequate treatments. We proposed that personalized nutritional interventions can improve the symptomatology of patients and their quality of life.Methods: Fifty FM patients with clinical suspicion of food sensitivities were included in the study. Data were collected from nutritional questionnaries, the Fibromyalgia Impact Questionnaire (FIQ) and the ratio of mitochondrial mass and selective autophagy. Mitochondrial markers were measured pre and post nutritional interventions with the objective of evaluating if personalized diets improved clinical symptoms.Results: Rich antioxidant diets decreased radical oxygen species (ROS) levels in blood and improved significantly clinical symptoms. In addition, a balanced, personalized diet for each patient improved the ratio between mitochondrial mass and selective autophagy.Conclusions: FM patients presents several associated comorbidities such as food sensitivities which need more attention and research. The adequate consumption of antioxidants and other micronutrients is important in FM management. Therefore,  personalized antioxidant diets could be a promising approach for the improvement of clinical symptoms in FM

Is Inflammation a Mitochondrial Dysfunction-Dependent Event in Fibromyalgia?

Fibromyalgia (FM) is a complex disorder that affects up to 5% of the general population worldwide. Both mitochondrial dysfunction and inflammation have been implicated in the pathophysiology of FM. We have investigated the possible relationship between mitochondrial dysfunction, oxidative stress, and inflammation in FM. We studied 30 women diagnosed with FM and 20 healthy women. Blood mononuclear cells (BMCs) from FM patients showed reduced level of coenzyme Q10 (CoQ10) and mtDNA contents and high level of mitochondrial reactive oxygen species (ROS) and serum tumor necrosis factor (TNF)-alpha and transcript levels. A significant negative correlation between CoQ10 and TNF-alpha levels (r= -0.588; p < 0.01), and a positive correlation between ROS and TNF-alpha levels (r = 0.791; p < 0.001) were observed accompanied by a significant correlation of visual analogical scale with serum TNF-alpha and transcript levels (r = 0.4507; p < 0.05 and r = 0.7089; p < 0.001, respectively). TNF-alpha release was observed in an in vitro (BMCs) and in vivo (mice) CoQ10 deficiency model. Oral CoQ10 supplementation restored biochemical parameters and induced a significant improvement in clinical symptoms ( p < 0.001). These results lead to the hypothesis that inflammation could be a mitochondrial dysfunction-dependent event implicated in the pathophysiology of FM in several patients indicating at mitochondria as a possible new therapeutic target.Unión Europea FIS PI10/00543Servicio Andaluz de Salud Junta de Andalucía SAS 111242Junta de Andalucía CTS-572

Microstructural Characterization of Silver Nanoparticles for Bioimaging Applications

Junta de Andalucía FQM-6615, PI0070, FQM31

Fibroblasts derived from nemaline myopathy patients: a useful cellular model for studying the pathophysiological mechanisms implicated in disease's development and for pharmacological screenings

Motivation: Nemaline myopathy (NM) is one of the most common forms of congenital myopathy and it is identified by the presence of inclusions in muscle fibers called "nemaline bodies" or rods which are considered to be derived from Z lines because they have a similar structure and express similar proteins. Clinical features include hypotonia and muscle weakness, nevertheless, the clinical spectrum varies between lethal neonatal forms and less severe forms of later onset. This pathology is heterogeneous from a genetic point of view, its inheritance can be autosomal-dominant(AD), autosomal-recessive(AR) or sporadic. Although most cases of NM result from mutations in the genes encoding skeletal α-actin (ACTA1) and nebulin (NEB), mutations have been found in more than 10 genes that cause the disease, including genes encoding proteins for sarcomeric thin filament components (NEB, ACTA1, TPM2, TPM3, TNNT1, CFL2, LMOD3), Kelch domain-associated proteins (KBTBD13, KLHL40 and KLHL41) and an unconventional myosin (MYO18B). Unfortunately, there is no curative treatment for NM patients and the pathogenetic mechanisms remains unclear, therefore, studying fibroblasts derived from patients can be useful to understand the pathophysiological alterations of NM and to identify potential therapies. Methods: We study the pathophysiological alterations in NM using fibroblasts from patients with mutations in ACTA1 and NEB genes which are obtained by performing a skin biopsy. As a screening strategy, fibroblasts are treated with different compounds for a week and after that, they are stained with rhodamine-phalloidin in order to analyze the status of the cytoskeletal actin filaments by fluorescence microscopy techniques using DeltaVision system. Positive compounds are those that improve the formation of actin filaments. Results: Fibroblasts from NM patients showed incorrect actin filament formation compared to control fibroblasts. Moreover, we identified two compounds that improved the correct formation of actin filaments with an increase in the length of the filaments compared to untreated fibroblasts from patients (p&lt;0,01). Conclusions: Our results suggest that fibroblasts derived from NM patients can be a useful cellular model to study the pathophysiological mechanisms involved in NM and to find new therapies. However, further studies are needed to elucidate the mechanism of action of the two positive compounds identified and to determine their therapeutic applications

Mitochondrial membrane Protein-Associated Neurodegeneration (MPAN): Pathophysiological characterization and pharmacological screening for potential therapies

Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of different rare disorders in which iron is accumulated in the brain, especially in the basal ganglia and substantia nigra. Prevalent symptoms are dystonia, spasticity and neuropsychiatric disorders. In the current study, we focused on Mitochondrial-membrane Protein-Associated Neurodegeneration (MPAN) which is a subtype of NBIA caused by&nbsp; mutations in the C19orf12 gene. This gene encodes a protein located in the external membrane of the mitochondrial of unknown function. However, it is proposed that C19orf12 could be implicated in fatty acid biosynthesis, calcium exchange, coenzyme A biosynthesis or mitophagy. From a genetic point of view, the disease is autosomal-recessive, although cases of autosomal-dominant inheritance have been described. Motivation: Nowadays, there is not an effective treatment for these patients, therefore it is very important to study the pathophysiology of MPAN and search for potential therapies. Methods: We studied the pathophysiological alterations in MPAN using fibroblasts derived from three confirmed patients identified as MPAN1, MPANH and MPANB, the first two with the same mutation (p.Gly69Arg) and MPANB with another one (p.Gly58Arg) but all of them from different countries. In order to achieve this aim, pathophysiological mechanisms will be studied examining protein expression levels of several cellular pathways by Western blotting and iron accumulation by Prussian Blue staining. Results: We found iron accumulation and alteration of several pathways in the cellular models. In addition, we found two pharmacological cocktails that both reduced iron accumulation and corrected all pathological alterations.&nbsp; Conclusions: Fibroblast cell cultures derived from patients are interesting cellular models for both disease modelling and pharmacological screenings but it is neccesary further studies to confirm the results and to establish the mechanism of action of the two pharmacological cocktails and their use as medical therapies

PEGylated versus non-PEGylated magnetic nanoparticles as camptothecin delivery system

This is an Open Access article under the terms of the Creative Commons Attribution License.Camptothecin (CPT; (S)-(+)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione) is a highly cytotoxic natural alkaloid that has not yet found use as chemotherapeutic agent due to its poor water-solubility and chemical instability and, as a consequence, no effective administration means have been designed. In this work, camptothecin has been successfully loaded into iron oxide superparamagnetic nanoparticles with an average size of 14 nm. It was found that surface modification of the nanoparticles by polyethylene glycol enables loading a large amount of camptothecin. While the unloaded nanoparticles do not induce apoptosis in the H460 lung cancer cell line, the camptothecin-loaded nanoparticle formulations exhibit remarkable proapoptotic activity. These results indicate that camptothecin retains its biological activity after loading onto the magnetic nanoparticles. The proposed materials represent novel materials based on naturally occurring bioactive molecules loaded onto nanoparticles to be used as chemotherapeutic formulations. The procedure seems apt to be extended to other active molecules extracted from natural products. In addition, these materials offer the potential of being further implemented for combined imaging and therapeutics, as magnetic nanoparticles are known to be multifunctional tools for biomedicine.This work was supported by Fundación Progreso y Salud, Consejería de Salud (PI0070) and Proyecto de Investigación de Excelencia de la Junta de Andalucía (P10-FQM-6615).Peer Reviewe