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To describe the prevalence of Peripheral Artery Disease (PAD) in a random population sample and to evaluate its relationship with Mediterranean diet and with other potential cardiovascular risk factors such as serum uric acid and pulse pressure in individuals ranged 45 to 74 years.Cross-sectional analysis of 1568 subjects (mean age 6.5 years, 43% males), randomly selected from the population. A fasting blood sample was obtained to determine glucose, lipids, and HbA1C levels. An oral glucose tolerance test was performed in non-diabetic subjects. PAD was evaluated by ankle-brachial index and/or having a prior diagnosis.PAD prevalence was 3.81% (95% CI, 2.97-4.87) for all participants. In men, PAD prevalence was significantly higher than in women [5.17% (95% CI, 3.74-7.11) vs. 2.78% (95% CI, 1.89-4.07); p = 0.014]. Serum uric acid in the upper quartile was associated with the highest odds ratio (OR) of PAD (for uric acid > 6.1 mg/dl, OR = 4.31; 95% CI, 1.49-12.44). The remaining variables more strongly associated with PAD were: Heart rate >90 bpm (OR = 4.16; 95%CI, 1.62-10.65), pulse pressure in the upper quartile (≥ 54 mmHg) (OR = 3.82; 95%CI, 1.50-9.71), adherence to Mediterranean diet (OR = 2.73; 95% CI, 1.48-5.04), and former smoker status (OR = 2.04; 95%CI, 1.00-4.16).Our results show the existence of a low prevalence of peripheral artery disease in a population aged 45-74 years. Serum uric acid, pulse pressure and heart rate >90 bpm were strongly associated with peripheral artery disease. The direct association between Mediterranean diet and peripheral artery disease that we have found should be evaluated through a follow-up study under clinical practice conditions.

To evaluate the performance of the Finnish Diabetes Risk Score (FINDRISC) and a simplified FINDRISC score (MADRISC) in screening for undiagnosed type 2 diabetes mellitus (UT2DM) and dysglycaemia.A population-based, cross-sectional, descriptive study was carried out with participants with UT2DM, ranged between 45-74 years and lived in two districts in the north of metropolitan Madrid (Spain). The FINDRISC and MADRISC scores were evaluated using the area under the receiver operating characteristic curve method (ROC-AUC). Four different gold standards were used for UT2DM and any dysglycaemia, as follows: fasting plasma glucose (FPG), oral glucose tolerance test (OGTT), HbA1c, and OGTT or HbA1c. Dysglycaemia and UT2DM were defined according to American Diabetes Association criteria.The study population comprised 1,426 participants (832 females and 594 males) with a mean age of 62 years (SD = 6.1). When HbA1c or OGTT criteria were used, the prevalence of UT2DM was 7.4% (10.4% in men and 5.2% in women; p

SARS-CoV-2 has continued spreading around the world in recent years since the initial outbreak in 2019, frequently developing into new variants with greater human infectious capacity. SARS-CoV-2 and its mutants use the angiotensin-converting enzyme 2 (ACE2) as a cellular entry receptor, which has triggered several therapeutic strategies against COVID-19 relying on the use of ACE2 recombinant proteins as decoy receptors. In this work, we propose an ACE2 silent Fc fusion protein (ACE2-hFcLALA) as a candidate therapy against COVID-19. This fusion protein was able to block the binding of SARS-CoV-2 RBD to ACE2 receptor as measured by ELISA and flow cytometry inhibition assays. Moreover, we used classical neutralization assays and a progeny neutralization assay to show that the ACE2-hFcLALA fusion protein is capable of neutralizing the authentic virus. Additionally, we found that this fusion protein was more effective in preventing in vitro infection with different variants of interest (alpha, beta, delta, and omicron) compared to the D614G strain. Our results suggest the potential of this molecule to be used in both therapeutic and preventive settings against current and emerging mutants that use ACE2 as a gateway to human cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Bermúdez-Abreut et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The aim of this study was to investigate the reliability and validity of an affordable wireless force sensor in measuring mean and peak forces during resistance training.A Suiff Pro wireless force sensor (Suiff, Spain) and a MuscleLab force platform (Ergotest, Norway) were used concurrently to assess tensile load and the ground reaction force resulting from an upright row exercise. Thirteen participants (28.2 ± 5.7 years, 76.2 ± 9.6 kg, 178.2 ± 9.2 cm) performed the exercise under three velocity conditions and isometrically. Each condition involved three sets of exercise. Mean (Fmean) and peak (Fpeak) force values from both sensors were collected and compared.Suiff Pro exhibited excellent reliability for Fmean and Fpeak (ICCs = 0.99). When compared to the criterion measures, Suiff Pro showed trivial standardized bias for Fmean (Mean = 0.00 [CI 95% = 0.00 to 0.01]) and Fpeak (-0.02 [-0.04 to 0.00]). The standardized typical error was also trivial for Fmean (0.03 [0.02 to 0.03]) and Fpeak (0.07 [0.05 to 0.09]). Correlations with the MuscleLab force platform were nearly perfect: Fmean (0.97 [0.94 to 0.98]; p<0.001); Fpeak (0.96 [0.92 to 0.97]; p<0.001).The findings demonstrate that the Suiff Pro sensor is reliable and valid device for measuring force during isometric and dynamic resistance training exercises. Therefore, practitioners can confidently use this device to monitor kinematic variables of resistance training exercises and to obtain real-time augmented feedback during a training session., Competing Interests: The company, Suiff, received funding for product development from the INEFC of Barcelona. Two of the authors, Jorge Castizo-Olier and Adrián Garcia-Fresneda, are affiliated with the INEFC of Barcelona as educators. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Illera-Domínguez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The aim of this study was to determine whether whole body vibration increases movement variability while performing a half squat with different ballasts and rhythms through entropy. A total of 12 male athletes (age: 21.24 ± 2.35 years, height: 176.83 ± 5.80 cm, body mass: 70.63 ± 8.58 kg) performed a half squat with weighted vest, dumbbells and bar with weights suspended with elastic bands, with and without vibration at the squat rhythm of 40 and 60 bpm. Each ballast corresponded to 15% of the body mass. The movement variability was analysed by calculating the sample entropy of the acceleration signal, recorded at the waist using an accelerometer. With vibration, differences were found between weighted vest and dumbbells (t(121) = -8.81, p < 0.001 at 40 bpm; t(121) = -8.18, p < 0.001 at 60 bpm) and between weighted vest and bar at both rhythms (t(121) = -8.96, p < 0.001 at 40 bpm; t(121) = -8.83, p < 0.001 at 60 bpm). Furthermore, a higher sample entropy was obtained at 40 bpm with all ballasts (t(121) = 5.65, p < 0.001 with weighted vest; t(121) = 6.27, p < 0.001 with dumbbells; t(121) = 5.78, p < 0.001 with bar). No differences were found without vibration. These findings reveal that adding mechanical vibration to a half squat produces a non-proportional increase in movement variability, being larger when the ballast is placed on the upper limbs and when performed at a slow rhythm., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tuyà Viñas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The stigma and negative attitudes surrounding Alzheimer's disease (AD) are reinforced by the prevalence of their negative representations. This study aimed to determine how AD framing influences attitudes towards AD and whether this influence differs between younger and older people. Additionally, the elaboration likelihood model (ELM) was used to examine the mediating role that emotions induced by different frames may have in bringing about attitude change. Posters with framed messages on AD (dualism and unity) were designed and shown to 136 participants (68 younger and 68 older adults). Both the younger and older participants were randomly divided into two sub-groups. Each sub-group was shown posters of a campaign with different AD frames: one group viewed posters with messages of the dualism frame and the other group viewed posters with messages of the unity frame. To analyse the effect of the different frames, a mixed design of repeated measures (ANOVA) was used in which attitudes towards AD were measured on two occasions. Both the impact and the emotions produced by exposure to the messages were recorded after the presentation of the posters and a MANOVA test was performed on them. Attitudes, impact and emotions experienced by the younger and older participants were compared. Older adults displayed positive attitudes towards AD but less than younger people. Unity-framed messages produced a positive change in attitudes regardless of the audience's age and led to higher levels of happiness, whereas dualism-framed messages had a greater impact and produced feelings of sadness, anger and fear but did not change attitudes. These findings suggest that reframing of AD may be essential to achieve a positive attitudinal change in both younger and older populations and foster positive emotions. The use of unity-framed messages should be considered when developing and implementing policies targeted at communication and awareness of AD in order to reduce the stigma associated with this form of dementia., Competing Interests: The authors have declared that no competing interests exist.

Leishmaniasis is endemic and a mandatory reporting disease in Spain since 1982. However, between 1996 and 2014, surveillance on public health was decentralized and only some autonomous regions monitored the disease. The aim of this study is to estimate the incidence of leishmaniasis and to evaluate the extent of underreporting in Spain. A capture-recapture (CRC) study was conducted to calculate the incidence of human leishmaniasis using reports from the National Surveillance Network (RENAVE) and the Hospital Discharge Records of the National Health System (CMBD) for 2016 and 2017. During the study period, 802 cases were reported to RENAVE and there were 1,149 incident hospitalizations related to leishmaniasis. The estimated incidence rates through the CRC study were 0.79 per 100,000 inhabitants for visceral leishmaniasis (VL), 0.88 (cutaneous leishmaniasis (CL)) and 0.12 (mucocutaneous leishmaniasis (MCL)) in 2016 and 0.86 (VL), 1.04 (CL) and 0.12 (MCL) in 2017. An underreporting of 14.7-20.2% for VL and 50.4-55.1% for CL was found. The CRC method has helped us to assess the sensitivity and representativeness of leishmaniasis surveillance in Spain, being a useful tool to assess whether the generalization of leishmaniasis surveillance throughout the Spanish territory achieves a reduction in underreporting., Competing Interests: The authors have declared that no competing interests exist.

Asthma is one of the most common chronic diseases characterized by sex disparities. Gender bias is a well-documented issue detected in the design of published clinical trials (CTs). International guidelines encourage researchers to analyze clinical data by sex, gender, or both where appropriate. The objective of this work was to evaluate gender bias in the published CTs of biological agents for the treatment of severe asthma. A systematic review of randomized controlled CTs of the biological agents (omalizumab, benralizumab, reslizumab, mepolizumab or dupilumab) for the treatment of severe asthma was conducted. The literature search was performed using PubMed and EMBASE without language restrictions. This study followed the corresponding international recommendations. We identified a total of 426 articles, of which 37 were finally included. Women represented 60.4% of patients included. The mean percentage of women in these trials was 59.9%, ranged from 40.8% to 76.7%. The separate analysis by sex of the main variable was only performed in 5 of the 37 publications included, and none of the trials analyzed secondary variables by sex. Only 1 of the articles discussed the results separately by sex. No study included the concept of gender in the text or analyzed the results separately by gender. The proportion of women included in CTs was higher compared to publications of other disciplines, where women were under-represented. The analysis of the main and secondary variables by sex or gender, even the discussion separately by sex, was insufficient. This gives rise to potential gender bias in these CTs., Competing Interests: The authors have declared that no competing interests exist.

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1, SF3B1, ATM, TP53, BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM (rituximab en mantenimiento) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment., Competing Interests: J.A. Garcia-Marco has received honoraria for advisory board and speaker’s bureau from Mundipharma, Glaxo, AbbVie, Roche, Gilead, and Janssen, and research support from Hoffman-La Roche and Janssen. Jaime Pérez de Oteyza declares a consulting and advisory role for Hoffman-La Roche. The other authors have no conflicts of interest to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose: The aim of this study was to describe the physical demands during U18 elite basketball games according to the game quarter and to identify a smaller subset of variables and threshold scores that distinguish players' physical performance in each quarter., Methods: Data was collected from ninety-four players who participated in the study (age: 17.4 ± 0.74 years; height: 199.0 ± 0.1 cm; body mass: 87.1 ± 13.1 kg) competing in the Euroleague Basketball Next Generation Tournament. Players' movements during the games were measured using a portable local positioning system (LPS) (WIMU PRO®, Realtrack Systems SL, Almería, Spain) and included relative distance (total distance / playing duration), relative distance in established speed zones, high-intensity running (18.1-24.0 km·h-1) and sprinting (> 24.1 km·h-1). player load, peak speed (km·h-1) and peak acceleration (m·s-2) number of total accelerations and total decelerations, high intensity accelerations (> 2 m·s-2) and decelerations (< -2 m·s-2)., Results: There was an overall decrease in distance covered, player load, number of high intensity accelerations and decelerations between the first and last quarter of the games in all playing positions. A classification tree analysis showed that the first quarter had much influence of distance covered (above 69.0 meters), distance covered <6.0 km·h-1 and accelerations (> 2 m·s-2), whereas the fourth quarter performance had much influence of distance covered (below 69.0) and distance covered 12.1-18.0 km·h-1., Conclusions: A significant reduction in physical demands occurs during basketball, especially between first and last quarter for players in all playing positions during basketball games of under 18 elite players., Competing Interests: The authors have declared that no competing interests exist.

Autoimmune diseases have a higher prevalence of cardiovascular events compared to the general population. The objective of this study was to investigate the genetic basis of cardiovascular disease (CVD) risk in autoimmunity. We analyzed genome-wide genotyping data from 6,485 patients from six autoimmune diseases that are associated with a high socio-economic impact. First, for each disease, we tested the association of established CVD risk loci. Second, we analyzed the association of autoimmune disease susceptibility loci with CVD. Finally, to identify genetic patterns associated with CVD risk, we applied the cross-phenotype meta-analysis approach (CPMA) on the genome-wide data. A total of 17 established CVD risk loci were significantly associated with CVD in the autoimmune patient cohorts. From these, four loci were found to have significantly different genetic effects across autoimmune diseases. Six autoimmune susceptibility loci were also found to be associated with CVD risk. Genome-wide CPMA analysis identified 10 genetic clusters strongly associated with CVD risk across all autoimmune diseases. Two of these clusters are highly enriched in pathways previously associated with autoimmune disease etiology (TNFα and IFNγ cytokine pathways). The results of this study support the presence of specific genetic variation associated with the increase of CVD risk observed in autoimmunity.

Bicuspid aortic valve (BAV) is the most prevalent human congenital cardiac malformation. It may appear isolated, associated with other cardiovascular malformations, or forming part of syndromes. Cranial neural crest (NC) defects are supposed to be the cause of the spectrum of disorders associated with syndromic BAV. Experimental studies with an inbred hamster model of isolated BAV showed that alterations in the migration or differentiation of the cardiac NC cells in the embryonic cardiac outflow tract are most probably responsible for the development of this congenital valvular defect. We hypothesize that isolated BAV is not the result of local, but of early alterations in the behavior of the NC cells, thus also affecting other cranial NC-derived structures. Therefore, we tested whether morphological variation of the aortic valve is linked to phenotypic variation of the mandible and the thymus in the hamster model of isolated BAV, compared to a control strain. Our results show significant differences in the size and shape of the mandible as well as in the cellular composition of the thymus between the two strains, and in mandible shape regarding the morphology of the aortic valve. Given that both the mandible and the thymus are cranial NC derivatives, and that the cardiac NC belongs to the cephalic domain, we propose that the causal defect leading to isolated BAV during embryonic development is not restricted to local alterations of the cardiac NC cells in the cardiac outflow tract, but it is of pleiotropic or polytopic nature. Our results suggest that isolated BAV may be the forme fruste of a polytopic syndrome involving the cranial NC in the hamster model and in a proportion of affected patients.

MPTP-mouse model constitutes a well-known model of neuroinflammation and mitochondrial failure occurring in Parkinson's disease (PD). Although it has been extensively reported that nitric oxide (NO●) plays a key role in the pathogenesis of PD, the relative roles of nitric oxide synthase isoforms iNOS and nNOS in the nigrostriatal pathway remains, however, unclear. Here, the participation of iNOS/nNOS isoforms in the mitochondrial dysfunction was analyzed in iNOS and nNOS deficient mice. Our results showed that MPTP increased iNOS activity in substantia nigra and striatum, whereas it sharply reduced complex I activity and mitochondrial bioenergetics in all strains. In the presence of MPTP, mice lacking iNOS showed similar restricted mitochondrial function than wild type or mice lacking nNOS. These results suggest that iNOS-dependent elevated nitric oxide, a major pathological hallmark of neuroinflammation in PD, does not contribute to mitochondrial impairment. Therefore, neuroinflammation and mitochondrial dysregulation seem to act in parallel in the MPTP model of PD. Melatonin administration, with well-reported neuroprotective properties, counteracted these effects, preventing from the drastic changes in mitochondrial oxygen consumption, increased NOS activity and prevented reduced locomotor activity induced by MPTP. The protective effects of melatonin on mitochondria are also independent of its anti-inflammatory properties, but both effects are required for an effective anti-parkinsonian activity of the indoleamine as reported in this study.

Radiotherapy-induced gut toxicity is among the most prevalent dose-limiting toxicities following radiotherapy. Prevention of radiation enteropathy requires protection of the small intestine. However, despite the prevalence and burden of this pathology, there are currently no effective treatments for radiotherapy-induced gut toxicity, and this pathology remains unclear. The present study aimed to investigate the changes induced in the rat small intestine after external irradiation of the tongue, and to explore the potential radio-protective effects of melatonin gel. Male Wistar rats were subjected to irradiation of their tongues with an X-Ray YXLON Y.Tu 320-D03 irradiator, receiving a dose of 7.5 Gy/day for 5 days. For 21 days post-irradiation, rats were treated with 45 mg/day melatonin gel or vehicle, by local application into their mouths. Our results showed that mitochondrial oxidative stress, bioenergetic impairment, and subsequent NLRP3 inflammasome activation were involved in the development of radiotherapy-induced gut toxicity. Oral treatment with melatonin gel had a protective effect in the small intestine, which was associated with mitochondrial protection and, consequently, with a reduced inflammatory response, blunting the NF-κB/NLRP3 inflammasome signaling activation. Thus, rats treated with melatonin gel showed reduced intestinal apoptosis, relieving mucosal dysfunction and facilitating intestinal mucosa recovery. Our findings suggest that oral treatment with melatonin gel may be a potential preventive therapy for radiotherapy-induced gut toxicity in cancer patients.

Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Osteoarthritis (OA) is characterized by altered homeostasis of joint cartilage and bone, whose functional properties rely on chondrocytes and osteoblasts, belonging to mesenchymal stem cells (MSCs). WNT signaling acts as a hub integrating and crosstalking with other signaling pathways leading to the regulation of MSC functions. The aim of this study was to evaluate the existence of a differential signaling between Healthy and OA-MSCs during osteogenesis., Methods: MSCs of seven OA patients and six healthy controls were isolated, characterised and expanded. During in vitro osteogenesis, cells were recovered at days 1, 10 and 21. RNA and protein content was obtained. Expression of WNT pathway genes was evaluated using RT-qPCR. Functional studies were also performed to study the MSC osteogenic commitment and functional and post-traslational status of β-catenin and several receptor tyrosine kinases., Results: Several genes were downregulated in OA-MSCs during osteogenesis in vitro. These included soluble Wnts, inhibitors, receptors, co-receptors, several kinases and transcription factors. Basal levels of β-catenin were higher in OA-MSCs, but calcium deposition and expression of osteogenic genes was similar between Healthy and OA-MSCs. Interestingly an increased phosphorylation of p44/42 MAPK (ERK1/2) signaling node was present in OA-MSCs., Conclusion: Our results point to the existence in OA-MSCs of alterations in expression of Wnt pathway components during in vitro osteogenesis that are partially compensated by post-translational mechanisms modulating the function of other pathways. We also point the relevance of other signaling pathways in OA pathophysiology suggesting their role in the maintenance of joint homeostasis through modulation of MSC osteogenic potential.

Objective: Anti-TNF therapies have been highly efficacious in the management of rheumatoid arthritis (RA), but 25-30% of patients do not show a significant clinical response. There is increasing evidence that genetic variation at the Fc receptor FCGR2A is associated with the response to anti-TNF therapy. We aimed to validate this genetic association in a patient cohort from the Spanish population, and also to identify new genes functionally related to FCGR2A that are also associated with anti-TNF response., Methods: A total of 348 RA patients treated with an anti-TNF therapy were included and genotyped for FCGR2A polymorphism rs1081274. Response to therapy was determined at 12 weeks, and was tested for association globally and independently for each anti-TNF drug (infliximab, etanercept and adalimumab). Using gene expression profiles from macrophages obtained from synovial fluid of RA patients, we searched for genes highly correlated with FCGR2A expression. Tag SNPs were selected from each candidate gene and tested for association with the response to therapy., Results: We found a significant association between FCGR2A and the response to adalimumab (P=0.022). Analyzing the subset of anti-CCP positive RA patients (78%), we also found a significant association between FCGR2A and the response to infliximab (P=0.035). DHX32 and RGS12 were the most consistently correlated genes with FCGR2A expression in RA synovial fluid macrophages (P<0.001). We found a significant association between the genetic variation at DHX32 (rs12356233, corrected P=0.019) and a nominally significant association between RGS12 and the response to adalimumab (rs4690093, uncorrected P=0.040). In the anti-CCP positive group of patients, we also found a nominally significant association between RGS12 and the response to infliximab (rs2857859, uncorrected P=0.042)., Conclusions: In the present study we have validated the FCGR2A association in an independent population, and we have identified new genes associated with the response to anti-TNF therapy in RA.

Objective: Uncontrolled studies of mesenchymal stem cells (MSCs) in multiple sclerosis suggested some beneficial effect. In this randomized, double-blind, placebo-controlled, crossover phase II study we investigated their safety and efficacy in relapsing-remitting multiple sclerosis patients. Efficacy was evaluated in terms of cumulative number of gadolinium-enhancing lesions (GEL) on magnetic resonance imaging (MRI) at 6 months and at the end of the study., Methods: Patients unresponsive to conventional therapy, defined by at least 1 relapse and/or GEL on MRI scan in past 12 months, disease duration 2 to 10 years and Expanded Disability Status Scale (EDSS) 3.0-6.5 were randomized to receive IV 1-2×10(6) bone-marrow-derived-MSCs/Kg or placebo. After 6 months, the treatment was reversed and patients were followed-up for another 6 months. Secondary endpoints were clinical outcomes (relapses and disability by EDSS and MS Functional Composite), and several brain MRI and optical coherence tomography measures. Immunological tests were explored to assess the immunomodulatory effects., Results: At baseline 9 patients were randomized to receive MSCs (n = 5) or placebo (n = 4). One patient on placebo withdrew after having 3 relapses in the first 5 months. We did not identify any serious adverse events. At 6 months, patients treated with MSCs had a trend to lower mean cumulative number of GEL (3.1, 95% CI = 1.1-8.8 vs 12.3, 95% CI = 4.4-34.5, p = 0.064), and at the end of study to reduced mean GEL (-2.8±5.9 vs 3±5.4, p = 0.075). No significant treatment differences were detected in the secondary endpoints. We observed a non-significant decrease of the frequency of Th1 (CD4+ IFN-γ+) cells in blood of MSCs treated patients., Conclusion: Bone-marrow-MSCs are safe and may reduce inflammatory MRI parameters supporting their immunomodulatory properties. ClinicalTrials.gov NCT01228266.

Introduction: Rheumatoid arthritis is an inflammatory disease with high incidence of cardiovascular disease due to accelerated atherosclerosis. Osteoprotegerin (OPG) has been associated with increased risk of atherosclerotic disease in the general population. Several polymorphisms in the OPG gene with functional effects on cardiovascular disease in non-rheumatic individuals have been described. Therefore, we aimed to analyze the effect of three of these functional OPG polymorphisms on the risk of cardiovascular disease in a large and well-characterized cohort of Spanish patients with rheumatoid arthritis., Methods: Three OPG gene variants (rs3134063, rs2073618 and rs3134069) were genotyped by TaqMan assays in 2027 Spanish patients with rheumatoid arthritis. Anti-cyclic citrullinated peptide (anti-CCP) antibody testing was positive in 997 of 1714 tested. Also, 18.3% of the whole series had experienced cardiovascular events, including 5.4% with cerebrovascular accidents. The relationship between OPG variants and cardiovascular events was assessed using Cox regression., Results: No association between OPG gene variants and cardiovascular disease was observed in the whole group of rheumatoid arthritis patients or in anti-CCP positive patients. Nevertheless, a protective effect of CGA haplotype on the risk of cardiovascular disease in general, and specifically in the risk of cerebrovascular complications after adjusting for sex, age at disease diagnosis and traditional cardiovascular risk factors was disclosed in anti-CCP negative patients (HR = 0.54; 95%CI: 0.31-0.95; p = 0.032 and HR = 0.17; 95%CI: 0.04-0.78; p = 0.022, respectively)., Conclusion: Our results indicate a protective effect of the OPG CGA haplotype on cardiovascular risk, mainly due to a protective effect against cerebrovascular events in anti-CCP negative rheumatoid arthritis patients.

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.

Background: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo., Methods: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy., Results: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region., Conclusion: MTA may potentially offer therapeutic neuroprotection.

Unlabelled: Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased risk of cardiovascular (CV) disease. Previous genome-wide association studies have described SMAD3 rs17228212 polymorphism as an important signal associated with CV events. The aim of the present study was to evaluate for the first time the relationship between this gene polymorphism and the susceptibility to CV manifestations and its potential association with the presence of subclinical atherosclerosis assessed by the evaluation of carotid intima-media thickness (cIMT) in patients with RA., Methods: One thousand eight hundred and ninety-seven patients fulfilling classification criteria for RA were genotyped for SMAD3 rs17228212 gene polymorphism through TaqMan genotyping assay. Also, subclinical atherosclerosis determined by the assessment of cIMT was analyzed in a subgroup of these patients by carotid ultrasonography., Results: No statistically significant differences were observed when allele frequencies of RA patients with or without CV events were compared. Nevertheless, when RA patients were stratified according to anti-cyclic citrullinated peptide (anti-CCP) status, we found that in RA patients who were negative for anti-CCP antibodies, the presence of C allele of SMAD3 rs17228212 polymorphism conferred a protective effect against the risk of cerebrovascular accident (CVA) after adjustment for demographic and classic CV risk factors (HR [95%CI]=0.36 [0.14-0.94], p=0.038) in a Cox regression model. Additionally, correlation between the presence of C allele of SMAD3 rs17228212 polymorphism and lower values of cIMT was found after adjustment for demographic and classic CV risk factors (p-value=0.0094) in the anti-CCP negative RA patients., Conclusions: Our results revealed that SMAD3 rs17228212 gene variant is associated with lower risk of CVA and less severe subclinical atherosclerosis in RA patients negative for anti-CCP antibodies. These findings may have importance to establish predictive models of CV disease in RA patients according to anti-CCP status.

Objective: To describe the prevalence of dementia and subtypes in a general elderly population in northwestern Spain and to analyze the influence of socio-demographic factors., Methods: Cross-sectional, two-phase, door-to-door, population-based study. A total of 870 individuals from a rural region and 2,119 individuals from an urban region of Valladolid, Spain, were involved. The seven-minute screen neurocognitive battery was used in the screening phase. A control group was included., Results: A total of 2,170 individuals aged 65 to 104 years (57% women) were assessed. There were 184 subjects diagnosed with dementia. The crude prevalence was 8.5% (95% CI: 7.3-9.7). Age- and sex-adjusted prevalence was 5.5 (95% CI: 4.5-6.5). Main subtypes of dementia were: Alzheimer's disease (AD) 77.7%, Lewy Body disease, 7.6% and vascular dementia (VD) 5.9%. Crude prevalences were 6.6% (AD), 0.6% (Lewy Body disease), and 0.5% (VD). Dementia was associated with age (OR 1.14 for 1-year increase in age), female sex (OR 1.79) and the absence of formal education (OR 2.53 compared to subjects with primary education or more)., Conclusion: The prevalence of dementia in the study population was lower than the most recent estimates for Western Europe. There was a high proportion of AD among all dementia cases and very low prevalence of VD. Old age, female sex, and low education level were independent risk factors for dementia and AD.

The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release.

Background: BANK1 and BLK belong to the pleiotropic autoimmune genes; recently, epistasis between BANK1 and BLK was detected in systemic lupus erythematosus. Although BLK has been reproducibly identified as a risk factor in rheumatoid arthritis (RA), reports are conflicting about the contribution of BANK1 to RA susceptibility. To ascertain the real impact of BANK1 on RA genetic susceptibility, we performed a large meta-analysis including our original data and tested for an epistatic interaction between BANK1 and BLK in RA susceptibility., Patients and Methods: We investigated data for 1,915 RA patients and 1,915 ethnically matched healthy controls genotyped for BANK1 rs10516487 and rs3733197 and BLK rs13277113. The association of each SNP and RA was tested by logistic regression. Multivariate analysis was then used with an interaction term to test for an epistatic interaction between the SNPs in the 2 genes., Results: None of the SNPs tested individually was significantly associated with RA in the genotyped samples. However, we detected an epistatic interaction between BANK1 rs3733197 and BLK rs13277113 (P(interaction)  = 0.037). In individuals carrying the BLK rs13277113 GG genotype, presence of the BANK1 rs3733197 G allele increased the risk of RA (odds ratio 1.21 [95% confidence interval 1.04-1.41], P = 0.015. Combining our results with those of all other studies in a large trans-ethnic meta-analysis revealed an association of the BANK1 rs3733197 G allele and RA (1.11 [1.02-1.21], P = 0.012)., Conclusion: This study confirms BANK1 as an RA susceptibility gene and for the first time provides evidence for epistasis between BANK1 and BLK in RA. Our results illustrate the concept of pleiotropic epistatic interaction, suggesting that BANK1 and BLK might play a role in RA pathogenesis.

Background: Human endogenous retroviruses (HERVs) are genomic sequences that resulted from ancestral germ-line infections by exogenous retroviruses and therefore are transmitted in a Mendelian fashion. Increased HERV expression and antibodies to HERV antigens have been found in various autoimmune diseases. HERV-K18 in chromosome 1 was previously associated with type one diabetes and multiple sclerosis (MS). The etiology of these complex conditions has not been completely elucidated even after the powerful genome wide association studies (GWAS) performed. Nonetheless, this approach does not scrutinize the repetitive sequences within the genome, and part of the missing heritability could lie behind these sequences. We aimed at evaluating the role of HERV-K18 in chromosome 1 on autoimmune disease susceptibility., Methods: Two HERV-K18 SNPs (97Y/C and 154W/Stop substitutions) conforming three haplotypes were genotyped in Spanish cohorts of multiple sclerosis (n = 942), rheumatoid arthritis (n = 462) and ethnically matched controls (n = 601). Our findings were pooled in a meta-analysis including 5312 autoimmune patients and 4032 controls., Results: Significant associations of both HERV-K18 polymorphisms in chromosome 1 with MS patients stratified by HLA-DRB1*15:01 were observed [97Y/C p = 0.02; OR (95% CI) = 1.5 (1.04-2.17) and 154W/Stop: p = 0.001; OR (95% CI) = 1.6 (1.19-2.16)]. Combined meta-analysis of the previously published association studies of HERV-K18 with different autoimmune diseases, together with data derived from Spanish cohorts, yielded a significant association of the HERV-K18.3 haplotype [97Y-154W: p(M-H) = 0.0008; OR(M-H) (95% CI) = 1.22 (1.09-1.38)]., Conclusion: Association of the HERV-K18.3 haplotype in chromosome 1 with autoimmune-disease susceptibility was confirmed through meta-analysis.

Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased cardiovascular (CV) morbidity and mortality. Since interferon-gamma (IFN-γ) has a direct effect on inflammation, in this study we assessed the potential association of the IFNG functional gene variant rs2430561 with CV disease in patients with RA., Methods: One thousand six hundred and thirty-five patients fulfilling the 1987 American College of Rheumatology classification criteria for RA were genotyped for the IFNG (rs2430561, +874T/A) gene polymorphism using TaqMan genotyping assay. Patients were stratified according to the presence of CV events or not. Logistic regression models to explain the presence of CV disease according to the IFNG rs2430561 allele distribution were performed. The potential influence of this variant in the development of subclinical atherosclerosis was also analyzed in a subgroup of patients with no history of CV events to determine carotid artery intima-media thickness (IMT) (n = 286) and presence of carotid plaques. Levels of the cytokine were determined in a subgroup of patients by ELISA., Results: Adjusted logistic regression model disclosed that presence of the minor allele A was not associated with increased risk of suffering CV events in RA patients. Besides, differences did not achieve statistical significance regarding carotid IMT and presence of carotid plaques in RA patients carrying IFNG rs2430561 variant allele. Levels of IFN-γ were higher in patients who had suffered CV events compared to patients who did not., Conclusion: Our results do not support a role of IFNG rs2430561 (+874T/A) functional gene variant in the development of CV disease in RA patients.

Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased cardiovascular (CV) mortality. Since CD40-CD154 binding has direct consequences on inflammation process initiation, we aimed to replicate previous findings related to disease susceptibility in Spanish RA population. Furthermore, as the major complication in RA disease patients is the development of CV events due to accelerated atherosclerosis, and elevated levels of CD40L/CD154 are present in patients with acute myocardial infarction, we assessed the potential association of CD40 and CD154/CD40L gene variants with CV risk in Spanish RA patients., Methods: One thousand five hundred and seventy-five patients fulfilling the 1987 ACR classification criteria for RA and 1600 matched controls were genotyped for the CD40 rs1883832, rs4810485 and rs1535045 and CD154 rs3092952 and rs3092920 gene polymorphisms, using predesigned TaqMan single nucleotide polymorphism genotyping assays. Afterwards, we investigated the influence of CD40-CD154 gene variants in the development of CV events. Also, in a subgroup of 273 patients without history of CV events, we assessed the influence of these polymorphisms in the risk of subclinical atherosclerosis determined by carotid ultrasonography., Results: Nominally significant differences in the allele frequencies for the rs1883832 CD40 gene polymorphism between RA patients and controls were found (p=0.038). Although we did not observe a significant association of CD40-CD154 gene variants with the development of CV events, an ANCOVA model adjusted for sex, age at the time of the ultrasonography assessment, follow-up time, traditional CV risk factors and anti-cyclic citrullinated peptide antibodies disclosed a significant association (p=0.0047) between CD40 rs1535045 polymorphism and carotid intima media thickness, a surrogate marker of atherosclerosis., Conclusion: Data from our pilot study indicate a potential association of rs1883832 CD40 gene polymorphism with susceptibility to RA. Also, the CD40 rs1535045 gene variant may influence development of subclinical atherosclerosis in RA patients.

Glutamate is the main excitatory neurotransmitter involved in spinal cord circuits in vertebrates, but in most groups the distribution of glutamatergic spinal neurons is still unknown. Lampreys have been extensively used as a model to investigate the neuronal circuits underlying locomotion. Glutamatergic circuits have been characterized on the basis of the excitatory responses elicited in postsynaptic neurons. However, the presence of glutamatergic neurochemical markers in spinal neurons has not been investigated. In this study, we report for the first time the expression of a vesicular glutamate transporter (VGLUT) in the spinal cord of the sea lamprey. We also study the distribution of glutamate in perikarya and fibers. The largest glutamatergic neurons found were the dorsal cells and caudal giant cells. Two additional VGLUT-positive gray matter populations, one dorsomedial consisting of small cells and another one lateral consisting of small and large cells were observed. Some cerebrospinal fluid-contacting cells also expressed VGLUT. In the white matter, some edge cells and some cells associated with giant axons (Müller and Mauthner axons) and the dorsolateral funiculus expressed VGLUT. Large lateral cells and the cells associated with reticulospinal axons are in a key position to receive descending inputs involved in the control of locomotion. We also compared the distribution of glutamate immunoreactivity with that of γ-aminobutyric acid (GABA) and glycine. Colocalization of glutamate and GABA or glycine was observed in some small spinal cells. These results confirm the glutamatergic nature of various neuronal populations, and reveal new small-celled glutamatergic populations, predicting that some glutamatergic neurons would exert complex actions on postsynaptic neurons.

Metastatic colorectal cancer (mCRC) relies on the detachment of aggressive malignant cells from the primary tumor into the bloodstream and, concordantly, the presence of these Circulating Tumor Cells (CTC) is associated with a poor prognosis. In this work, the molecular characterization of CTC from mCRC patients was approached, with the aim of understanding their biology and improving their clinical utility in the management of colorectal cancer patients. For this, EpCAM-based immunoisolation of CTC was combined with whole transcriptome amplification and hybridization onto cDNA microarrays. Gene expression data from mCRC patients, once the background of unspecific immunoisolation from a group of controls had been subtracted, resulted in 410 genes that characterized the CTC population. Bioinformatics were used for the biological interpretation of the data, revealing that CTC are characterized by genes related to cell movement and adhesion, cell death and proliferation, and cell signalling and interaction. RTqPCR on an independent series of mCRC patients and controls was used for the validation of a number of genes related to the main cellular functions characterizing the CTC population. Comparison between primary carcinomas and lung and liver metastases further involved the CTC-genes in the promotion of metastasis. Moreover, the correlation of CTC-gene expression with clinical parameters demonstrated detection and prognosis significance. In conclusion, the molecular characterization of CTC from mCRC patients and the identification of diagnostic and prognostic biomarkers represent an innovative and promising approach in the clinical management of this type of patients.