Synthetic Pulmonary Surfactant Preparations: New developments and future trends

Pulmonary surfactant is a lipid-protein complex that coats the interior of the alveoli and enables the lungs to function properly. Upon its synthesis, lung surfactant adsorbs at the interface between the air and the hypophase, a capillary aqueous layer covering the alveoli. By lowering and modulating surface tension during breathing, lung surfactant reduces respiratory work of expansion, and stabilises alveoli against collapse during expiration. Pulmonary surfactant deficiency, or dysfunction, contributes to several respiratory pathologies, such as infant respiratory distress syndrome (IRDS) in premature neonates, and acute respiratory distress syndrome (ARDS) in children and adults. The main clinical exogenous surfactants currently in use to treat some of these pathologies are essentially organic extracts obtained from animal lungs. Although very efficient, natural surfactants bear serious defects: i) they could vary in composition from batch to batch; ii) their production involves relatively high costs, and sources are limited; and iii) they carry a potential risk of transmission of animal infectious agents and the possibility of immunological reaction. All these caveats justify the necessity for a highly controlled synthetic material. In the present review the efforts aimed at new surfactant development, including the modification of existing exogenous surfactants by adding molecules that can enhance their activity, and the progress achieved in the production of completely new preparations, are discussed

Obtención de células pluripotentes inducidas específicas de pacientes con retinosis pigmentaria

La terapia celular ha demostrado ser una alternativa revolucionaria para la degeneración de retina mediante el trasplante de células. Este enfoque ha sido posible gracias a la generación de células de pluripotencia inducida (iPS) como fuente de células específica de pacientes y de ciertas enfermedades con la ventaja de eludir la respuesta inmune y evitar los problemas éticos relacionados con el uso de células madre embrionarias. Nuestro objetivo consiste en la optimización del proceso de reprogramación para la obtención de células específicas de pacientes con distrofias retinianas (DR) como retinitis pigmentosa (RP) o Amaurosis Congenita de Leber. Las DR hereditarias representan individualmente enfermedades raras y constituyen la forma más común de ceguera hereditaria debido a una degeneración de los fotorreceptores. Actualmente no existen tratamientos eficaces para las DR lo que nos conduce a impulsar el desarrollo de nuevos enfoques terapéuticos. La metodología principal se fundamenta en varios procesos de reprogramación exentos de integración en el genoma y con eficiencia relevante. En los estudios iniciales realizados, se han generado células iPS a partir de fibroblastos de pacientes, siendo uno de ellos portador de una mutación responsable de la RP, a través de tecnologías no integrativas de reprogramación por mARN (Yakubov E et al, 2010), vectores episomales (Junying Yu et al, 2009) y Sendai virus (Fusaki N et al. 2009) como portadores de los factores de transcripción claves para activar la pluripotencia. Los resultados obtenidos nos permiten comparar la eficiencia de las distintas tecnologías aplicadas. Las líneas de iPS obtenidas se están caracterizando por criterios morfológicos y expresión de marcadores de pluripotencia (NANOG, OCT4, SOX2, SSEA-4, TR-60-1 y TR-1-81) mediante inmunocitoquímica, ensayos de fosfatasa alcalina y expresión génica, además son capaces de diferenciarse en las tres capas germinales cuando se someten a ensayos de diferenciación in vitro (Lowry et al, 2008). Hasta el momento y en el transcurso de nuestros experimentos se ha demostrado que la posibilidad de reprogramar células somáticas constituye una herramienta única para la obtención de células paciente-específicas. Tras finalizar la caracterización de las líneas celulares pluripotentes obtenidas mediante cada una de la tecnologías aplicadas se seleccionará la que sea más eficaz para su uso en el futuro

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimer- isation motif that mediates transmembrane (TM) helix associa- tion, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Fur- ther, we have used this minimised GpA motif to assess the influ- ence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix-helix association and/or dissociation between TM fragments

Generation of an iPSC line from a retinitis pigmentosa patient carrying a homozygous mutation in CERKL and a healthy sibling

Dermal fibroblasts from an autosomal recessive retinitis pigmentosa (RP) patient, homozygous for the mutation c.769 C>T, p.Arg257Ter, in CERKL (Ceramide Kinase-Like) gene, and a healthy sibling were derived and reprogrammed by Sendai virus. The generated human induced pluripotent stem cell (hiPSC) lines RP3-FiPS4F1 and Ctrl3-FiPS4F1, were free of genomically integrated reprogramming genes, showed stable karyotypes, expressed pluripotency markers and could be differentiated towards the three germ layers in vitro. These hiPSC lines offer a useful resource to study RP pathomechanisms, drug testing and therapeutic opportunities

Retinal Organoids derived from hiPSCs of an AIPL1-LCA Patient Maintain Cytoarchitecture despite Reduced levels of Mutant AIPL1

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor-specific chaperone that stabilizes the effector enzyme of phototransduction, cGMP phosphodiesterase 6 (PDE6). Mutations in the AIPL1 gene cause a severe inherited retinal dystrophy, Leber congenital amaurosis type 4 (LCA4), that manifests as the loss of vision during the first year of life. In this study, we generated three-dimensional (3D) retinal organoids (ROs) from human induced pluripotent stem cells (hiPSCs) derived from an LCA4 patient carrying a Cys89Arg mutation in AIPL1. This study aimed to (i) explore whether the patient hiPSC-derived ROs recapitulate LCA4 disease phenotype, and (ii) generate a clinically relevant resource to investigate the molecular mechanism of disease and safely test novel therapies for LCA4 in vitro. We demonstrate reduced levels of the mutant AIPL1 and PDE6 proteins in patient organoids, corroborating the findings in animal models; however, patient-derived organoids maintained retinal cell cytoarchitecture despite significantly reduced levels of AIPL1.This work was supported by Institute of Health Carlos III (ISCIII)/ ERDF (European Research Development Fund), Spain, ((PI16/00409 (DL); DL, AAC, and SE are members of PRB3 supported by a grant (PT17/0019/0024) of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. The work was also supported by ISCIII-ERDF (PI16/00425), CIBERER 06/07/0036, IIS-FJD Biobank PT13/0010/0012, RAREGENOMICS funded by Regional Government of Madrid, (CAM, B2017/BMD3721) and ERDF, the University Chair UAM-IIS-FJD of Genomic Medicine, the Spanish National Organization of the Blind (ONCE), the Spanish Fighting Blindness Foundation (FUNDALUCE), and the Ramon Areces Foundation. MC is supported by the Miguel Servet Program (CPII17_00006) from ISCIII. DL is supported by Miguel Servet I Program (CP18/00033). VR is supported by National Institute of Health (R01 EY028035, R01 EY025536). Transcriptome profiling and analyses were supported by the Intramural Research Program of the National Eye Institute (ZIAEY000450, ZIAEY000474) and utilized the high-performance computational capabilities of the Biowulf Linux cluster at NIH (http://biowulf.nih.gov)

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities.This work was supported by Institute of Health Carlos III/ERDF (European Regional Development Fund), Spain [PI16/00409 (DL), PI20/01119 (DL), CP18/00033 (DL), PI15/00227 (MC), CPII16/00037 (SE), and PI18-00286 (SE)], Platform for Proteomics, Genotyping and Cell Lines; PRB3 of ISCIII (PT17/0019/0024); National Science Foundation GACR 18-04393S and the project “Centre of Reconstructive Neuroscience”, registration number CZ.02. 1.01/0.0./0.0/15_003/0000419PI15/00227; Spanish Ministry of Economy and Competitiveness grant BES-2016-076994 (ÁA-L); and Academy of Finland (HS)

Human iPSC derived disease model of MERTK-associated retinitis pigmentosa

Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches.This work was supported by Andalusian Health Council (PI-0324-2013), Instituto de Salud Carlos III (PI13/01331), Spanish Ministry of Economy and Competitiveness-FEDER BFU2012-36845, Instituto de Salud Carlos III RETICS RD12/0034/0010 and Academy of Finland (218050; 272808)

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich α helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% α-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression π-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations

Recombinant production and characterization of surfactant protein C (SP-C)

La proteina SP-C forma parte del surfactante pulmonar, una mezcla de lípidos y proteínas que recubre los alvéolos, indispensable para la respiración. la SP-C e5 un componente crítico en las preparaciones usadas para el tratamiento del sindrome de distrés respiratorio en los fetos prematuros. El objetivo principal de esta tesis ha sido la puesta a punto de un nuevo método de producción de la proteína SP-C, mediante tecnología recombinante que podría servir como base para la obtención de nuevos surfactantes sintéticos, asi como una herramienta inmejorable en la cararterización de determinantes estructura-función con el objetivo último de intentar mejorar las propiedades tensioactivas de la proteína. Hemos descrito un nuevo método de obtención de la proteína SP-C recombinante de cultivos bacterianos, que está basado en una estrategia de sobreexpresión de proteínas transmembrana en procariotas y en las extracciones orgánicas empleadas en el aislamiento de la SP-C nativa a partir de lavados pulmonares. Este método resultó altamente eficaz en la preservación de la estructura helicoidal de la proteína, la principal caraderistica de su actividad. Además hemos diseñado variantes de la SP-C con dos triptófanos y dos cisteinas en las posiciones 5 y 6 para estudiar el efecto de los resíduos aromáticos en el segmento N-terminal de la proteína. En este caso el objetivo era comprobar hasta que punto una mayor actividad de la proteína podía estar definida par una mayor afinidad del segmento N-terminal hacia las interfases comparativamente con la secuencia utilizada inicialmente que presenta dos resíduos de fenidalarina en las posiciones mencionadas. Según esta hipótesis la variante con dos triptófanos podría mostrar una actividad aún mayor que la forma de SP-C que posee fenilalaninas, mientras que la que pasee doS cisteínas podría mostrar una actividad inferior. Los resultados experimentales de los análisis estructurales mostraron qu