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Fernandez-Tajes J.,Complejo Hospitalario Universitario runa CHUAC | Soto-Hermida A.,Complejo Hospitalario Universitario runa CHUAC | Vazquez-Mosquera M.E.,Complejo Hospitalario Universitario runa CHUAC | Cortes-Pereira E.,Complejo Hospitalario Universitario runa CHUAC | And 8 more authors.
Annals of the Rheumatic Diseases | Year: 2014

Objective: Alterations in DNA methylation patterns have been found to correlate with several diseases including osteoarthritis (OA). The aim of this study was to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA cartilage and healthy control cartilage samples. Methods: DNA methylation profiling was performed using Illumina Infinium HumanMethylation27 in 25 patients with OA and 20 healthy controls. Subsequent validation was performed by genome-wide expression analysis using the Affymetrix Human Gene 1.1 ST array in an independent cohort of 24 patients with OA. Finally, the most consistent genes in both assays were amplified by quantitative reverse transcriptase PCR in a validation cohort of 48 patients using microfluidic real-time quantitative PCR. Appropriate bioinformatics analyses were carried out using R bioconductor software packages and qBase plus software from Biogazelle. Results: We found 91 differentially methylated (DM) probes, which permitted us to separate patients with OA from healthy controls. Among the patients with OA, we detected 1357 DM probes that identified a tight cluster of seven patients who were different from the rest. This cluster was also identified by genome-wide expression in which 450 genes were differentially expressed. Further validation of the most consistent genes in an independent cohort of patients with OA permitted us to identify this cluster, which was characterised by increased inflammatory processes. Conclusions: We were able to identify a tight subgroup of patients with OA, characterised by an increased inflammatory response that could be regulated by epigenetics. The identification and isolation of this subgroup may be critical for the development of effective treatment and disease prevention.


Santiso R.,Centro Oncologico Of Galicia | Santiso R.,Complejo Hospitalario Universitario runa CHUAC | Tamayo M.,Centro Oncologico Of Galicia | Tamayo M.,Complejo Hospitalario Universitario runa CHUAC | And 7 more authors.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | Year: 2012

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41°C and 45°C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24. h), or acute (1. h) exposure to each treatment followed by incubation at 37°C over a period of 24. h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24. h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF. © 2012 Elsevier B.V..


Bou G.,Complejo Hospitalario Universitario runa CHUAC | Otero F.M.,Complejo Hospitalario Universitario runa CHUAC | Otero F.M.,Centro Oncologico Of Galicia | Santiso R.,Complejo Hospitalario Universitario runa CHUAC | And 8 more authors.
Journal of Clinical Microbiology | Year: 2012

Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settings. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Tamayo M.,Complejo Hospitalario Universitario runa CHUAC | Tamayo M.,Centro Oncologico Of Galicia | Mosquera A.,Complejo Hospitalario Universitario runa CHUAC | Rego I.,Complejo Hospitalario Universitario runa CHUAC | And 4 more authors.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | Year: 2011

Length of telomeric DNA sequences and numerical chromosome aberrations from uncultured human osteoarthritic (OA) articular chondrocytes were compared with those from peripheral blood leukocytes (PBL) from the same individual and from chondrocytes and PBL from control subjects. Cells were both obtained from 39 OA patients (age range: 43-80 years) and from 20 control subjects (age range: 39-94 years). Mean length of telomeric DNA sequences was determined using a quantitative real-time polymerase chain reaction (qPCR) assay and numerical chromosome aberrations were identified in interphase nuclei by Fluorescence In Situ Hybridization (FISH) using cocktails of specific DNA probes for chromosomes 7, 8 and for 18, X and Y. Chondrocytes revealed higher telomere size than PBL, both in control subjects and in OA patients, being 2 and 1.6 times higher respectively, thus revealing cell type specific differences. However, chondrocytes from OA patients showed significantly shorter telomere size than chondrocytes from control subjects (T/S ratio 1.64 ± 0.41 vs. 1.99 ± 0.54; mean ± sd; p=0.008). Regarding the percentage of numerical chromosome aberrations, OA chondrocytes showed 1.7 times higher than chondrocytes from control subjects (19.80 ± 3.31 vs.11.48 ± 4.11; p<0.01) and 1.5 times average higher than that from PBL from the own OA patient (13.06 ± 1.45; p<0.001). Moreover, PBL from OA patients also showed 1.4 times more anomalies than PBL from controls (13.06 ± 1.45 vs. 9.54 ± 1.61; p<0.001). No significant differences were found between chondrocytes and PBL in control subjects. Chromosome loss was the more frequent aneuploidy, mainly monosomy 18. The decreased telomere size and increased chromosome instability in chondrocytes from OA affected joints may imply a local advanced senescence that could contribute to the pathogenesis or progression of the degenerative articular disease. Moreover, the increased chromosomal abnormalities in PBL from OA patients suggest a more general accelerated senescence phenotype that could promote the age-related degenerative joint pathology. © 2011 Elsevier B.V.


PubMed | Autonomous University of Madrid and Complejo Hospitalario Universitario runa CHUAC
Type: | Journal: Mutation research | Year: 2015

Telomere length was sequentially determined in peripheral blood leukocytes (PBL) from patients with ankylosing spondylitis (AS; n = 44) and psoriatic arthritis (PsA; n = 42) followed through 2.93 0.99 years, using a quantitative PCR (qPCR) assay. The initial telomere size from PsA patients was higher than those with cutaneous psoriasis only (n = 53), possibly due to the inflammatory condition. The qPCR assay was sensitive enough to evidence a significant telomere length shortening in PBL from practically all subjects and PsA patients showed a higher rate of loss of telomere sequence than patients with AS during the follow-up time.


Fernandez J.L.,Complejo Hospitalario Universitario runa CHUAC | Fernandez J.L.,Centro Oncologico Of Galicia
Journal of Theoretical Biology | Year: 2012

Two procedures for detection of Sister-Chromatid Exchanges (SCEs) in interphase cells within specific DNA sequence areas are delineated. Proliferating cells cultured with 5-bromodeoxyuridine (BrdU) for one cell cycle are subjected to cytokinesis-block to produce binucleated cells. Then, the BrdU-substituted DNA strands are removed as in the chromosome orientation-FISH (CO-FISH) procedure. In one case, the intact unsubstituted DNA strands may be hybridized with differentially labelled direct and reverse single-stranded DNA (ssDNA) probes from the target area. After formation of a SCE during the first cycle, specifically within the target area, three signals should be visualized in each nucleus of the binucleated cell. Two of them, of different color, must appear colocalized or adjacent simultaneously in both sister nuclei, corresponding to the SCE site, being accompanied by a signal from the other homologous locus showing a complementary color pattern in both sister nuclei. Other possibility is hybridizing two separated direct ssDNA probes flanking the target sequence area labelled with the same color and the two reverse complementary ssDNA probes labelled with other color. The presence of three signals with similar color and one of different color in each nucleus from the binucleated cell, following a complementary color pattern between both nuclei, would indicate the presence of a SCE within the DNA sequence area flanked by the DNA probes. The availability of complementary ssDNA probes specific for single locus should make the Interphase SCE-FISH a suitable procedure. © 2012 Elsevier Ltd.


de Andres M.C.,Complejo Hospitalario Universitario runa CHUAC | Maneiro E.,Complejo Hospitalario Universitario runa CHUAC | Martin M.A.,Institute Investigacion Hospital 12 Of Octubre I12 | Arenas J.,Institute Investigacion Hospital 12 Of Octubre I12 | Blanco F.J.,Complejo Hospitalario Universitario runa CHUAC
Arthritis Research and Therapy | Year: 2013

Introduction: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way. Methods: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[3H]glucose (2-[3H]DG). Statistical analysis was performed using the Mann-Whitney U test. Results: NOC-12 liberates approximately ten times more NO2 -than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[3H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[3H]DG than healthy cells. Conclusions: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO. © 2013 de Andrés et al.; licensee BioMed Central Ltd.


PubMed | Complejo Hospitalario Universitario runa CHUAC
Type: | Journal: Journal of theoretical biology | Year: 2012

Two procedures for detection of Sister-Chromatid Exchanges (SCEs) in interphase cells within specific DNA sequence areas are delineated. Proliferating cells cultured with 5-bromodeoxyuridine (BrdU) for one cell cycle are subjected to cytokinesis-block to produce binucleated cells. Then, the BrdU-substituted DNA strands are removed as in the chromosome orientation-FISH (CO-FISH) procedure. In one case, the intact unsubstituted DNA strands may be hybridized with differentially labelled direct and reverse single-stranded DNA (ssDNA) probes from the target area. After formation of a SCE during the first cycle, specifically within the target area, three signals should be visualized in each nucleus of the binucleated cell. Two of them, of different color, must appear colocalized or adjacent simultaneously in both sister nuclei, corresponding to the SCE site, being accompanied by a signal from the other homologous locus showing a complementary color pattern in both sister nuclei. Other possibility is hybridizing two separated direct ssDNA probes flanking the target sequence area labelled with the same color and the two reverse complementary ssDNA probes labelled with other color. The presence of three signals with similar color and one of different color in each nucleus from the binucleated cell, following a complementary color pattern between both nuclei, would indicate the presence of a SCE within the DNA sequence area flanked by the DNA probes. The availability of complementary ssDNA probes specific for single locus should make the Interphase SCE-FISH a suitable procedure.


PubMed | Complejo Hospitalario Universitario runa CHUAC
Type: Journal Article | Journal: Annals of the rheumatic diseases | Year: 2014

Alterations in DNA methylation patterns have been found to correlate with several diseases including osteoarthritis (OA). The aim of this study was to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA cartilage and healthy control cartilage samples.DNA methylation profiling was performed using Illumina Infinium HumanMethylation27 in 25 patients with OA and 20 healthy controls. Subsequent validation was performed by genome-wide expression analysis using the Affymetrix Human Gene 1.1 ST array in an independent cohort of 24 patients with OA. Finally, the most consistent genes in both assays were amplified by quantitative reverse transcriptase PCR in a validation cohort of 48 patients using microfluidic real-time quantitative PCR. Appropriate bioinformatics analyses were carried out using R bioconductor software packages and qBase plus software from Biogazelle.We found 91 differentially methylated (DM) probes, which permitted us to separate patients with OA from healthy controls. Among the patients with OA, we detected 1357 DM probes that identified a tight cluster of seven patients who were different from the rest. This cluster was also identified by genome-wide expression in which 450 genes were differentially expressed. Further validation of the most consistent genes in an independent cohort of patients with OA permitted us to identify this cluster, which was characterised by increased inflammatory processes.We were able to identify a tight subgroup of patients with OA, characterised by an increased inflammatory response that could be regulated by epigenetics. The identification and isolation of this subgroup may be critical for the development of effective treatment and disease prevention.


PubMed | Complejo Hospitalario Universitario runa CHUAC
Type: | Journal: BMC musculoskeletal disorders | Year: 2012

Oxidative stress due to the overproduction of nitric oxide (NO) and other oxygen reactive species (ROS), play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. Therefore, the goal of this work is to describe the difference in telomere length of peripheral blood leukocytes (PBLs) and Nitric Oxide (NO) production between mitochondrial DNA (mtDNA) haplogroup J and non-J carriers, as indirect approaches of oxidative stress.The telomere length of PBL was analyzed in DNA samples from 166 healthy controls (114 J and 52 non-J) and 79 OA patients (41 J and 38 non-J) by means of a validated qPCR method. The NO production was assessed in 7 carriers of the haplogroup J and 27 non-J carriers, by means of the colorimetric reaction of the Griess reagent in supernatants of cultured chondrocytes. Inducible nitric oxide synthase (iNOS) mRNA from these samples was analyzed by qPCR. Appropiated statistical analyses were performedCarriers of the haplogroup J showed a significantly longer telomere length of PBLs than non-J carriers, regardless of age, gender and diagnosis (p = 0.025). Cultured chondrocytes carrying the mtDNA haplogroup J also showed a lower NO production than non-J carriers (p = 0.043). No significant correlations between age and telomore length of PBLs were detected neither for carriers of the haplogroup J nor for non-J carriers. A strong positive correlation between NO production and iNOS expression was also observed (correlation coefficient = 0.791, p < 0.001).The protective effect of the mtDNA haplogroup J in the OA disease arise from a lower oxidative stress in carriers of this haplogroup, since this haplogroup is related to lower NO production and hence longer telomere length of PBLs too.

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