Aislamiento y cultivo de células osteoblásticas: Interés para la investigación en cirugía ortopédica y traumatología

Describimos un método de aislamiento y caracterización de células osteoblásticas procedentes de la superficie de trabéculas óseas de explantes obtenidos de metafisis de fémures y tibias de conejo y de cabezas femorales humanas de pacientes sometidos a cirugía protésica de cadera. Después de eliminar las células de la médula ósea de los explantes, estos se despositaron sobre dos tipos de mallas, de 80 y 100 µm., obteniéndose un cultivo en monocarpa que se mostró confluente a partir de los 20 días. La población celular aislada presentó un fenotipo osteoblástico utilizando métodos colorimétricos para determinación de la actividad de fosfatasa alcalina (FA) y morfológicos por medio de la observación con microscopía electrónica de barrido. Las células cultivadas exhibieron una morfología diferente según el grado de confluencia de cultivo, siendo en todo momento positivas las sucesivas determinaciones colorimétricas de FA realizadas a lo largo de la experiencia. Consideramos el método de gran interés en investigación en ortopédica, pues permite el estudio de comportamiento de estas células cuando están sometidas a cualquier tipo de estímulo externo, así como cuando se enfrentan a nuevas generaciones de biomateriales, etc.We describe a method of isolation and characterization of osteoblastic cells taken from the surface of bone trabeculae of explants obtained from femur and tibia metaphysis in rabbits and human femoral head in patients undergoing orthopaedic surgery. Afther removing the cells from the bone marrow of explants, they were placed over two mesh, 80 and 100 µmn, obtaining then a culture in monolayer which showed to be confluent after 20 days. The isolated cell population showed an osteoblastic phenotype using colourmetric methods to determinate the alkaline phosphatase (AF) activity and morphologic methods by means of scanning. The cultivated cell showed a different morphology according to the degree of confluence of the culture, being always positive the sucesive colourmetric determinations of AF along the experiment. We consider the method of a great interest for orthopaedic investigations, since it allows the study of the behaviour of these cells when they are subjet to different types of external stimulus as well as meet they the new generations of biomaterials

Small-animal PET registration method with intrinsic validation designed for large datasets

Proceeding of: 2007 IEEE Nuclear Science Symposium Conference Record (NSS'07), Honolulu, Hawaii, USA, Oct. 27 - Nov. 3, 2007We present a procedure to validate the results of small animal Positron Emission Tomography (PET) image registration by means of consistency measures. Small animal 2-Deoxy-2-[F-18]fluoro-D-glucose (FDG) PET studies do not show the same intensity distribution even for images acquired in similar conditions, as the resulting image is influenced by several variables which are not always completely under control. Because of these difficulties, the results from automatic registration methods have to be visually inspected to detect failures. We propose a method to automate this validation process. Two reference images from the dataset are selected by an expert user avoiding images with poor contrast, animal movement or low quality, and both are co-registered using anatomical landmarks. All the remaining images in the dataset are then registered to every reference with an automatic two-step algorithm based on Mutual Information. The known transformation relating both references allows measuring the registration consistency, which is a good estimator of the accuracy of the alignment process, for every image in the dataset. This value can be used to assess the quality of the registration and therefore detect the incorrect results. We have applied this validation process on a large dataset of 120 FDG-PET rat brain images obtained with a rotating PET scanner. The registration consistency was calculated for every image in the dataset and values below 1.65 mm (PET image resolution) were considered as successful registrations. 116 images were correctly registered with an average error of 0.839 mm, while in four images the proposed method detected a registration failure. Two of these failures were due to very low image quality and these studies were discarded from the study, while the other two were correctly aligned after applying a manual pre-alignment step. Our approach requires minimal user interaction and provides automatic assessment of the registration error, making it unnecessary to visually inspect and check every registration result.This work was supported by projects CIBER CB06/01/0079 (Ministerio de Sanidad y Consumo) and CDTEAM (CENIT program, Ministerio de Industria)

Can metacognitive interventions improve insight in schizophrenia spectrum disorders? A systematic review and meta-analysis

Background: Patients with schizophrenia spectrum disorders (SSD) tend to lack insight, which is linked to poor outcomes. The effect size of previous treatments on insight changes in SSD has been small. Metacognitive interventions may improve insight in SSD, although this remains unproved. Methods: We carried out a systematic review and meta-analysis of randomized controlled trials (RCTs) to examine the effects of metacognitive interventions designed for SSD, namely Metacognitive Training (MCT) and Metacognitive Reflection and Insight Therapy (MERIT), on changes in cognitive and clinical insight at post-treatment and at follow-up. Results: Twelve RCTs, including 10 MCT RCTs (n = 717 participants) and two MERIT trials (n = 90), were selected, totalling N = 807 participants. Regarding cognitive insight six RCTs (n = 443) highlighted a medium effect of MCT on self-reflectiveness at post-treatment, d = 0.46, p < 0.01, and at follow-up, d = 0.30, p < 0.01. There was a small effect of MCT on self-certainty at post-treatment, d = −0.23, p = 0.03, but not at follow-up. MCT was superior to controls on an overall Composite Index of cognitive insight at post-treatment, d = 1.11, p < 0.01, and at follow-up, d = 0.86, p = 0.03, although we found evidence of heterogeneity. Of five MCT trials on clinical insight (n = 244 participants), which could not be meta-analysed, four of them favoured MCT compared v. control. The two MERIT trials reported conflicting results. Conclusions: Metacognitive interventions, particularly Metacognitive Training, appear to improve insight in patients with SSD, especially cognitive insight shortly after treatment. Further long-term RCTs are needed to establish whether these metacognitive interventions-related insight changes are sustained over a longer time period and result in better outcomes

IL6/sIL6R Regulates TNFα-Inflammatory Response in Synovial Fibroblasts Through Modulation of Transcriptional and Post-Transcriptional Mechanisms

[Abstract] Introduction: The clinical efficacy of specific interleukin-6 inhibitors has confirmed the central role of IL6 in rheumatoid arthritis (RA). However the local role of IL6, in particular in synovial fibroblasts (SF) as a direct cellular target to IL6/sIL6R signal is not well characterized. The purpose of the study was to characterize the crosstalk between TNFα and IL6/sIL6R signaling to the effector pro-inflammatory response of SF. Methods: SF lines were stimulated with either TNFα, IL6/sIL6R, or both together, for the time and dose indicated for each experiment, and where indicated, cells were treated with inhibitors actinomycin D, adalimumab, ruxolitinib and cycloheximide. mRNA expression of cytokines, chemokines and matrix metalloproteases (MMPs) were analyzed by quantitative RT-PCR. Level of IL8/CXCL8 and CCL8 in culture supernatants was measured by ELISA. Mononuclear and polymorphonuclear cells migration assays were assessed by transwell using conditioned medium from SF cultures. Statistical analyses were performed as indicated in the corresponding figure legends and a p-value < 0.05 was considered statistically significant. Results: The stimulation of SF with IL6/sIL6R and TNFα, cooperatively promotes the expression of mono- and lymphocytic chemokines such as IL6, CCL8 and CCL2, as well as matrix degrading enzymes such as MMP1, while inhibiting the induction of central neutrophil chemokines such as IL8/CXCL8. These changes in the pattern of chemokines expression resulted in reduced polymorphonuclear (PMN) and increased mononuclear cells (MNC) chemoattraction by SF. Mechanistic analyses of the temporal expression of genes demonstrated that the cooperative regulation mediated by these two factors is mostly induced through de novo transcriptional mechanisms activated by IL6/sIL6R. Furthermore, we also demonstrate that TNFα and IL6/sIL6R cooperation is partially mediated by the expression of secondary factors signaling through JAK/STAT pathways. Conclusions: These results point out to a highly orchestrated response to IL6 in TNFα-induced SF and provide additional insights into the role of IL6/sIL6R in the context of RA, highlighting the contribution of IL6/sIL6R to the interplay of SF with other inflammatory cells.Instituto de Salud Carlos III; FIS 16/00032Instituto de Salud Carlos III; RETICS RD16/0012 RIE

Compilation of parameterized seismogenic sources in Iberia for the SHARE European-scale seismic source model.

Abstract: SHARE (Seismic Hazard Harmonization in Europe) is an EC-funded project (FP7) that aims to evaluate European seismic hazards using an integrated, standardized approach. In the context of SHARE, we are compiling a fully-parameterized active fault database for Iberia and the nearby offshore region. The principal goal of this initiative is for fault sources in the Iberian region to be represented in SHARE and incorporated into the source model that will be used to produce seismic hazard maps at the European scale. The SHARE project relies heavily on input from many regional experts throughout the Euro-Mediterranean region. At the SHARE regional meeting for Iberia, the 2010 Working Group on Iberian Seismogenic Sources (WGISS) was established; these researchers are contributing to this large effort by providing their data to the Iberian regional integrators in a standardized format. The development of the SHARE Iberian active fault database is occurring in parallel with IBERFAULT, another ongoing effort to compile a database of active faults in the Iberian region. The SHARE Iberian active fault database synthesizes a wide range of geological and geophysical observations on active seismogenic sources, and incorporates existing compilations (e.g., Cabral, 1995; Silva et al., 2008), original data contributed directly from researchers, data compiled from the literature, parameters estimated using empirical and analytical relationships, and, where necessary, parameters derived using expert judgment. The Iberian seismogenic source model derived for SHARE will be the first regional-scale source model for Iberia that includes fault data and follows an internationally standardized approach (Basili et al., 2008; 2009). This model can be used in both seismic hazard and risk analyses and will be appropriate for use in Iberian- and European-scale assessments

Cost-efficiency assessment of Advanced Life Support (ALS) courses based on the comparison of advanced simulators with conventional manikins

<p>Abstract</p> <p>Background</p> <p>Simulation is an essential tool in modern medical education. The object of this study was to assess, in cost-effective measures, the introduction of new generation simulators in an adult life support (ALS) education program.</p> <p>Methods</p> <p>Two hundred fifty primary care physicians and nurses were admitted to ten ALS courses (25 students per course). Students were distributed at random in two groups (125 each). Group A candidates were trained and tested with standard ALS manikins and Group B ones with new generation emergency and life support integrated simulator systems.</p> <p>Results</p> <p>In group A, 98 (78%) candidates passed the course, compared with 110 (88%) in group B (p < 0.01). The total cost of conventional courses was €7689 per course and the cost of the advanced simulator courses was €29034 per course (p < 0.001). Cost per passed student was €392 in group A and €1320 in group B (p < 0.001).</p> <p>Conclusion</p> <p>Although ALS advanced simulator systems may slightly increase the rate of students who pass the course, the cost-effectiveness of ALS courses with standard manikins is clearly superior.</p

Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) a-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions

Copper-catalyzed diastereo- and enantioselective desymmetrization of cyclopropenes: Synthesis of cyclopropylboronates

This document is the accepted manuscript version of a Published Work that appeared in final form in Journal of American Chemical Society 136.45, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see DOI: 10.1021/ja510419zA novel Cu-catalyzed diastereo- and enantioselective desymmetrization of cyclopropenes to afford nonracemic cyclopropylboronates is described. Trapping the cyclopropylcopper intermediate with electrophilic amines allows for the synthesis of cyclopropylaminoboronic esters and demonstrates the potential of the approach for the synthesis of functionalized cyclopropanesWe thank the European Research Council (ERC-337776) and MINECO (CTQ2012-35957) for financial support. M. T. and A. P. thank MICINN for RyC and JdC contract