Estudios genéticos, fisiopatológicos y con oligonucleótidos antisentido en enfermedades neurometabólicas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-07-2015In this work, we have characterized the molecular basis of the defect in fibroblasts samples from 10 patients diagnosed with propionic acidemia (PA), one of the most common organic acidemias. In addition, we have investigated oxidative stress and cellular damage in PA patients´ fibroblasts. Furthermore, using the hypomorph mouse model of PA we have characterized the mitochondrial dysfunction and oxidative damage and studied the behavior and motor coordination. Finally, we have tested in vivo the efficiency of splice-modulation using antisense oligonucleotides (AONs), which are a promising therapy for specific splicing defects. To provide molecular insight into the pathogenicity of PA variants we have analyzed the structural and functional effects of five point mutations in PCCA and PCCB genes, by using homology modeling with the recently solved crystal structure of the PCC holoenzyme and an eukaryotic expression system. The results confirmed the pathogenic nature of these mutations, showing a more severe effect in those ones located near the active site in the a subunit compared to mutations located between b subunits that showed higher residual PCC activity. We have performed in silico and functional analysis using minigenes to analyze a point mutation in the last nucleotide of the exon 7 of the PCCB gene, revealing it is a partial splicing defect and underscoring the need of carefully evaluating every nucleotide change for a possible splicing defect. Moreover, we have investigated oxidative stress analyzing intracellular ROS levels, antioxidant enzymes´ levels and apoptosis parameters in PA patients-derived fibroblasts. Our results indicate that oxidative stress may be an important contributing factor to the cellular damage. Additionally, we have shown that different antioxidants successfully scavenge ROS production in these patients´ cells. These results establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA. To gain further insights into the physiopathology of PA, we have used the mouse model of PA to study the expression of several proteins involved in mitochondrial function by Reverse Phase Protein Microarrays (RPPMa), OXPHOS activities, mtDNA copy number and oxidative damage. The results showed impairment of oxidative phosphorylation, increased ROS production, lipid peroxidation and mtDNA depletion in relevant tissues. To characterize behavioral and locomotive defects, we have carried out open-field test and rota-rod performance. The results indicate age-dependent alterations in locomotion, coordination and endurance. These data support the hypothesis that mitochondrial dysfunction may contribute to the physiopathology of PA. Finally, in the heterozygous PKU mouse model and in wildtype mice we have tested AONs using different dosing, mode of injection and treatment regimes. The results showed that consecutive intravenous injections of optimal doses result in transient hyperphenylalaninemia correlating with complete exon skipping and absence of PAH protein and enzyme activity. These results provide a sensitive in vivo assay to test the ability of AONs to modulate splicing, as well as a possible method to generate murine models for genetic liver diseasesEn este trabajo, se han caracterizado algunas mutaciones identificadas en fibroblastos de 10 pacientes diagnosticados con acidemia propiónica (AP), una de las acidemias orgánicas más comunes. Además, se ha investigado el estrés oxidativo y el daño celular en los fibroblastos de estos pacientes. Asimismo, utilizando el modelo hipomorfo murino de AP se ha caracterizado el daño oxidativo y la disfunción mitocondrial y estudiado el comportamiento y la coordinación motora. Por último, se ha probado la eficiencia in vivo de la modulación de splicing mediante el uso de oligonucleótidos antisentido (AONs), que constituyen una terapia prometedora para los defectos específicos de splicing. Para proporcionar un mejor entendimiento molecular de la patogenicidad de variantes en AP se ha analizado el efecto estructural y funcional de cinco mutaciones puntuales en los genes PCCA y PCCB, mediante el modelado por homología de la estructura cristalina recientemente resuelta de la holoenzima PCC y un sistema de expresión eucariota. Los resultados confirmaron la naturaleza patogénica de estas mutaciones, que muestran un efecto más severo en aquellas variantes situadas cerca del centro activo en la subunidad α en comparación con mutaciones localizadas entre las subunidades β que mostraron una mayor actividad PCC residual. Se ha llevado a cabo el análisis in silico y funcional empleando minigenes para analizar una mutación puntual en el último nucleótido del exon 7 del gen PCCB, mostrando un defecto de splicing parcial que subraya la necesidad de evaluar cuidadosamente todos los cambios nucleotídicos como posibles defectos de splicing. Por otra parte, se ha investigado el estrés oxidativo analizando los niveles intracelulares de ROS y de enzimas antioxidantes y parámetros relacionados con la apoptosis en los fibroblastos de pacientes con AP. Nuestros resultados indican que el estrés oxidativo puede ser un importante factor que contribuye al daño celular. Además, se ha demostrado con éxito que diferentes antioxidantes neutralizan la producción de ROS en las células de estos pacientes. Estos resultados establecen la prueba de concepto del posible uso de antioxidantes como una terapia adyuvante en AP. Para obtener un mayor conocimiento en la fisiopatología de la AP, se ha utilizado el modelo de ratón de AP para estudiar la expresión de varias proteínas implicadas en la función mitocondrial mediante microarrays de proteínas de fase reversa, midiendo las actividades OXPHOS, el número de copias de DNA mitocondrial y el daño oxidativo. Los resultados mostraron una alteración en la fosforilación oxidativa, un aumento de la producción de ROS y de lipoperoxidación y una depleción de DNA mitocondrial en tejidos relevantes. Para caracterizar defectos locomotores y de comportamiento, se han llevado a cabo pruebas de campo abierto y pruebas de rota-rod. Los resultados sugieren alteraciones dependientes de la edad en la locomoción, la coordinación y la resistencia. Todos estos datos apoyan la hipótesis de que la disfunción mitocondrial puede contribuir a la fisiopatología de la AP. Finalmente, se han contrastado diferentes dosis, modos de inyección y pautas de administración de oligonucleótidos antisentido en el modelo de ratón PKU heterocigoto y en ratones wildtype. El estudio mostró que inyecciones intravenosas consecutivas de dosis óptimas resultan en una hiperfenilalaninemia transitoria que se correlaciona con un completo exon skipping y con la ausencia de proteína y actividad enzimática PAH. Estos resultados proporcionan un ensayo eficaz in vivo para probar la capacidad de los AONs para modular el splicing, así como un posible método para generar modelos murinos para enfermedades genéticas hepáticas.Este trabajo ha sido posible gracias a una ayuda de posgrado para la Formación de Personal Investigador (BES-2011-045011) y a una ayuda para estancias breves (EEBB-I-2012-04671

Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort

Background Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster.Methods Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3.Results Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3.Conclusions During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Repair of Rat Mandibular Bone Defects by Alveolar Osteoblasts in a Novel Plasma-Derived Albumin Scaffold

9 páginas, 6 figuras, 1 tabla.-- et al.Repair of bone deficiencies in the craniofacial skeleton remains a difficult clinical problem. The aim of this study was to evaluate a novel albumin scaffold seeded with human alveolar osteoblasts and implanted into experimental mandibular defects. An experimental solid protein scaffold was prepared with human plasmatic albumin crossed with a glutaraldehyde-type agent. Microstructure of scaffold and mechanical properties were examined using scanning electron microscopy and a stress-controlled rheometer. Bilateral critical mandibular defects were created in eight immunodeficient rats. Defects of the right side of the mandibles received the cell–scaffold construct in all animals. All left mandibular defects were left untreated as blank controls. Sections of the defects were collected at 5, 8, and 11 weeks postsurgery and processed for histological and immunohistochemical observation, computed tomography examination, and computed tomography digital analysis. Histologically, bone formation was observed in both groups at 5 weeks postsurgery, and the engineered bone became more mature after 8 and 11 weeks, which was similar to normal bone. The origin of bone-forming cells within the defects and the localization of implanted human osteoblasts were confirmed by human vimentin expression. No bone formation could be observed at any control defect. Bone density after 8 weeks was significantly higher than that of the 5-week group (p = 0.02), and significant differences were also observed between 8 and 11 weeks (p < 0.01). The results indicate the clinical feasibility of albumin scaffold loaded with human alveolar cells and that it can be used as a good alternative for bone regeneration.This study was supported by the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (PI070174).Peer reviewe

Segmental uniparental disomy leading to homozygosity for a pathogenic mutation in three recessive metabolic diseases

Ministerio de Ciencia e Innovación (SAF2010-17272)Peer Reviewe

Treatment of Cystathionine β-Synthase Deficiency in Mice Using a Minicircle-Based Naked DNA Vector

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs$^{-/-}$) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs$^{-/-}$ mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 μM before injection to 176 μM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency