Nucleic Acid Delivery by Solid Lipid Nanoparticles Containing Switchable Lipids: Plasmid DNA vs. Messenger RNA

The development of safe and effective nucleic acid delivery systems remains a challenge, with solid lipid nanoparticle (SLN)-based vectors as one of the most studied systems. In this work, different SLNs were developed, by combination of cationic and ionizable lipids, for delivery of mRNA and pDNA. The influence of formulation factors on transfection efficacy, protein expression and intracellular disposition of the nucleic acid was evaluated in human retinal pigment epithelial cells (ARPE-19) and human embryonic kidney cells (HEK-293). A long-term stability study of the vectors was also performed. The mRNA formulations induced a higher percentage of transfected cells than those containing pDNA, mainly in ARPE-19 cells; however, the pDNA formulations induced a greater protein production per cell in this cell line. Protein production was conditioned by energy-dependent or independent entry mechanisms, depending on the cell line, SLN composition and kind of nucleic acid delivered. Vectors containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as unique cationic lipid showed better stability after seven months, which improved with the addition of a polysaccharide to the vectors. Transfection efficacy and long-term stability of mRNA vectors were more influenced by formulation-related factors than those containing pDNA; in particular, the SLNs containing only DOTAP were the most promising formulations for nucleic acid delivery.This research was funded by the MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the UPV/EHU (GIU17/032)

MMP-9 Downregulation with Lipid Nanoparticles for Inhibiting Corneal Neovascularization by Gene Silencing

Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.This research was funded by the Ministerio de Economía y Competitividad (SAF2014-53092-R), by FEDER funds from the EU, and by the UPV/EHU (PPG17/65, GIU17/032). J Torrecilla and I Gómez-Aguado thank UPV/EHU for their research grants

α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.This research was funded by Merck Salud Foundation, MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU17/032)

mRNA-Based Nanomedicinal Products to Address Corneal Inflammation by Interleukin-10 Supplementation

The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated in vitro in human corneal epithelial (HCE-2) cells. Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.This research was funded by MCIU/AEI/FEDER, UE (SAF2014-53092-R), the UPV/EHU (GIU 20/048) and by the Università degli Studi di Torino (Ricerca Locale 2019)

Green spaces and cognitive development in primary schoolchildren

© 2015, National Academy of Sciences. All rights reserved. Exposure to green space has been associated with better physical and mental health. Although this exposure could also influence cognitive development in children, available epidemiological evidence on such an impact is scarce. This study aimed to assess the association between exposure to green space and measures of cognitive development in primary schoolchildren. This study was based on 2,593 schoolchildren in the second to fourth grades (7-10 y) of 36 primary schools in Barcelona, Spain (2012-2013). Cognitive development was assessed as 12-mo change in developmental trajectory of working memory, superior working memory, and inattentiveness by using four repeated (every 3 mo) computerized cognitive tests for each outcome. We assessed exposure to green space by characterizing outdoor surrounding greenness at home and school and during commuting by using high-resolution (5 m x5 m) satellite data on greenness (normalized difference vegetation index). Multilevel modeling was used to estimate the associations between green spaces and cognitive development. We observed an enhanced 12-mo progress in working memory and superior working memory and a greater 12-mo reduction in inattentiveness associated with greenness within and surrounding school boundaries and with total surrounding greenness index (including greenness surrounding home, commuting route, and school). Adding a traffic-related air pollutant (elemental carbon) to models explained 20-65% of our estimated associations between school greenness and 12-mo cognitive development. Our study showed a beneficial association between exposure to green space and cognitive development among schoolchildren that was partly mediated by reduction in exposure to air pollution

Gene-terapia: ikuspegi terapeutiko berria begietako gaitzen tratamenduan

Gene-terapia etorkizun handiko tresna bezala sortu da tratamendurik ez duten asaldurentzat. Azido nukleiko terapeutikoen administrazioan oinarritzen da gaixotasunak tratatzeko. Gene-terapia arrakastatsua izan dadin material genetikoaren askapen eraginkorra bermatu behar da itu-zeluletan. Geneen administrazio-sistemen artean, bektore biralak asko erabili dira ahalbidetzen duten transferentzia genikorako gaitasun onarengatik. Hala ere, horien arrisku nagusien ondorioz (immunogenizitatea eta mutagenesia), bektore ez-biralen diseinua sustatu da. Bektore ez-biralak seguruagoak dira eta ekoizpena errazagoa da, baina hauen muga nagusia transfekzio-eraginkortasun baxua da. Gene-terapiarako organo interesgarri bat begia da, eskuragarria eta aztertzeko erraza baita. Gainera, immunitate-sistematik babestuta dago. Gene-terapia entsegu kliniko guztien % 1,3 bakarrik tratatzen dituzte begietako gaitzak, baina etorkizun handia aurkeztu dute azken urteetan. Izan ere, 2018an Estatu Batuetan eta Europan gene-terapian oinarritutako begirako lehen medikamentuaren komertzializazioa onartu zen, Luxturna®, Sortzetiko Leberren Amaurosiaren tratamendurako. Berrikuspen honetan, begiko administrazio-bideak, gene-terapia estrategiak eta transferentzia geniko eraginkorrerako gainditu behar diren begiko mugak aurkezten dira. Halaber, gene-terapiaren bidez tratatzeko hautagai diren begietako gaitz ezberdinak ere biltzen dira. Gene-terapiaren inguruan egindako ahaleginei eta aurrerapenei esker, begietako gaitzak tratatzeko medikamentu berriak garatu dira. Horietako asko entsegu klinikoetan ebaluatzen ari dira oraindik, baina beste batzuk jada merkatura eta pazienteetara iritsi dira.; Gene therapy has emerged as a promising tool for disorders that have no cure. It consists in the administration of therapeutic nucleic acids into patients for treating diseases. The success of gene therapy relies on the efficient delivery of the genetic material to target cells. Among gene delivery systems, viral vectors have been widely used due to their good gene transference efficacy. However, their potential risk associated with immunogenicity and mutagenesis has promoted the design of non-viral vectors. Non-viral vectors are safer and easier to produce, but their main limitation remains lower transfection efficacy. An attractive candidate for gene therapy is the eye, since it is easily accessible, easily examined and relatively immune privileged. Only 1,3% of all gene therapy clinical trials treat ocular disorders, but they have shown great potential in recent years. In fact, in 2018 the Food and Drug Administration and the European Medicine Agency approved the commercialization of the first gene therapy based ocular drug, Luxturna®, for the treatment of Leber Congenital Amaurosis. In this review, we present the main administration routes to eye, gene therapy strategies and ocular barriers to overcome for successful gene transfer. Different ocular disease candidates to be treated by gene therapy are also reviewed. The efforts and advances made in the field of gene therapy have led to the development of new drugs to treat eye diseases. Many of them are still being evaluated in clinical trials, but some have already reached the market and patients

La enseñanza del derecho en el siglo XX: Homenaje a Mariano Peset

Este libro cierra el tracto que se inició, en octubre de 2002 con la investidura como doctor honoris causa del profesor Mariano Peset. El hecho de que la historia de las universidades haya sido una línea ininterrumpida a lo largo de su investigación y el que hayamos tratado de materializar sus enseñanzas de tantos años en el Instituto Antonio de Nebrija de Estudios sobre la Universidad explican que le hayamos dedicado este homenaje científico en un seminario que tuvo lugar en el campus de Colmenarejo de la Universidad Carlos III durante los días 22 y 23 de septiembre de 2003. Quisimos que el encuentro científico tuviera carácter monográfico, para centrar el interés de nuestro encuentro y evitar la dispersión de los discursos. Y pensamos que tratar de la enseñanza del derecho en el siglo XX era un buen tema por varias razones, entre las que subrayo dos: porque la centuria pasada ya puede ser tratada históricamente y, sobre todo, para continuar la labor de Mariano Peset que, en su día, hizo lo propio para el siglo XIX, con incursiones también en el que nos ocupa en este libro.Prólogo / Adela Mora Cañada. -- La enseñanza de la historia del derecho y la LRU / Ramon Aznar i Garcia y Manuel Martínez Neira. -- Vicente Santamaría de Paredes, político y administrativista / Yolanda Blasco Gil. -- La enseñanza del Derecho en Valencia durante la autonomía de César Silió (1919-1921) / Daniel Comas Caraballo. -- Críticas a la codificación foral prevista en el código civil a la luz de la doctrina jurídico-civil contemporánea / Manuel Vicente Febrer Romaguera. -- La Facultad de Derecho de Valencia en el primer tercio del siglo xx (1900-1938) / Jorge Correa y Fco. Javier Palao. -- La política desde una asignatura: el Derecho Natural / Eusebio Fernández García. -- La institución del Senado en los catedráticos de Derecho Político (1900-1940) / Pilar García Trobat. -- Catedráticos de derecho en la Asamblea Nacional Consultiva de 1927 / María Pilar Hernando Serra. -- El Derecho político en Valencia, con especial referencia a Mariano Gómez González (1915-1932) / M.ª Fernanda Mancebo. -- Fe católica y razón liberal en el Derecho político. La anónima relevancia de Juan de Dios Vico y Brabo (1845-1908) / Sebastián Martín. -- Una polémica profesional: catedráticos y magistrados durante la II República / Pascual Marzal Rodríguez. -- Centro y periferia: el Doctorado en Derecho durante el siglo XX / Antonio Merchán. -- La irrupción de nuevos sectores: el Derecho Laboral / Miguel C. Rodríguez-Piñero Royo. -- El trabajo de cátedra de Adolfo Posada / Mónica Soria. -- Sobre los inicios en la enseñanza del Derecho del Trabajo / Carlos Tormo Camallonga. -- La enseñanza del mercantil a principios del siglo XX. El manual de Lorenzo Benito y Endara / Sergio Villamarín Gómez. – Clausura / Mariano Pese

The nuclear receptor LXR limits bacterial infection of host macrophages through a mechanism that impacts cellular NAD metabolism

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of nonopsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bonemarrow- derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway

Correction : Chaparro et al. Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain: Large-Scale Epidemiological Study. J. Clin. Med. 2021, 10, 2885

The authors wish to make the following corrections to this paper [...]

Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain : Large-Scale Epidemiological Study

(1) Aims: To assess the incidence of inflammatory bowel disease (IBD) in Spain, to describe the main epidemiological and clinical characteristics at diagnosis and the evolution of the disease, and to explore the use of drug treatments. (2) Methods: Prospective, population-based nationwide registry. Adult patients diagnosed with IBD-Crohn's disease (CD), ulcerative colitis (UC) or IBD unclassified (IBD-U)-during 2017 in Spain were included and were followed-up for 1 year. (3) Results: We identified 3611 incident cases of IBD diagnosed during 2017 in 108 hospitals covering over 22 million inhabitants. The overall incidence (cases/100,000 person-years) was 16 for IBD, 7.5 for CD, 8 for UC, and 0.5 for IBD-U; 53% of patients were male and median age was 43 years (interquartile range = 31-56 years). During a median 12-month follow-up, 34% of patients were treated with systemic steroids, 25% with immunomodulators, 15% with biologics and 5.6% underwent surgery. The percentage of patients under these treatments was significantly higher in CD than UC and IBD-U. Use of systemic steroids and biologics was significantly higher in hospitals with high resources. In total, 28% of patients were hospitalized (35% CD and 22% UC patients, p < 0.01). (4) Conclusion: The incidence of IBD in Spain is rather high and similar to that reported in Northern Europe. IBD patients require substantial therapeutic resources, which are greater in CD and in hospitals with high resources, and much higher than previously reported. One third of patients are hospitalized in the first year after diagnosis and a relevant proportion undergo surgery