Interferon Action: A. Studies of the Effect of Interferons on SV40 Replication. B. Studies of the 2\u27-5\u27 A System

This thesis is divided into two parts: the first deals with studies that focus on the effect of interferons on the expression of foreign genetic material. The second part deals with the interferon-regulated (2\u275\u27)oligoadenylate system. This system is most probably an effector arm of the anti-viral anti-mitogenic actions of interferons.I show here that interferons inhibit the expression of both early SV40 RNA and SV40 Tag, and SV40 DNA replication in SV40 infected cells. Interferons selectively inhibit the expression of the Tag encoded by superinfecting SV40 virions in SV40-transformed cells superinfected with SV40. The effect of interferon was seen when cells were transfected with SV40 DNA or infected with SV40 virions.Data shown indicates that interferons inhibit the expression of bacterial genes driven by the SV40 early promoter-enhancer elements. Moreover, interferon treatment inhibits the expression of a bacterial gene driven by cellular promoter-enhancer elements, when newly transfected into cells. Thus, it seems that interferons can distinguish between stably integrated genes and newly arriving genes.The (2\u275\u27)oligoadenylate system is composed of the (2\u275\u27)oligoadenylate synthetases, the RNase L, a (2\u275\u27)phosphodiesterase, and the (2\u275\u27)oligoadenylates. In the presence of double stranded RNA the (2\u275\u27)oligoadenylate synthetases convert ATP into (2\u275\u27)oligoadenylates. The RNase L is a latent endonuclease that when activated by binding a (2\u275\u27)oligoadenylate cleaves on single stranded regions of RNA. A scheme for the partial purification of the RNase L from calf spleens is presented. Furthermore, the discovery of a (2\u275\u27)oligoadenylate binding protein in wheat is described.Growth conditions profoundly affect the (2\u275\u27)oligoadenylate system. I show here that Platelet-derived growth factor increases the levels of (2\u275\u27)oligoadenylate synthetase transcripts and of (2\u275\u27)oligoadenylate synthetases in Balb/c-3T3 cells. Moreover, Platelet-derived growth factor decreases the levels of RNase L in these cells. Interferon treatment inhibits the mitogenic action of Platelet-derived growth factor without inhibiting the expression of c-myc RNA.Finally I present a hypothesis implicating endogenous double stranded RNAs in the control of cell growth

Síndrome compartimental en joven con alteración hemática

El síndrome compartimental es una patología bien conocida que se produce por un aumento de la presión dentro de un compartimiento miofascial. Presentamos el caso de un paciente con trombopenia que sufrió un traumatismo banal. Necesitó fasciotomía del compartimiento anteroexterno de la pierna y, más tarde, esplenectomía. El diagnóstico y tratamiento tardíos de esta complicación, así como una descomprensión inadecuada, pueden conducir a la pérdida de función en una extremidad.The compartment syndrome is a well described cli- nical entity that results from increased pressure within a myo- fascial compartment. An unusual case of a patient with thrombopenia and a minimal traumatism, is reported. He required fasciotomy of the anterolateral compartment of the leg and, later, splenectomy. Late recognition and treatment of this complication, as well as inadequate decompression, can lead to loss of limb

Changes in Circulating Lysyl Oxidase-Like-2 (LOXL2) Levels, HOMA, and Fibrosis after Sustained Virological Response by Direct Antiviral Therapy

Background: we aimed to assess the influence of metabolic syndrome on fibrosis regression (using liver-stiffness measurement (LSM) and serological scores) and the relationship with the expression of lysyl oxidase-like-2 as a potential goal of antifibrotic therapy. Methods: We included 271 patients treated with Direct Antiviral Therapy (DAAs) in our hospital who achieved a sustained virological response (SVR); physical examination, blood tests, and LSM were made at baseline (B) and 24 months (24 M) after SVR. Hemodynamic studies and transjugular liver biopsies were performed on 13 patients. Results: At B, 68 patients were F1 (25.1%); F2 n = 59 (21.7%); F3 n = 44 (16.05%); and 100 were F4 (36.9%). Although the LSM (absolute value) improved in 82% of patients (n = 222), it progressed in 17.5% of patients (n = 48). At 24 M, 48 patients met the metabolic syndrome (MetS) criteria and there was an increase in patients with a BMI of >25 kg/m2 (p 25 kg/m2 is a risk factor for significant fibrosis or steatosis at 24 M (p 9 kPa vs. <9 kPa (p = 0.046). Conclusion: Regression of LSM was reached in 82% of patients. Downregulated LOXL2 was demonstrated post-SVR, with overexpression in cirrhotic patients being a potential therapy goal in selected patients.Funding: This study was supported by the Health Research Institute Marqués de Valdecilla. IDIVAL. Santander. NEXT VAL15/12 grant to Dra Angela Puente Sanchez: Regresión de la fibrosis hepática tras erradicación del virus de la hepatitis C. Papel de la LOXL2. Acknowledgments: This study has been supported by competitive grants from the Instituto de Salud Carlos III (Spanish Ministries of Health and of Economy; PIE15/00079 and PI15/02138)

Circulating Cell Biomarkers in Pulmonary Arterial Hypertension: Relationship with Clinical Heterogeneity and Therapeutic Response

Biomarcadores; Disfunción endotelial; Células progenitorasBiomarcadors; Disfunció endotelial; Cèl·lules progenitoresBiomarkers; Endothelial dysfunction; Progenitor cellsBackground: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment. Methods: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31+CD42b−) and activated (CD31+CD42b−CD62E+), as well as circulating PCs (CD34+CD133+CD45low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy. Results: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH.This research was funded by grants PI12/00510, PI15/00582 and PI18/00960 from the Institute of Health Carlos III (ISCiii), Spain, co-funded by the European Union (ERDF/ESF); the Catalan Society of Respiratory Medicine (SOCAP); and the Fundación Contra la Hipertensión Pulmonar (FCHP). OTC is the former recipient of a Marie Curie Post-Doctoral Fellowship Award BIOTRACK-IDIBAPS, and the current recipient of a Miguel Servet contract from ISCiii (CP17/00114)

Electrogeneration of Gold Nanoparticles on Porous-Carbon PaperBased Electrodes and Application to Inorganic Arsenic Analysis in White Wines by Chronoamperometric Stripping

This paper describes the development of simple, sustainable, and low-cost strategies for signal enhancement on paper-based carbon platforms through gold nanoparticles electrogenerated from small volumes of tetrachloroauric (III) acid solutions. Carbon ink is deposited on a hydrophilic working area of the paper delimited with hydrophobic wax. This maskless procedure is fast and cuts down ink waste. The connection of this working electrode to the potentiostat is ensured with the use of screen-printed electrodes (SPEs). Close contact of the whole area of both carbon electrodes improves the precision of the nanostructuration. Resulting gold-modified paper-based carbon working electrodes (AuNPs-PCWEs) were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and electron dispersion X-ray spectrometry (SEM/EDX). This methodology was applied for the first time to the inorganic arsenic determination in commercial white wines by chronoamperometric stripping of the electrodeposited As(0). In an optimized system, As(III) was reduced and deposited as As(0) on the nanostructured surface by applying a potential of −0.3 V during 180 s. Then, anodic stripping chronoamperometry was performed at +0.4 V. The analytical signal was the current recorded at 30 s. On the other hand, As(V) was chemically reduced to As(III) with 0.2 M KI, and total determination of arsenic could be carried out. As(V) was determined as the difference between total As and As(III).Then, this fast, simple and low-cost method can be employed for speciation purposes. Limits of detection for As(III) and total arsenic (in the presence of KI) are 2.2 μg L−1 and 2.4 μg L−1, respectively, and indicate that this method is suitable for regulated quality control

Molecular Profiling of Decompensated Cirrhosis by a Novel MicroRNA Signature

Noninvasive staging of decompensated cirrhosis is an unmet clinical need. The aims of this study were to characterize and validate a novel microRNA (miRNA) signature to stage decompensated cirrhosis and predict the portal pressure and systolic cardiac response to nonselective beta-blockers (NSBBs). Serum samples from patients with decompensated cirrhosis (n = 36) and healthy controls (n = 36) were tested for a novel signature of five miRNAs (miR-452-5p, miR-429, miR-885-5p, miR-181b-5p, and miR-122-5p) identified in the secretome of primary human hepatocytes and for three miRNAs (miR-192-5p, miR-34a-5p, and miR-29a-5p) previously discovered as biomarkers of chronic liver disease. All patients had ascites, which was refractory in 18 (50%), and were placed on NSBBs for variceal bleeding prophylaxis. In all patients, serum miRNAs, hepatic venous pressure gradient, and an echocardiogram study were performed before and 1 month after NSBBs. Patients with cirrhosis had lower serum levels of miR-429, miR-885-5p, miR-181b-5p, miR-122-5p, miR-192-5p, and miR-29a-5p (P < 0.05). Baseline serum miR-452-5p and miR-429 levels were lower in NSBB responders (P = 0.006). miR-181b-5p levels were greater in refractory ascites than in diuretic-sensitive ascites (P = 0.008) and correlated with serum creatinine. miR-452-5p and miR-885-5p were inversely correlated with baseline systemic vascular resistance (ρ = −0.46, P = 0.007; and ρ = −0.41, P = 0.01, respectively) and with diminished systolic contractility (ρ = −0.55, P = 0.02; and ρ = −0.55, P = 0.02, respectively) in patients with refractory ascites after NSBBs. Conclusion: Analysis of a miRNA signature in serum discriminates between patients with decompensated cirrhosis who show more severe systemic circulatory dysfunction and compromised systolic function after beta-blockade and those more likely to benefit from NSBBs

Clinical and molecular characterization of steatotic liver disease in the setting of immune-mediated inflammatory diseases

Background & Aims: Growing evidence suggests an increased prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) in the context of immune-mediated inflammatory diseases (IMIDs). We aimed to clinically and mechanistically characterize steatotic liver disease (SLD) in a prospective cohort of patients with IMID compared to controls. Methods: Cross-sectional, case-control study including a subset of patients with IMID. Controls from the general population were age-, sex-, type 2 diabetes-, and BMI-matched at a 1:2 ratio. SLD was established using controlled attenuation parameter. Liver biopsies were obtained when significant liver fibrosis was suspected. Total RNA was extracted from freshly frozen cases and analyzed by RNA-seq. Differential gene expression was performed with ‘limma-voom’. Gene set-enrichment analysis was per formed using the fgsea R package with a preranked “limma t-statistic” gene list. Results: A total of 1,456 patients with IMID and 2,945 controls were included. Advanced SLD (liver stiffness measurement ≥9.7 kPa) (13.46% vs. 3.79%; p <0.001) and advanced MASLD (12.8% vs. 2.8%; p <0.001) prevalence were significantly higher among patients with IMID than controls. In multivariate analysis, concomitant IMID was an independent, and the strongest, predictor of advanced SLD (adjusted odds ratio 3.318; 95% CI 2.225-4.947; p <0.001). Transcriptomic data was obtained in 109 patients and showed 87 significant genes differentially expressed between IMID-MASLD and control-MASLD. IMID-MASLD cases displayed an enriched expression of genes implicated in pro-tumoral activities or the control of the cell cycle concomitant with a negative expression of genes related to metabolism. Conclusions: The prevalence of advanced SLD and MASLD is disproportionately elevated in IMID cohorts. Our findings suggest that IMIDs may catalyze a distinct MASLD pathway, divergent from classical metabolic routes, highlighting the need for tailored clinical management strategies.Acknowledgments: This work was fully suported by grants from the Spanish Instituto de Salud Carlos III (ISCIII-FEDER). Grant numbers: PI18/01304, PI20/01279, and PI19/00204This work was fully suported by grants from the Spanish Instituto de Salud Carlos III (ISCIII-FEDER). Grant numbers: PI18/01304, PI20/01279, and PI19/00204

Assessment of Exercise Capacity in Post-COVID-19 Patients: How Is the Appropriate Test Chosen?

There is a wide range of sequelae affecting COVID-19 survivors, including impaired physical capacity. These sequelae can affect the quality of life and return to work of the active population. Therefore, one of the pillars of following-up is the evaluation of physical capacity, which can be assessed with field tests (such as the six-minute walk test, the one-minute standing test, the Chester step test, and the shuttle walking test) or laboratory tests (such as the cardiopulmonary exercise test). These tests can be performed in different contexts and have amply demonstrated their usefulness in the assessment of physical capacity both in post-COVID-19 patients and in other chronic respiratory, metabolic, cardiologic, or neurologic diseases. However, when traditional tests cannot be performed, physical function can be a good substitute, especially for assessing the effects of an intervention. For example, the Short Physical Performance Battery assessment and the Timed Up and Go assessment are widely accepted in older adults. Thus, the test should be chosen according to the characteristics of each subject

Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids

Exosomes are cell-secreted nanovesicles (40–200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.FICYT; Ministerio de Educación; Ministerio de Economía y Competitividad; Gobierno Regional de Asturias; Gobierno Regional de Madri