The effect of cellular differentiation on HSV-1 infection of oligodendrocytic cells

Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs) cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLsThe work was partially supported by the Ministerio de Economía y Competitividad-MINECO (SAF2012-40023 and RD12-0032-12 -partially funded by FEDER- European Union/Una manera de hacer Europa). F de C is hired by Gobierno de Castilla-La Mancha-SESCAM. CK is supported by Public Health Service grant AI-097171 from the National Institute of Allergy and Infectious Disease

Cardiovascular disease in immune-mediated inflammatory diseases: A cross-sectional analysis of 6 cohorts

To analyze in several immune-mediated inflammatory diseases (IMIDs) the influence of demographic and clinical-related variables on the prevalence of cardiovascular disease (CVD), and compare their standardized prevalences.Cross-sectional study, including consecutive patients diagnosed with rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic lupus erythematosus, Crohn disease, or ulcerative colitis, from rheumatology, gastroenterology, and dermatology tertiary care outpatient clinics located throughout Spain, between 2007 and 2010. Our main outcome was defined as previous diagnosis of angina, myocardial infarction, peripheral vascular disease, and/or stroke. Bivariate and multivariate logistic and mixed-effects logistic regression models were performed for each condition and the overall cohort, respectively. Standardized prevalences (in subjects per 100 patients, with 95% confidence intervals) were calculated using marginal analysis.We included 9951 patients. For each IMID, traditional cardiovascular risk factors had a different contribution to CVD. Overall, older age, longer disease duration, presence of traditional cardiovascular risk factors, and male sex were independently associated with a higher CVD prevalence. After adjusting for demographic and traditional cardiovascular risk factors, systemic lupus erythematosus exhibited the highest CVD standardized prevalence, followed by rheumatoid arthritis, psoriasis, Crohn disease, psoriatic arthritis, and ulcerative colitis (4.5 [95% confidence interval (CI): 2.2, 6.8], 1.3 [95% CI: 0.8, 1.8], 0.9 [95% CI: 0.5, 1.2], 0.8 [95% CI: 0.2, 1.3], 0.6 [95% CI: 0.2, 1.0], and 0.5 [95% CI: 0.1, 0.8], respectively).Systemic lupus erythematosus, rheumatoid arthritis, and psoriasis are associated with higher prevalence of CVD compared with other IMIDs. Specific prevention programs should be established in subjects affected with these conditions to prevent CVD

Interventions in health organisations to reduce the impact of adverse events in second and third victims

Background Adverse events (AE) are also the cause of suffering in health professionals involved. This study was designed to identify and analyse organization-level strategies adopted in both primary care and hospitals in Spain to address the impact of serious AE on second and third victims. Methods A cross-sectional study was conducted in healthcare organizations assessing: safety culture; health organization crisis management plans for serious AE; actions planned to ensure transparency in communication with patients (and relatives) who experience an AE; support for second victims; and protective measures to safeguard the institution’s reputation (the third victim). Results A total of 406 managers and patient safety coordinators replied to the survey. Deficient provision of support for second victims was acknowledged by 71 and 61 % of the participants from hospitals and primary care respectively; these respondents reported there was no support protocol for second victims in place in their organizations. Regarding third victim initiatives, 35 % of hospital and 43 % of primary care professionals indicated no crisis management plan for serious AE existed in their organization, and in the case of primary care, there was no crisis committee in 34 % of cases. The degree of implementation of second and third victim support interventions was perceived to be greater in hospitals (mean 14.1, SD 3.5) than in primary care (mean 11.8, SD 3.1) (p?<?0.001). Conclusions Many Spanish health organizations do not have a second and third victim support or a crisis management plan in place to respond to serious AEs

The aftermath of adverse events in spanish primary care and hospital health professionals

Background Adverse events (AEs) cause harm in patients and disturbance for the professionals involved in the event (second victims). This study assessed the impact of AEs in primary care (PC) and hospitals in Spain on second victims. Methods A cross-sectional study was conducted. We carried out a survey based on a random sample of doctors and nurses from PC and hospital settings in Spain. A total of 1087 health professionals responded, 610 from PC and 477 from hospitals. Results A total of 430 health professionals (39.6%) had informed a patient of an error. Reporting to patients was carried out by those with the strongest safety culture (Odds Ratio –OR- 1.1, 95% Confidence Interval –CI- 1.0-1.2), nurses (OR 1.9, 95% CI 1.5-2.3), those under 50 years of age (OR 0.7, 95% CI 0.6-0.9) and primary care staff (OR 0.6, 95% CI 0.5-0.9). A total of 381 (62.5%, 95% CI 59-66%) and 346 (72.5%, IC95% 69-77%) primary care and hospital health professionals, respectively, reported having gone through the second-victim experience, either directly or through a colleague, in the previous 5 years. The emotional responses were: feelings of guilt (521, 58.8%), anxiety (426, 49.6%), re-living the event (360, 42.2%), tiredness (341, 39.4%), insomnia (317, 38.0%) and persistent feelings of insecurity (284, 32.8%). In doctors, the most common responses were: feelings of guilt (OR 0.7 IC95% 0.6-0.8), re-living the event (OR 0.7, IC95% o.6-0.8), and anxiety (OR 0.8, IC95% 0.6-0.9), while nurses showed greater solidarity in terms of supporting the second victim, in both PC (p?=?0.019) and hospital (p?=?0.019) settings. Conclusions Adverse events cause guilt, anxiety, and loss of confidence in health professionals. Most are involved in such events as second victims at least once in their careers. They rarely receive any training or education on coping strategies for this phenomenon

Genetic variation associated with cardiovascular risk in autoimmune diseases

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 (TNF? and IFN? cytokine pathways). The results of this study support the presence of specific genetic variation associated with the increase of CVD risk observed in autoimmunity

A deletion at Adamts9-magi1 Locus is associated with psoriatic arthritis risk

Objective: Copy number variants (CNVs) have been associated with the risk to develop multiple autoimmune diseases. Our objective was to identify CNVs associated with the risk to develop psoriatic arthritis (PsA) using a genome-wide analysis approach. Methods: A total of 835 patients with PsA and 1498 healthy controls were genotyped for CNVs using the Illumina HumanHap610 BeadChip genotyping platform. Genomic CNVs were characterised using CNstream analysis software and analysed for association using the χ2 test. The most significant genomic CNV associations with PsA risk were independently tested in a validation sample of 1133 patients with PsA and 1831 healthy controls. In order to test for the specificity of the variants with PsA aetiology, we also analysed the association to a cohort of 822 patients with purely cutaneous psoriasis (PsC). Results: A total of 165 common CNVs were identified in the genome-wide analysis. We found a highly significant association of an intergenic deletion between ADAMTS9 and MAGI1 genes on chromosome 3p14.1 (p=0.00014). Using the independent patient and control cohort, we validated the association between ADAMTS9-MAGI1 deletion and PsA risk (p=0.032). Using next-generation sequencing, we characterised the 26 kb associated deletion. Finally, analysing the PsC cohort we found a lower frequency of the deletion compared with the PsA cohort (p=0.0088) and a similar frequency to that of healthy controls (p>0.3). Conclusions: The present genome-wide scan for CNVs associated with PsA risk has identified a new deletion associated with disease risk and which is also differential from PsC risk

Evolutionary Analyses of Entire Genomes Do Not Support the Association of mtDNA Mutations with Ras/MAPK Pathway Syndromes

BACKGROUND: There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS) and related disorders (such as LEOPARD, neurofibromatosis type 1), although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA) genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM), which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45), most of them classified as NS patients (n = 42). METHODS/PRINCIPAL FINDINGS: The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg) patterns of a typical Iberian dataset (including hgs H, T, J, and U). Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5) are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. CONCLUSIONS/SIGNIFICANCE: As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS

Efecto de la infección de oligodendrocitos humanos por HSV-1: Papel de la diferenciación celular y valoración del ácido valproico como posible antiherpético

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 02-07-2015Herpesvirus humano de tipo 1 (HSV-1) es un agente infectivo ubicuo. Se considera que más de la mitad de la población mundial presenta anticuerpos a los 15 años, elevándose al 90% la cifra en adultos que muestran evidencias serológicas de la infección. A pesar de que existen indicios de infecciones por miembros de la familia Herpesviridae desde el neolítico y que se cuentan por decenas de miles las publicaciones científicas que aparecen anualmente referentes a estos virus, cuestiones tan importantes como los mecanismos moleculares de entrada del HSV-1, proteínas víricas y celulares implicadas en el proceso y desarrollo del ciclo infectivo siguen sin tener respuesta firme. HSV-1 es un virus neurotrópico capaz de inducir infecciones agudas y latentes en el sistema nervioso central (SNC) y periférico (SNP). No obstante, aunque HSV-1 puede infectar neuronas, oligodendrocitos (OLs), astrocitos y microglía, y persiste en el SNC indefinidamente, su papel en enfermedades desmielinizantes como la esclerosis múltiple (EM) sigue siendo materia de especulación. En el presente trabajo se ha estudiado la susceptibilidad a HSV-1 de células oligodendrocíticas, tanto en líneas humanas establecidas como en cultivo primario de ratón. Para ello, se han utilizado diferentes técnicas y aproximaciones, las cuales concluyen con un claro aumento de la infección viral tras la diferenciación celular. Asimismo, se observó que tras la infección se producían algunos cambios celulares compatibles con un proceso directo de diferenciación celular. Ensayos en este sentido mostraron un incremento de los marcadores moleculares típicos de OLs diferenciados. Además, las células infectadas poseían prolongaciones (procesos) desarrollados y las denominadas estructuras semejantes a las vainas de mielina (MLS) en cultivo in vitro. Por ello, todos estos resultados sugieren que el estado diferenciado de los OLs genera un significativo aumento en la producción viral y que HSV-1, per sé, promueve ese mismo estado de diferenciación en las células. En la actualidad, están surgiendo virus resistentes a los antiherpéticos que se utilizan como tratamiento paliativo. Por ello, hemos caracterizado el efecto que produce el ácido valproico (VPA) sobre las infecciones de HSV-1 en OLs. Nuestros resultados permiten concluir que dicho fármaco posee un gran efecto inhibitorio sobre la producción viral en OLs, generado por una disminución de la expresión de los genes virales, principalmente los inmediatamente tempranos. También observamos una disminución de hasta cuatro órdenes de magnitud de la producción de la progenie viral extracelular, lo cual apunta, tal y como ya había sido publicado en otros sistemas, que VPA podría estar afectando también a la salida viral. Por todo ello, y debido a que este agente inhibidor se está administrando a millones de personas en todo el mundo con desórdenes neurológicos, discutimos sobre la posibilidad de que VPA podría, en un futuro, actuar como fármaco antiherpético capaz de ser administrado en pacientes con resistencias antivirales

Role of the small GTPase Rab27a during Herpes simplex virus infection of oligodendrocytic cells

Abstract Background The morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection. Results Herein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells. Conclusions Altogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.Peer Reviewe

The transcriptome of Leishmania major in the axenic promastigote stage: transcript annotation and relative expression levels by RNA-seq

Abstract Background Although the genome sequence of the protozoan parasite Leishmania major was determined several years ago, the knowledge of its transcriptome was incomplete, both regarding the real number of genes and their primary structure. Results Here, we describe the first comprehensive transcriptome analysis of a parasite from the genus Leishmania. Using high-throughput RNA sequencing (RNA-seq), a total of 10285 transcripts were identified, of which 1884 were considered novel, as they did not match previously annotated genes. In addition, our data indicate that current annotations should be modified for many of the genes. The detailed analysis of the transcript processing sites revealed extensive heterogeneity in the spliced leader (SL) and polyadenylation addition sites. As a result, around 50% of the genes presented multiple transcripts differing in the length of the UTRs, sometimes in the order of hundreds of nucleotides. This transcript heterogeneity could provide an additional source for regulation as the different sizes of UTRs could modify RNA stability and/or influence the efficiency of RNA translation. In addition, for the first time for the Leishmania major promastigote stage, we are providing relative expression transcript levels. Conclusions This study provides a concise view of the global transcriptome of the L. major promastigote stage, providing the basis for future comparative analysis with other development stages or other Leishmania species.This work was funded by Ministerio de Ciencia y Tecnología [BFU2009-08986 to J.M.R., BFU 2008-03126 to B.A.], Comunidad Autonoma de Madrid (S2010/BMD-2361 to J.M.R.), and the Fondo de Investigaciones Sanitarias [ISCIII-RETIC RD06/0021/0008-FEDER to J.M.R. and R.M.R]. A.R. holded a postgraduate fellowship (FPU) from the Ministerio de Educación y Ciencia. Also, an institutional grant from Fundación Ramón Areces is acknowledged.Peer Reviewe