'Ex vivo' gene correction of PRPF31 c.165G>A mutation causing retinitis pigmentosa

Motivation: Retinitis pigmentosa (RP) is the most common form of retinal dystrophy, a group of blinding diseases characterized by progressive photoreceptor death, with a prevalence of 1 in 4000. RP is highly-heterogeneous, with 15% of autosomal dominant cases caused by mutations in the pre-mRNA processing factors (PRPFs), components of the spliceosome.To date, there are no effective treatments for RP. Gene editing is a rapidly evolving field that may in the future, allow the repair of a mutated endogenous locus. CRISPR/Cas9 system has a mechanism of action based on nucleotide recognition of target DNA by engineered single-guide RNA (sgRNA) and Cas9 endonuclease activity. Genomic edition of patient-derived induced pluripotent stem cells (iPSCs) would allow autologous transplantation of repaired cells, once differentiated to retinal cell types.Methods: iPSCs obtained from a RP patient with a PRPF31 c.165G>A mutation were the starting biological material. Pluripotency of the iPSCs was checked by inmunofluorescence (IF) analysis.Disease phenotyping of the cell line was performed by IF for PRPF31 and for the ciliary protein ARL13B, as PRPF31 mutations have been previously described to affect cilia.sgRNAs directed to the mutation were designed using the web crispor.tefor.net. The best sgRNA and a ssODN template, covering the mutation site, were synthesized by IDT. The sgRNA-CRISPR/Cas9 complex was assembled and co-transfected with the ssODN into the iPSCs. FACs was used to measure the efficiency and to select transfected cells. A bulk transfected cell population was analyzed by Sanger sequencing to check for HR-mediated knock-in. Selection of individual iPSC clones and genotyping is being performed to search  for corrected clones.Results: Positive labeling for OCT4, NANOG, SSEA3, SSEA4 and TRA-1-81 showed pluripotency of the iPSC line. PRPF31 immunolocalization and quantificacion have been used to phenotype the iPSC line compared to a healthy control. Sanger sequencing of the genomic DNA showed successful editing of the mutation in the bulk population of transfected cells. Different culture conditions were tested for iPSC clonal selection. Best conditions provided a 0.8 % of efficiency. The CRISPR/Cas9-corrected iPSC clone will be differentiated to retinal pigment epithelium (RPE) and photoreceptors, in parallel with uncorrected PRPF31-iPSCs, to establish if in situ gene editing restores key celular and functional phenotypes associated with this type of RP

European and comparative law study regarding family’s legal role in deceased organ procurement

Several European countries are approving legislative reforms moving to a presumed consent system in order to increase organ donation rates. Nevertheless, irrespective of the consent system in force, family's decisional capacity probably causes a greater impact on such rates. In this contribution we have developed a systematic methodology in order to analyse and compare European organ procurement laws, and we clarify the weight given by each European law to relatives' decisional capacity over individual's preferences (expressed or not while alive) regarding the destination of his or her organs after death. In this sense, the results constitute the first comprehensive and comparative legislative mapping on European transplantation laws

IGFBPs mediate IGF-1's functions in retinal lamination and photoreceptor development during pluripotent stem cell differentiation to retinal organoids

Abstract Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner

La confiscación de órganos a la luz del derecho constitucional a la protección de la salud

This paper analyses the arguments for and against what we have called automatic organ procurement model in relation to the organs of the deceased. For this purpose, this work provides empirical evidence to assess the potential impact of this model on donation rates and on public opinion. Specifically, we examine first the reasons supporting this model, with special reference to utilitarian and justice arguments. On the other hand, we analyse both the approaches based on the violation of pre mortem and post mortem interests opposing this theoretical model and the rejection the model would generate in the population. Finally, we point out the aspects that, in our opinion, should be exhaustively regulated if this model were implemented. In particular, we refer to the legal status of the human body, the treatments for end-of-life patients, the incentives for health professionals and the recognition of the right to conscientious objectio

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: a Comprehensive Validation Using Drugs and Compounds Affecting the Retina

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs

Incorporating microglia‐like cells in human induced pluripotent stem cell‐derived retinal organoids

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches

Rede de Aerobiologia da Extremadura

The development of aerobiological networks in Spain has been made at the level of the autonomous communities. In Extremadura the sampling is conducted by the University of Extremadura. It began in 1993 with a station in Badajoz (BA). Two towns have had sampling on a temporary basis: Cáceres (CC 1996-2001) and Merida (ME 1996-1998). Currently there are 3 more sampling stations, which have been running since 2011: Plasencia (PL), Don Benito (DB) and Zafra (ZA), and CC again recently. The Annual Pollen Index (annual daily amounts) has shown an average of more than 50.000 pollen grains/m3 (23.532-92.655). In Badajoz (23 years) the results show a downward trend. The maximum daily concentration peaks were reached in 1997, with values of 6.102 grains/ m3 (CC 21/3) and 5.041 grains/m3 (BA 23/3). The five most important pollen types represent 78% of all the pollen registered. Their importance varies from one station to another. Quercus, Poaceae, Olea, Cupressaceae and Platanus, in this order, are the most abundant pollen types in all stations, except Platanus, which is the second most abundant in DB, and Olea, which is second in ZA. The data have been available on the research group’s website (www.aerouex.es) since 2006, and the record of visitors to the site shows a signi cant correlation with the concentration of pollen. Extremadura stands out for its high pollen concentrations of Poaceae and Quercus, due to its wide expanses of oak and cork trees. Pollen from ornamental sources –Cupressaceae and Platanus— shows a strong dependence on their abundance and distribution.El desarrollo de redes aerobiológicas en España se ha realizado a nivel de las comunidades autónomas. En Extremadura el muestreo llevado a cabo por la Universidad de Extremadura comenzó en 1993 con la estación de Badajoz (BA). Dos localidades han tenido muestreo de forma temporal, Cáceres (CC 1996-2001) y Mérida (ME 1996-1998). En la actualidad se cuenta con 3 estaciones de muestreo más, funcionando desde 2011: Plasencia (PL), Don Benito (DB) y Zafra (ZA) y, de forma reciente, nuevamente CC. El Índice Polínico Anual (la suma de las concentraciones de polen diarias para un año) ha mostrado un promedio de más de 50.000 granos/m3 (23.532-92.655). Para Badajoz (23 años) se aprecia una tendencia a la reducción. Los picos de concentración diaria máxima se alcanzaron en 1997 con valores de 6.102 granos/m3 (CC 21/3) y 5.041 granos/m3 (BA 23/3). Los cinco tipos polínicos más relevantes representan el 78% del total de polen registrado. Su importancia varía de una estación a otra. Quercus, Poaceae, Olea, Cupressaceae y Platanus, en este orden, son los tipos más abundantes en todas las estaciones, excepto Platanus que es el segundo en DB y Olea que es el segundo en ZA. Desde 2006 los datos están disponibles a través de la página web del grupo de investigación (www.aerouex.es) y el registro de los accesos a dicho sitio muestra una correlación significativa con la concentración de polen. Extremadura se destaca por los altos valores de concentración de polen de Quercus y Poaceae, debido a la gran extensión de encinares y alcornocales. El polen de fuentes ornamentales, Cupressaceae y Platanus, muestra una importante dependencia de su abundancia y distribución en las localidades estudiadas.O desenvolvimento das redes de aerobiologia em Espanha foi realizado ao nível das comunidades autónomas. Na Extremadura a amostragem levada a cabo pela Universidade de Extremadura começou em 1993 com a estação de Badajoz (BA). Duas localidades foram temporariamente estudadas como pontos de amostragem, Cáceres (CC 1996-2001) e Mérida (ME 1996-1998). Existem, atualmente, em execução desde 2011, mais 3 estações de amostragem: Plasencia (PL), Don Benito (DB) e Zafra (ZA) e, recentemente, de novo CC. O Índice Polínico Anual (somas diárias anuais) mostrou uma média de mais de 50.000 grãos/m3 (23.532-92.655). Em Badajoz (23 anos) verifica-se uma tendência para a redução da concentração. Os picos de concentração máximos diários foram alcançados em 1997 com os valores de 6.102 grãos/m3 (CC 21/3) e 5.041 grãos/m3 (BA 23/3). Os cinco tipos polínicos mais importantes representam 78% de pólen total registrado. A sua importância varia de uma estação para outra. Quercus, Poaceae, Olea, Cupressaceae e Platanus, nesta ordem, são os tipos mais abundantes em todas as estações, exceto Platanus que é o segundo em DB e Olea que é o segundo em ZA. Os dados estão disponíveis desde 2006 através do site do grupo de investigação (www. aerouex.es) e o registro de acessos mostra uma correlação significativa com a concentração de pólen. A Extremadura destaca-se pelos valores elevados de concentração de pólen de Poaceae e de Quercus, devido à grande extensão de azinheiras e sobreiros. O pólen de origens ornamentais, Cupressaceae e Platanus, mostra uma dependência significativa de sua abundância e distribuição nas localidades estudadas

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe