Search

[Abstract] Despite the efforts made to improve the care of cardiogenic shock (CS) patients, including the development of mechanical circulatory support (MCS), the prognosis of these patients continues to be poor. In this context, CS code initiatives arise, based on providing adequate, rapid, and quality care to these patients. In this multidisciplinary document we try to justify the need to implement the SC code, defining its structure/organization, activation criteria, patient flow according to care level, and quality indicators. Our specific purposes are: a) to present the peculiarities of this condition and the lessons of infarction code and previous experiences in CS; b) to detail the structure of the teams, their logistics and the bases for the management of these patients, the choice of the type of MCS, and the moment of its implantation, and c) to address challenges to SC code implementation, including the uniqueness of the pediatric SC code. There is an urgent need to develop protocolized, multidisciplinary, and centralized care in hospitals with a large volume and experience that will minimize inequity in access to the MCS and improve the survival of these patients. Only institutional and structural support from the different administrations will allow optimizing care for CS. [Resumen] Pese a los esfuerzos realizados para mejorar la atención al shock cardiogénico (SC), incluyendo el desarrollo de dispositivos de asistencia circulatoria mecánica (ACM), su pronóstico continúa siendo desfavorable. En este contexto surgen iniciativas de código SC, basadas en proporcionar una asistencia rápida y de calidad a estos pacientes. Este documento multidisciplinario trata de justificar la necesidad de implantar el código SC, definiendo su estructura/organización, criterios de activación, flujo de pacientes según nivel asistencial e indicadores de calidad. Sus propósitos concretos son: a) presentar las peculiaridades de esta enfermedad y el aprendizaje del código infarto y de experiencias previas en SC; b) detallar las bases para el abordaje de estos pacientes, la estructura de los equipos, su logística, la elección del tipo de ACM y el momento de su implante, y c) abordar los desafíos para la implantación del código SC, como la singularidad del código SC pediátrico. Urge desarrollar una asistencia protocolizada, multidisciplinaria y centralizada en hospitales con gran volumen y experiencia que permita minimizar la inequidad en el acceso a la ACM y mejorar la supervivencia de estos enfermos. Solo el apoyo institucional y estructural de las distintas administraciones permitirá optimizar la atención al SC.

Introduction and objectives HCN4 variants have been reported to cause combined sick sinus syndrome (SSS) and left ventricular noncompaction (LVNC) cardiomyopathy. This relationship has been proven in few cases and no previous patients have associated left atrial dilatation (LAD). Our objective was to study a familial disorder characterized by SSS, LAD, and hypertrabeculation/LVNC and to identify the underlying genetic and electrophysiological characteristics. Methods A family with SSS and LVNC underwent a clinical, genetic, and electrophysiological assessment. They were studied via electrocardiography, Holter recording, echocardiography , and exercise stress tests; cardiac magnetic resonance imaging was additionally performed in affected individuals. Genetic testing was undertaken with targeted next-generation sequencing , as well as a functional study of the candidate variant in Chinese hamster ovary cells . Results Twelve members of the family had sinus bradycardia , associated with complete criteria of LVNC in 4 members and hypertrabeculation in 6 others, as well as LAD in 9 members. A HCN4 c.1123C >T;(p.R375C) variant was present in heterozygosis in all affected patients and absent in unaffected individuals. Electrophysiological analyses showed that the amplitude and densities of the HCN4 currents (I HCN4) generated by mutant p.R375C HCN4 channels were significantly lower than those generated by wild-type channels. Conclusions The combined phenotype of SSS, LAD, and LVNC is associated with the heritable HCN4 c.1123C >T;(p.R375C) variant. HCN4 variants should be included in the genetic diagnosis of LVNC cardiomyopathy and of patients with familial forms of SSS, as well as of individuals with sinus bradycardia and LAD.

Background Weaning from venoarterial extracorporeal membrane oxygenation (VA-ECMO) support fails in 30% to 70% of patients. Objective To explore the utility of echocardiographic parameters in predicting successful disconnection from VA-ECMO. Methods Patients receiving VA-ECMO in a referral hospital were included. The relationships between echocardiographic parameters during the weaning trial and weaning success (survival > 24 hours after VA-ECMO explant and no death from cardiogenic shock, heart failure, or cardiac arrest during the hospital stay) and survival were evaluated. Results Of 85 patients included, 61% had successful weaning. Parameters significantly related to weaning success were higher left ventricular ejection fraction (LVEF; 40% in patients with weaning success vs 30% in patients with weaning failure, P = .01), left ventricular outflow tract velocity time integral (15 cm vs 11 cm, P = .01), aortic valve opening in every cycle (98% vs 91% of patients, P = .01), and normal qualitative right ventricular function (60% vs 42% of patients, P = .02). The LVEF remained as an independent predictor of weaning success (hazard ratio, 0.938; 95% CI, 0.888-0.991; P = .02). An LVEF >33.4% was the optimal cutoff value to discriminate patients with successful weaning (area under the curve, 0.808; sensitivity, 93%; specificity, 72%) and was related to higher survival at discharge (60% vs 20%, P < .001). Conclusion Among weaning trial echocardiographic parameters, LVEF was the only independent predictor of successful VA-ECMO weaning. An LVEF >33.4% was the optimal cutoff value to discriminate patients with successful weaning and was related to final survival.

The liver is a key organ involved in a wide range of functions, whose damage can lead to chronic liver disease (CLD). CLD accounts for more than two million deaths worldwide, becoming a social and economic burden for most countries. Among the different factors that can cause CLD, alcohol abuse, viruses, drug treatments, and unhealthy dietary patterns top the list. These conditions prompt and perpetuate an inflammatory environment and oxidative stress imbalance that favor the development of hepatic fibrogenesis. High stages of fibrosis can eventually lead to cirrhosis or hepatocellular carcinoma (HCC). Despite the advances achieved in this field, new approaches are needed for the prevention, diagnosis, treatment, and prognosis of CLD. In this context, the scientific com-munity is using machine learning (ML) algorithms to integrate and process vast amounts of data with unprecedented performance. ML techniques allow the integration of anthropometric, genetic, clinical, biochemical, dietary, lifestyle and omics data, giving new insights to tackle CLD and bringing personalized medicine a step closer. This review summarizes the investigations where ML techniques have been applied to study new approaches that could be used in inflammatory-related, hepatitis viruses-induced, and coronavirus disease 2019-induced liver damage and enlighten the factors involved in CLD development.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

Introduction Postcardiotomy veno-arterial extracorporeal membrane oxygenation (V-A-ECMO) is associated with significant mortality. Identification of patients at very high risk for death is elusive and the decision to initiate V-A-ECMO is based on clinical judgment. The prognostic impact of pre-V-A-ECMO arterial lactate level in these critically ill patients has been herein evaluated. Methods A systematic review was conducted to identify studies on postcardiotomy VA-ECMO for the present individual patient data meta-analysis. Results Overall, 1269 patients selected from 10 studies were included in this analysis. Arterial lactate level at V-A-ECMO initiation was increased in patients who died during the index hospitalization compared to those who survived (9.3 vs 6.6 mmol/L, p < 0.0001). Accordingly, in hospital mortality increased along quintiles of pre-V-A-ECMO arterial lactate level (quintiles: 1, 54.9%; 2, 54.9%; 3, 67.3%; 4, 74.2%; 5, 82.2%, p < 0.0001). The best cut-off for arterial lactate was 6.8 mmol/L (in-hospital mortality, 76.7% vs. 55.7%, p < 0.0001). Multivariable multilevel mixed-effect logistic regression model including arterial lactate level significantly increased the area under the receiver operating characteristics curve (0.731, 95% CI 0.702–0.760 vs 0.679, 95% CI 0.648–0.711, DeLong test p < 0.0001). Classification and regression tree analysis showed the in-hospital mortality was 85.2% in patients aged more than 70 years with pre-V-A-ECMO arterial lactate level ≥6.8 mmol/L. Conclusions Among patients requiring postcardiotomy V-A-ECMO, hyperlactatemia was associated with a marked increase of in-hospital mortality. Arterial lactate may be useful in guiding the decision-making process and the timing of initiation of postcardiotomy V-A-ECMO.

Weaning failure and mortality rates in veno-arterial extracorporeal membrane oxygenation (VA-ECMO) supported patients are significant. Small studies suggest the possible usefulness of levosimendan in this environment, especially in postcardiotomy shock. We performed a retrospective analysis of VA-ECMO implants in a referral hospital comparing weaning failure and survival of patients treated with levosimendan with a control group. From 2013 to May 2020, 123 VA-ECMO for several indications were implanted. Levosimendan was administered in 23 patients (18.7%) with good tolerance. Levosimendan was used more frequently in cardiogenic shock due to acute coronary syndrome indication, and in patients with lower left ventricular ejection fraction (LVEF) at the implant. No significant differences were found in success of ECMO weaning (60.9% levosimendan group vs. 44% non-levosimendan group, P = .169) despite worse LVEF in levosimendan group. Survival at follow-up (20.6 [58] months) was higher in the group that received levosimendan, although without finding statistically significant differences (47.8% vs. 32.0%, log rank P = .124). Levosimendan can be safely administered during VA-ECMO support. Patients receiving levosimendan were weaned similarly from circulatory support despite worse LVEF. Its use did not influence in short- and medium-term survival. Randomized studies are needed to evaluate the levosimendan impact in this indication.

Context.— Food and Drug Administration–approved TRK inhibitors with impressive overall response rates are now available for patients with multiple cancer types that harbor NTRK rearrangements, yet the identification of NTRK fusions remains a difficult challenge. These alterations are highly recurrent in extremely rare malignancies or can be detected in exceedingly small subsets of common tumor types. A 2-step approach has been proposed, involving a screening by immunohistochemistry (IHC) followed by a confirmatory method (fluorescence in situ hybridization, reverse transcriptase–polymerase chain reaction, or next-generation sequencing) in cases expressing the protein. However, there is no interpretation guide for any of the available IHC clones. Objective.— To provide a pragmatic update on the use of pan-TRK IHC. Selected examples of the different IHC staining patterns across multiple histologies are shown. Data Sources.— Primary literature review with PubMed, combined with personal diagnostic and research experience. Conclusions.— In-depth knowledge of pan-TRK IHC will help pathologists implement a rational approach to the detection of NTRK fusions in human malignancies.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy (white matter disorder), which is mainly caused by defects in MLC1 or glial cell adhesion molecule (GlialCAM) proteins. In addition, autoantibodies to GlialCAM are involved in the pathology of multiple sclerosis. MLC1 and GLIALCAM genes encode for membrane proteins of unknown function, which has been linked to the regulation of different ion channels and transporters, such as the chloride channel VRAC (volume regulated anion channel), ClC-2 (chloride channel 2), and connexin 43 or the Na+/K+-ATPase pump. However, the mechanisms by which MLC proteins regulate these ion channels and transporters, as well as the exact function of MLC proteins remain obscure. It has been suggested that MLC proteins might regulate signalling pathways, but the mechanisms involved are, at present, unknown. With the aim of answering these questions, we have recently described the brain GlialCAM interactome. Within the identified proteins, we could validate the interaction with several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptors GPR37L1 and GPR37. In this review, we summarize new aspects of the pathophysiology of MLC disease and key aspects of the interaction between GPR37 receptors and MLC proteins.

Background: It is unclear whether peripheral arterial cannulation is superior to central arterial cannulation for postcardiotomy veno-arterial extracorporeal membrane oxygenation (VA-ECMO). Methods: A systematic review was conducted using PubMed, Scopus, and Google Scholar to identify studies on postcardiotomy VA-ECMO for the present individual patient data (IPD) meta-analysis. Analysis was performed according to the intention-to-treat principle. Results: The investigators of 10 studies agreed to participate in the present IPD meta-analysis. Overall, 1269 patients were included in the analysis. Crude rates of in-hospital mortality after central versus peripheral arterial cannulation for VA-ECMO were 70.7% vs. 63.7%, respectively (adjusted OR 1.38, 95% CI 1.08–1.75). Propensity score matching yielded 538 pairs of patients with balanced baseline characteristics and operative variables. Among these matched cohorts, central arterial cannulation VA-ECMO was associated with significantly higher in-hospital mortality compared to peripheral arterial cannulation VA-ECMO (64.5% vs. 70.8%, p = 0.027). These findings were confirmed by aggregate data meta-analysis, which showed that central arterial cannulation was associated with an increased risk of in-hospital mortality compared to peripheral arterial cannulation (OR 1.35, 95% CI 1.04–1.76, I2 21%). Conclusions: Among patients requiring postcardiotomy VA-ECMO, central arterial cannulation was associated with an increased risk of in-hospital mortality compared to peripheral arterial cannulation. This increased risk is of limited magnitude, and further studies are needed to confirm the present findings and to identify the mechanisms underlying the potential beneficial effects of peripheral VA-ECMO.

Bile acid (BA) synthesis from cholesterol by hepatocytes is inhibited by inflammatory cytokines. Whether liver inflammation also affects BA side chain shortening and conjugation was investigated. In human liver cell lines (IHH, HepG2, and HepaRG), agonists of nuclear receptors including the farnesoid X receptor (FXR), liver X receptor (LXR), and peroxisome proliferator-activated receptors (PPARs) did not affect the expression of BA-related peroxisomal enzymes. In contrast, hepatocyte nuclear factor 4¿ (HNF4¿) inhibition down-regulated acyl-CoA oxidase 2 (ACOX2). ACOX2 was repressed by fibroblast growth factor 19 (FGF19), which was prevented by extracellular signal-regulated kinase (ERK) pathway inhibition. These changes were paralleled by altered BA synthesis (HPLC-MS/MS). Cytokines able to down-regulate cholesterol-7¿-hydroxylase (CYP7A1) had little effect on peroxisomal enzymes involved in BA synthesis except for ACOX2 and bile acid-CoA:amino acid N-acyltransferase (BAAT), which were down-regulated, mainly by oncostatin M (OSM). This effect was prevented by Janus kinase (JAK) inhibition, which restored BA side chain shortening and conjugation. The binding of OSM to the extracellular matrix accounted for a persistent effect after culture medium replacement. In silico analysis of four databases (n = 201) and a validation cohort (n = 90) revealed an inverse relationship between liver inflammation and ACOX2/BAAT expression which was associated with changes in HNF4¿ levels. In conclusion, BA side chain shortening and conjugation are inhibited by inflammatory effectors. However, other mechanisms involved in BA homeostasis counterbalance any significant impact on the serum BA profile., This study was supported by the CIBERehd (EHD15PI05/2016) and Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (PI19/00819, PI20/00189, and PI20/01663 co-funded by European Regional Development Fund/European Social Fund, “Investing in your future”); Junta de Castilla y Leon (SA074P20); Fundació Marato TV3 (Ref. 201916/31), Spain; AECC Scientific Foundation (2017/2020), Spain; Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of Wuerzburg, Germany (Project A-E-384 to H.M.H.); grants PID2019-111669-RBI00, PID2020-115055RB-I00 from Agencia Estatal de Investigación (AEI), Spain; the AGAUR of the Generalidad de Cataluña SGR-2017-1112, Spain; and European Cooperation in Science & Technology (COST) Action CA17112. R.E.E was recipient of a predoctoral fellowship from “Junta de Castilla y León” and “Fondo Social Europeo” (EDU/574/2018). J.A. was recipient of a grant from Fundación Echebano (2020–2022).

Objective: The aim was to evaluate the value of microperimetry (MP) in the early detection of toxic maculopathy caused by HCQ treatment in patients with normal fundoscopy, as well as normal structural optical coherence tomography (OCT). Materials and methods: Microperimetry was performed in 13 patients under hydroxychloroquine treatment, who did not present fundoscopic or structural OCT alterations compatible with maculopathy. We used Nidek MP3s equipment (Nidek, Gamagori, Japan) with a 13-point pattern centered in fovea, in mesopic mode and in scotopic mode. Results: The mean retinal sensitivity (MRS) in the study group was 27.25 +/ - 2.80 dB (95% CI 26.09 to 28.41 dB) while in the group of healthy volunteers 29.34 +/ - 2.18 dB (95% CI 28.67 to 30.1 dB). In scotopic mode, the mean sensitivity was 13.38 +/ - 1.43 dB (95% CI 12.79 to 13.97 dB) for HCQ users and 14.40 +/ - 2.1 dB (95% CI 13.76 to 15.04 dB) in the non-user group. Central retinal sensitivity (CRS) was also lower in patients using HCQ 26.52 +/ -4.0 dB (95% CI 24.8 to 28.15 dB) vs. 29.06 +/ - 2.5 dB (95% CI 28.33 to 29.87 dB) in the control group in mesopic mode. The trend was repeated in scotopic CRS (10.85 +/ -1.84 dB vs. 12.16 +/ - 2.61 dB respectively). Discussion: Our results showed that MP, especially in its mesopic mode, is a useful method to detect retinal toxicity caused by HCQ consumption in patients without funduscopic alteration and with normal macular OCT. Conclusions: In mesopic mode, MRS was significantly lower in patients with long-term hydroxychloroquine treatment compared to those who did not use it, even in cases in which no fundoscopic or structural OCT alteration was detected. Abbreviations: HCQ = hydroxychloroquine, MP = microperimetry, OCT = optical coherence tomography, BCVA = best corrected visual acuity, CRS = central retinal sensitivity, RS = retinal sensitivity, GEE = generalized estimating equations, MRS = mean retinal sensitivity, MfERG = multifocal electroretinogram, AMD = age-related macular degeneration

BACKGROUND Age over 70 years seems to confer poor prognosis for patients under mechanical circulatory support (MCS). Advanced age is usually a relative contraindication. Our objective was to assess the impact of age on survival of patients with short-term MCS. METHODS Retrospective analysis of ≥70-year-old patients supported with veno-arterial extracorporeal membrane oxygenation (VA-ECMO) or Impella CP® due to cardiogenic shock and other situations of hemodynamic instability in a referral hospital (elderly group), compared with younger patients (

There is evidence of the role of milk components in the metabolic programming of offspring. Here, we aimed to investigate the effects of a diet during lactation on breast milk leptin, adiponectin, and related miRNAs’ expression, and their impact on dams and their offspring. Dams were fed a control diet (controls) or a diet enriched with oleic acid, betaine, and leucine (TX) throughout lactation. A TX diet promoted higher leptin at lactation day (LD) five and lower adiponectin on LD15 (vs. controls) in milk, resulting in increased leptin to adiponectin (L/A) ratio throughout lactation. Moreover, TX diet reduced milk levels of miR-27a, miR-103, miR-200a, and miR-222. Concerning TX offspring, higher body fat was early observed and maintained into adult life, accompanied by higher HOMA-IR than controls at three months of age. Offspring body fat content in adulthood correlated positively with milk L/A ratio at LD15 and negatively with miRNAs modulated by the TX diet. In conclusion, maternal diet during lactation can modulate leptin and adiponectin interplay with miRNAs in milk, setting up the metabolic programming of the offspring. Better knowledge about the influence of diet on this process is necessary to promote a healthy adult life in the progeny.

© The Author(s) 2020., Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of leukodystrophy characterized by white matter edema, and it is caused mainly by recessive mutations in MLC1 and GLIALCAM genes. These variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. In addition, dominant mutations in GLIALCAM have also been identified in a subtype of MLC patients with a remitting phenotype. This variant has been named MLC2B. GLIALCAM encodes for an adhesion protein containing two immunoglobulin (Ig) domains and it is needed for MLC1 targeting to astrocyte-astrocyte junctions. Most mutations identified in GLIALCAM abolish GlialCAM targeting to junctions. However, it is unclear why some mutations behave as recessive or dominant. Here, we used a combination of biochemistry methods with a new developed anti-GlialCAM nanobody, double-mutants and cysteine cross-links experiments, together with computer docking, to create a structural model of GlialCAM homo-interactions. Using this model, we suggest that dominant mutations affect different GlialCAM-GlialCAM interacting surfaces in the first Ig domain, which can occur between GlialCAM molecules present in the same cell (cis) or present in neighbouring cells (trans). Our results provide a framework that can be used to understand the molecular basis of pathogenesis of all identified GLIALCAM mutations., This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MICINN) (RTI2018-093493-B-I00 to RE) and BIO2016-79930-R to JFR. RE is a recipient of an ICREA Academia prize.

Aims: To study programmed death ligand 1 (PD-L1) expression, tumour-infiltrating T lymphocytes (TILs) and the molecular context in patients with early-stage squamous cell lung carcinomas (SCCs). Methods and results: The study included samples from 40 patients (discovery cohort) and 29 patients (validation cohort) diagnosed with early-stage SCC. PD-L1 immunohistochemistry (IHC) was performed with three commercially available clones (E1L3N, SP263 and SP142). CD8 + TILs were scored with a digital algorithm. All tumours were analysed with targeted next-generation sequencing (NGS). Additionally, TP53 mutations were investigated with direct sequencing. In both cohorts, we observed a significant association between CD8 + TILs density and high PD-L1 IHC expression in tumour cells (TCs). Furthermore, high SP142 PD-L1 expression in immune cells (ICs) was also associated significantly with CD8 + TILs density. Therefore, CD8 + TILs density discriminated between patients with high versus low PD-L1 IHC expression with excellent sensitivity and specificity. Interestingly, the highest percentages of PD-L1-positive TCs with the three antibodies were found in samples with cyclin-dependent kinase 6 (CDK6) amplification, with high amplification of proto-oncogene C-Myc (CMYC) or with cyclin D1–PI3 kinase subunit alpha (CCND1–PIK3CA) co-amplification. High SP142 PD-L1 IHC expression in ICs showed a non-significant correlation with TP53 mutations. Conversely, most cases with fibroblast growth factor receptor 1 (FGFR1) amplification were negative for all PD-L1 clones. Conclusions: Our preliminary results support the use of digital CD8 + TILs scoring and targeted NGS alongside PD-L1 expression. The approach presented herein could help define patients with SCCs candidates to immune checkpoints inhibitors.

Background Helicobacter pylori infection is associated with chronic gastritis, ulcers and gastric cancer. Antimicrobial resistance has increased worldwide affecting the efficacy of current treatments. Most guidelines recommend implementation of regional surveillance of primary antibiotic resistance of H pylori. Only a fraction of individuals infected with H pylori develop gastric diseases which are related to virulence factors of the bacteria. The aims of the study were to determine the primary antimicrobial resistance rates of H pylori and to know the virulence factors prevalence of strains circulating in Southern Europe. Materials and methods Susceptibility testing by Etest to clarithromycin, levofloxacin, metronidazole, amoxicillin and tetracycline was performed in 102 isolates (99 naive patients). The prevalence of virulence factors (cagA, vacA, oipA, babA and dupA) was evaluated in 102 H pylori isolates from patients with mild-disease symptoms and in 22 isolates from patients with severe-disease symptoms. Results Primary resistance rates were 12.1% to clarithromycin, 13.1% to levofloxacin, 24.2% to metronidazole and 0% to amoxicillin and tetracycline. Combined resistance to clarithromycin and levofloxacin was 3% and to clarithromycin and metronidazole 4%. Prevalence of virulence factors in the mild- and severe-disease group was 35.3% and 81.8% for cagA, 20.6% and 54.5% for cagA/vacAs1m1, 94.1% and 95.4% for babA2, 78.4% and 100% for oipA and 30.4% and 18.2% for dupA. Conclusions Primary antimicrobial resistance rates were under 15% for clarithromycin and levofloxacin. The prevalence of H pylori carrying the virulent genotype cagA/vacAs1m1 was higher than 20% in the mild-disease and 54% in the severe-disease symptom group.

Understanding why pathogenic Mycobacterium avium subsp. paratuberculosis (Map) isolates cause disparate disease outcomes with differing magnitudes of severity is important in designing and implementing new control strategies. We applied a suite of mathematical models: i) general linear, ii) and neurofuzzy logic, to explain how the host of origin of several Map isolates, Map genotype, host, macrophage-based in vitro model and time post-infection contributed to the infection. A logistic growth ordinary differential equation (ODE) model was applied to estimate within macrophage growth rates for the different Map isolates. The models revealed different susceptibilities of bovine and ovine macrophages to Map infection and confirmed distinct virulence profiles for the isolates, judged by their ability to grow within macrophages. Ovine macrophages were able to internalize Map isolates more efficiently than bovine macrophages. While bovine macrophages were able to internalize Map isolates from cattle with more efficiency, ovine macrophages were more efficient in internalizing ovine isolates. Overall, Map isolates from goat and sheep grew minimally within macrophages or did not grow but were able to persist by maintaining its initial population. In contrast, the ability of the bovine isolates and the non-domesticated animal isolates to grow to higher CFU numbers within macrophages suggests that these isolates are more virulent than the sheep and goat isolates, or that these isolates are better adapted to infect domestic ruminants. Overall, our study confirms the different virulence levels for the Map isolates and susceptibility profiles of host macrophages, which is crucial in increasing our understanding of Map infection.

Este artículo a texto completo en formato PDF puede consultarse y descargarse desde el siguiente enlace al índice de la revista: https://abanicoacademico.mx/revistasabanico/index.php/abanico-veterinario/article/view/152/117 Se ha registrado la manifestación de caída de 2.225 animales de 3 a 5 años, de la raza de Lidia, desde 1991 en diferentes plazas de toros de 1ª y 2ª categoría siguiendo la metodología y software de valoración etológica de Alonso et al. (1995a). Se parte de un 99,56% de los individuos que presentaron caídas durante los años 1991-1993 con problemas graves en el 17,16% de los animales (caídas tipo 4, 5 y 6) mejorando situación en la actualidad (2014-2016) con un 79,82% de individuos que manifiestan el síndrome, de los que sólo un 8,23% experimentan caídas tipo 4. La manifestación de caídas se acentúa con el avance de la lidia, siendo las formas más leves (tipos 1 y 2) los tipos de claudicaciones más comunes registradas, fundamentalmente en el tercio de muleta, fruto del incremento significativo de su duración. A su vez, se observa una disminución gradual de las formas más graves, llegando a ser prácticamente inexistentes las caídas tipo 5 y 6 en los últimos años. Todo ello asociado a una mejora en la selección y en la alimentación, unida a la implementación de la preparación física del animal previa a la lidia mediante entrenamientos estandarizados. / There has been registered 2,225 events of falling syndrome in fighting bulls 3 to 5 years old since 1991 in different bullrings of first and 2nd category the study was carried out observing the methodology and software by Alonso et al.(1995ª). The present work started with 99.56% of the individuals who showed falls during 1991-1993, with serious problems for the 17.16% of these animals (falls-type 4, 5 and 6) improving the present situation (2014-2016) with a 79.82% of individuals that manifested the syndrome and which only 8.23% experienced the fall-type-4. The manifestation of falls increases during the bullfight, being the milder forms (falls-type 1 and 2) the most common claudication recorded, mainly during the “Tercio de muleta”, generated by the significant increase of its duration. At the same time, it is observed a gradual decrease of the most critical forms of fall, becoming almost non-existent fall-type 5 and 6 in recent years. All of this has been achieved thanks to an improvement in genetic selection and feeding, along with the implementation of the animal's pre-fight physical preparation through standardized training.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.

Introduction Morbidity and mortality due to cardiovascular causes in patients with schizophrenia are higher than in the general population, a fact that has been observed more since second generation anti-psychotics came into general use. Objectives To determine the incidence of metabolic syndrome in patients with a previously untreated first psychotic episode, as well as the prospective changes in the parameters that define the criteria of metabolic syndrome. Method An observational study with a prospective cohort design including patients who were admitted to the Acute Unit of Donostia Hospital. Results A total of 21 patients were included in the study, of which 19 completed it. Just over one-quarter (26.3%) of the patients developed a metabolic syndrome at six months. Statistically significant differences were observed in the following parameters: (1) abdominal perimeter measurement with an increase of 14.6 cm at six months (P = .001); (2) triglyceride levels with a mean increase over the initial measurement of 48.99 mg/dl (P = .039); and (3) fasting blood glucose levels with a mean increase of 10.72 mg/dl (P = .001). Conclusions Significant changes were observed in metabolic parameters in a short period with the subsequent risk of associated cardiovascular events in a group of young patients. Actions are required to be directed at ensuring appropriate monitoring of these patients in order to measures to minimise the risks.

P. 35-46 El uso de la tecnología GPS en la monitorización de reses de Lidia nos permite disponer de datos de interés, en tiempo real, proporcionando información sobre los desplazamientos, territorio frecuentado, pautas de comportamiento, etc. En el presente trabajo se monitorizaron 9 animales de 3 ganaderías diferentes de la provincia de Salamanca (España), 3 en cada una, siendo sus características: madre (8-15 años) – hija (2 años) – independiente (4-5 años). Se diseñó una metodología que permitió extraer información de los datos de posición para profundizar en el conocimiento del comportamiento del ganado bovino en pastoreo, estudiando aspectos nada conocidos como el comportamiento social. Se calcularon las áreas de campeo de cada animal con una ocupación media del 92 % del espacio disponible en el cercado. Los animales tienden a iniciar su actividad diaria de pastoreo unas horas antes del amanecer, la cual se atenúa al anochecer para descansar durante la noche. En las distancias existentes entre las tres parejas de animales (madre-hija, madre-independiente e hija-independiente) no se apreciaron diferencias significativas que nos permitan confirmar la existencia de un vínculo materno -filial, que haría que los individuos de la misma familia mantuvieran una proximidad espacial. SI

Tumor invasion is paradigmatic of the complex interactions connecting a carcinoma with its environment, and a reflex of the cellular and molecular heterogeneity that defines the initiation of dissemination and metastasis. The hostile situation generated by a growing carcinoma and a reactive stroma is at the basis of the promotion of carcinoma invasion and metastasis, with oxidative stress emerging as a main player in the acquisition of an aggressive tumor phenotype. In this review, we present this complex scenario with a focus on the contribution of the reactive environment and the oxidative stress to the cellular and molecular events associated with carcinoma invasion and metastasis. We also discuss the potential of oxidative stress as a source of biomarkers of advance disease, and as supplier of a therapeutic armamentarium against the initial steps of metastatic dissemination.

We developed a suicidal DNA vaccine (pIRF1A-G-pMT-M) for salmonid fish susceptible to Infectious Hematopoietic Necrosis Virus (IHNV). The suicidal vaccine consists of two operons: i) an inducible fish promoter, the interferon regulatory factor 1A promoter (pIRF1A), driving the expression of the IHNV viral glycoprotein (G) gene that induces protection, and ii) a ZnCl(2) inducible fish promoter, the metallothionein promoter (pMT), driving the expression of the IHNV matrix (M) protein that induces apoptosis. The vaccine induces an immune response to the G protein and then induces the cell to undergo apoptosis to eliminate the DNA vaccine-containing cell. Also developed is another suicidal construct (pCMV-luc-pMT-M) for monitoring the persistence of luciferase (luc) expression after induction of apoptosis. In this study, we evaluated the inducibility of the MT promoter with ZnCl(2) and the capacity of cells transfected with the suicidal vector pCMV-luc-pMT-M to undergo apoptosis after ZnCl(2) addition. We also demonstrated the protective immunity elicited by the suicidal DNA vaccine pIRF1A-G-pMT-M, the survival of fish after treatment with ZnCl(2), and the elimination of the suicidal vector in fish after ZnCl(2) treatment.

The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

Introduction and aim Intestinal-type sinonasal adenocarcinomas (ITACs) are rare epithelial tumours, primarily originating in the nasal cavities and paranasal sinuses, and characterized by glandular structures. The aims of this study are: to determine the genetic alterations in ITACs by MLPA (Multiplex ligation-dependent probe amplification) and to correlate the findings to the clinical behaviour and follow-up information of the patients. Material and method We performed a longitudinal prospective study on 20 patients with ITAC, seen in our department between 1998 and 2004. DNA was extracted from primary tumour samples and analyzed by MLPA. Results The T stage of our series was T2: 4 (20%), T3: 6 (30%), T4a: 3 (15%), and T4b: 7 (35%). All cases initially were N0 and M0. Seventeen patients (85%) had professional exposure to wood dust. All patients underwent surgical intervention and 70% received complementary radiotherapy. Overall 5 and 10 year survival was 42% and 22%, respectively. Gains were found most frequently for PTP4A3 and PDCD8 (65%), TNRFSF7 (50%), RECQL4 and LMO2 (45%), and losses for BCL2 (70%), IL13 (55%), ABCB1 and RB1 (50%), PIK3CA and CDH1 (45%). Conclusions Losses of F3, MIF, and BRCA1 significantly correlated with the posterior development of metastases and with worse survival. Also gains of PIK3CA, UTY, and RELAcorrelated with poor clinical outcome. Losses of BRCA1 and F3 were significant in multivariate analysis.

Background: Lymph node metastasis is the mayor cause of mortality in patients with head and neck squamous cell carcinomas (45%). The genetic changes underlying metastasis are still largely unknown and genetic markers to predict lymph node positivity still need to be found. The aim of this study was to search such markers by using Multiplex Ligation-dependent Probe Amplification (MLPA), a semi-quantitative PCR technique to detect gene copy number alterations. Methods: Thirty-seven genes were analysed by MLPA in 34 larynx and 22 pharynx carcinomas. Results: Losses of CDKN2A (9p21) and MLH1 (3p22) and gains of CCND1, EMS1 (both at 11q13), RECQL4 and PTP4A3 (both at 8q24) were the most frequent aberrations in both larynx and pharynx carcinomas. Amplifications were detected at EMS1, CCND1 and ERBB2 (17q21). A correlation between loss of N33 (8p22) and poor survival was found (p=0.02). Gain of EMS1 had the same relation with survival but not significant (p=0.08). Lymph node positive tumors presented a specific pattern of genetic alterations, with losses of N33, STK11 (19p13) and TP53 (17p13), the latter especially in larynx tumors. Conclusion: We propose that these 3 genes might play a role in the development of metastasis in larynx and pharynx squamous cell carcinomas.

Introduction For most patients with squamous head and neck cancer (HN-SCC), locoregional tumour recurrence (TR) carries an extremely poor prognosis and is therapeutically challenging. Objective To define the clinical aspects of TR and their implication on the survival in patients with HN-SCC. Patients and method The clinical management and the outcome of 652 patients with HN-SCC were reviewed. Results The overall incidence of TR in this series of HN-SCC was 19.9% (n=130). The most frequent locations of the primary cancers were oropharynx (32%), hypopharynx (24%), and oral cavity (21%). The rates of recurrence were locoregional 50%, local 43% and stomal recurrence 7%. The appearance of a TR reduces the overall survival of patients with HN-SCC to 15%. Survival is better in glottic (38%) and supraglottic (27%), carcinomas, and worse in oro-hypopharynx tumours (2-4%). Conclusions RT are more frequent in pharyngeal tumours, especially locoregional recurrences. Patients with recurrence in pharynx were definitely associated with poor prognosis and in these cases salvage surgery seems not to improve survival rates.

Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10μg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100μg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also be observed. Although the protective effects of the oral pIRF1A-G vaccine after a challenge with IHNV were partial, significant differences in cumulative percent mortalities among the orally vaccinated fish and the unvaccinated or empty-plasmid vaccinated fish were observed. Similar levels of protection were obtained after the intramuscular administration of 5μg of pIRF1A-G or after the oral administration of a high dose of pIRF1A-G vaccine (100μg); with 70 and 56 relative percent survival values, respectively. When fish were vaccinated with alginate microspheres containing high doses of the pIRF1A-G vaccine (50 or 100μg), a significant increase in the production of anti-IHNV antibodies was detected in serum samples of the vaccinated fish compared with that in unvaccinated fish. At 10 days post-challenge, IHNV N gene expression was nearly undetectable in kidney and spleen of orally vaccinated fish which suggested that the vaccine effectively reduced the amount of virus in tissues of vaccinated fish that survived the challenge. In conclusion, our results demonstrated a significant increase in fish immune responses and resistance to an IHNV infection after the oral administration of increasing concentrations of a DNA vaccine against IHNV encapsulated into alginate microspheres., This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) and the Consejo Superior de Investigaciones Científicas (Spain) (AGL2010-18454 and 2010–20E084, respectively). N. Ballesteros is grateful to the MINECO for the award of a PhD student fellowship.

[ES] Durante los últimos años la concienciación de los consumidores sobre la importancia del bienestar de los animales destinados a la producción de alimentos ha ido en creciente aumento. Así, se han desarrollado estudios y sistemas para evaluar y controlar la calidad del bienestar principalmente en las especies explotadas en sistemas intensivos y se han publicado protocolos con diversos parámetros a determinar. Sin embargo, todos ellos presentan problemas no existiendo una metodología universalmente aceptada. Por ello, partiendo de criterios y parámetros basados en el animal y en los alojamientos y manejo utilizados en protocolos existentes y en otros considerados importantes para la salud y el de bienestar de las vacas de leche por profesores de los Dpts. de Producción Animal y de Medicina, Cirugía y Anatomía Veterinaria de la Facultad de Veterinaria de León, se ha desarrollado un programa de software libre (Happy Cow) por parte de alumnos de la Escu, [EN] During the past years the consumer’s awareness about the importance of welfare of animals used for food production is increasing. In this way, studies and systems to evaluate and monitor the quality of the welfare, mainly in intensive systems productions, have been developed and guidelines and protocols with parameters to be determined have been published. However, they all have various problems and there is no universally accepted methodology. Therefore, based on criteria and parameters based on the animal and on facilities and handling recommended on the aforementioned project and other variables considered important for dairy cows health and welfare by professors from the Departments of Animal Production and Medicine, Surgery and Veterinary Anatomy, Faculty of Veterinary Medicine of León, a free software program (Happy Cow) has developed by students of the School of Industrial Engineering and Computer ULE that achieves two objectives: to be used

Invasive head and neck squamous carcinomas are among the cytogenetically most complex tumors. Perhaps for this reason, there is little consensus on the prognostic value of specific chromosomal aberrations. Here we present Results of CGH analysis of 56 clinically well-characterized set of head and neck cancers, consisting of larynx and pharynx only. The aim was to find possible associations with clinical outcome. The major chromosome arms showing gains were (in decreasing order): 3q, 7q, 8q, 5p, 11q13, 17q and 18p, and losses occurred at 3p, 11qter, 4p, 18q, and 5q. The segments most frequently amplified were 3q26-qter, 11q13, 11q22, 3q12–13, 18p11.3, 18q11.2 and 8q24.3. Tumors with stages III and IV, and lymph node positive tumors had a worse clinical outcome. Surprisingly, no specific chromosomal abnormality correlated with disease-free survival. The only aberration that correlated to one of the clinico-pathological parameters was amplification 11q13, that occurred solely in lymph node positive, stage IV tumors. However 11q13 amplification did not correlate with disease-free survival. These Results seem to indicate that genetic alterations at the level of chromosomes have limited prognostic value in patients with invasive larynx and pharynx squamous cell carcinomas.

La qualité des espaces publics, leurs formes, la spatialisation et le fonctionnement de leurs équipements et dispositifs économiques, leurs ambiances, leurs modes d’intégration dans le système des centralités intra-urbaines sont au cœur de la réflexion sur les devenirs de la ville contemporaine. L’offre commerciale peut participer à la création d’un espace urbain attractif, intense et habitable dans les centres comme dans les zones périphériques. Quelle place doivent prendre les structures du commerce dans une nouvelle programmation urbaine permettant d’assurer une attractivité redéfinie des espaces urbains ? Et plus exactement, comment intégrer les acteurs du système commercial aux politiques de renouvellement des espaces publics dans une perspective d’urbanisme durable ? Le commerce est un des éléments structurant et modulant l’intensité urbaine, les pratiques de mobilité, le lien social, l’animation et l’identité des quartiers lausannois. On explore ici quelques pistes de réflexion relatives au modèle de régulation de l’urbanisme commercial lausannois et à la manière dont les activités commerciales peuvent participer à la mise en place de stratégies de qualification de l’espace public et d’organisation des centralités urbaines. The quality of urban space, its morphology, spatial planning and ambiences, as well as the way that these centralities are integrated within the urban network and the hierarchy of centrality nodes are at the core of the creation of and thought about the contemporary city. The breakthrough of retail policies in Lausanne could become an extremely helpful planning tool, oriented towards making proximate neighborhoods both residentially and economically attractive. How does vicinity of retail enhance urbanity, and how does it influence in urban intensity, and the livability of public space? More precisely, how can we integrate retail related role players in the enhancement of the public realm’s urbanity within a context of sustainable urban design? The present article carries out an enquiry of how retail activities take part in strategies to improve the quality of the public realm and the organization of urban centralities. It reveals some hints that could help urban practitioners when making decisions regarding retail oriented developments. Die Qualität der öffentlichen Räume, ihre Morphologie, ihre Raumordnung und die Organisation von ihren wirtschaftlichen Einrichtungen und Massnahmen, die Umgebung sowie die Art dieser Zentralitäten innerhalb des urbanischen Netzwerkes integriert wurden und die Hierarchie der Zentralitätsknoten sind im Mittelpunkt der Betrachtung bezüglich der Weiterentwicklung der zeitgenossischen Stadt. Der Handel ist einer der Bereiche, der die urbane Intensität, das Mobilitätsystem, den sozialen Kontakt, das Leben und die Identität der Viertels von Lausanne struckturiert und moduliert. Kaufmännliche Richtlinien in Lausanne könnten extrem hilfreiches urbanisches Mittel zur Entwicklung eines attraktiven und wirtschaftlichen urbanischen Raumes und Wohngebietes in Stadtmitten und an Stadträndern werden. Wie kann Handel im Nah Bereich die Urbanisierung aufwerten/verbessern und wie kann er die urbane Intensität und die Lebensqualität der öffentlichen Räume einwirken? Genauer gesagt, wie können wir einzelhandelbezogenen Rollenspieler in der Aufwertung der Urbanisierung der öffentlichen Räume im Zusammenhang mit einer nachhaltigen urbanen Perspektive einführen? Der vorliegende Artikel führt eine Untersuchung durch, die die Einwirkung der kommerziellen Urbaniserung auf die Srategien zur Qualitätsverbesserungen von dem öffentlichen Raum und zur Organisation/Ordnung von den Stadtzentralitäten betont. Es zeigt Anleitungen auf, die Stadtpläner weiterhelfen können, wenn sie Entscheidungen im Bereich einzelhandelbezogener Entwicklungen treffen müssen.

Mycobacterium tuberculosis,Mycobacterium leprae,Mycobacterium bovis,andMycobacterium aviumsubsp.paratuberculosiscan survive within host macrophages in a dormant state, encased within an organized aggregate of immune host cells called granuloma. Granulomas consist of uninfected macrophages, foamy macrophages, epithelioid cells, and T lymphocytes accumulated around infected macrophages. Within granulomas, activated macrophages can fuse to form multinucleated giant cells, also called giant Langhans cells. A rim of T lymphocytes surrounds the core, and a tight coat of fibroblast closes the structure. Severalin vivomodels have been used to study granuloma’s structure and function, but recently developedin vitromodels of granuloma show potential for closer observation of the early stages of host’s responses to live mycobacteria. This paper reviews culture conditions that resulted in three-dimensional granulomas, formed by the adhesion of cell populations in peripheral blood mononuclear cells infected with mycobacteria. The similarities of these models to granulomas encountered in clinical specimens include cellular composition, granulomas’ cytokine production, and cell surface antigens. A reliablein vitrodormancy model may serve as a useful platform to test whether drug candidates can kill dormant mycobacteria. Novel drugs that target dormancy-specific pathways may shorten the current long, difficult treatments necessary to cure mycobacterial diseases.

Plasmid vectors designed to facilitate the genetic manipulation of African swine fever virus (ASFV) are described. Our results demonstrate that the beta-glucuronidase enzyme (GUS) can be used to follow gene expression in ASFV-infected cells. Infectious plaques formed by ASFV expressing GUS are visually detectable, thus providing a simple and highly sensitive method for the selection of ASFV recombinants. These and previous results have allowed us to construct two chimeric gene cassettes that constitute the basic tools for the generation of vectors to carry out the deletion of multiple target sequences from the ASFV genome. These cassettes, formed by: (a) a virus promoter; (b) the coding sequence of a reporter gene, either Lac Z or gusA; and (c) a strong signal for the 3' end formation of ASFV mRNAs, can be easily isolated by endonuclease restriction from their corresponding plasmid vectors. A general insertion/coexpression plasmid vector, pEPV2, has also been constructed. pEPV2 facilitates the insertion of foreign genes, together with the Lac Z reporter, into the thymidine kinase locus of ASFV. The functionality of pEPV2 has been tested by generating a recombinant ASFV expressing the luciferase gene. The vectors presented in this report constitute the first reported set of tools for the genetic manipulation of ASFV.

Tumor growth and metastasis entangle the alteration and recruitment of non-malignant cells to the primary tumor, among them immune cells, constituting the tumor microenvironment (TME). Communication between tumor cells and their stroma has been shown as a fundamental driving force of the tumoral process. A great deal of effort has been focused on depicting their specific interactions and crosstalk. However, most research has been carried out in 2D conventional cultures that alter cell morphology and intracellular signaling processes. Considering these premises, we have developed a 3D cell co-culture model to mimic T cell infiltration into the tumor mass and explore tumor-immune cells interactions in the TME. Expression of specific cell markers and assessment of cell proliferation were carried out to characterize the proposed 3D co-culture model. Additionally, the study and profiling of the secretome revealed a subset of particular cancer-related inflammation proteins prompted upon 3D cultivation of tumor cells in presence of lymphocytes, pointing out an intercellular communication. Altogether, these results suggest that our 3D cell co-culture model can be a useful tool to identify and study critical factors mediating the crosstalk between tumor and immune cells in the TME. Finally, the potential of this model as a drug-screening platform has been explored using docetaxel as a model antitumoral compound.

Novel markers to accurately predict the risk of malignant transformation in laryngeal epithelial precursor lesions (EPL) are needed. We tried to identify some molecular alterations occurring in laryngeal tumorigenesis. In this study, 60 paraffin-embedded EPL and 17 metachronous invasive carcinomas were immunostained for markers associated with proliferation (Ki67), cell cycle control (p53, p21, p16, p27, cyclin D1), and cell adhesion and invasion (laminin and β-catenin). Aberrant expression of p16 and p53 and positivity at cytoplasm for β-catenin and cyclin D1 were detected significantly in EPL with progression to invasive laryngeal carcinoma. All cases with basal and suprabasal reactivity of p53 showed β-catenin overexpression. We found that β-catenin protein expression increased significantly with the grade of dysplasia. This is one of the studies with the largest number of laryngeal EPL and invasive carcinoma studied sequentially. Our data confirm the role of some cell cycle regulatory proteins in the development of laryngeal carcinoma. Cytoplasmic retention of β-catenin in EPL seems to be related with more aggressive biological behavior. Combined increased p53 and cytoplasmic β-catenin protein expression could be biologically important in laryngeal tumorigenesis. Further research is required to clarify the involvement of β-catenin in the mechanism associated with malignant transformation in laryngeal tissues.

Mycobacterium aviumsubsp.paratuberculosis(Map) is the causative agent of a chronic intestinal inflammation in ruminants named Johne's disease or paratuberculosis and a possible etiopathological agent of human Crohn's disease (CD). Analysis of macrophage transcriptomes in response toMapinfection is expected to provide key missing information in the understanding of the role of this pathogen in establishing an inappropriate and persistent infection in a susceptible host and of the molecular mechanisms that might underlie the early phases of CD. In this paper we summarize transcriptomic studies of human and bovine peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDMs), and macrophages-like cell linesin vitroinfected withMap. Most studies included in this paper consistently reported common gene expression signatures of bovine and human macrophages in response toMapsuch as enhanced expression of the anti-inflammatory cytokines IL-10 and IL-6, which promote bacterial survival. Overexpression of IL-10 could be responsible for theMap-associated reduction in the expression of the proapoptotic TNF-αgene observed in bovine and human macrophages.

This article reviews some of the recent patents on DNA vaccines against fish viruses, in particular against the novirhabdovirus infectious hematopoitic necrosis virus (IHNV). Although very effective in protecting fish against IHNV, only one DNA vaccine has been approved to date for use in Canada. In Europe and in US, its commercialization is restricted due to safety concerns.

Introduction and objectives The literature on the involvement of microsatellite instability in head and neck squamous cell carcinoma shows great variability, probably due to differences in the testing methods. Using a consensus detection system, we aimed to reach a reliable estimate of microsatellite instability prevalence in a subset of head and neck squamous cell carcinoma cases. Methods The microsatellite instability status of 43 patients with previously untreated primary laryngeal squamous cell carcinomas was analysed by a multiplex polymerase chain reaction assay including 5 mononucleotide repeat markers. Results Thirty-six cases showed a stable phenotype or a microsatellite stable phenotype (83.7%) and 7 cases (16.3%) showed a microsatellite instability-positive phenotype. One case showed instability in 3 of 5 markers, 1 case in 2 markers and 5 cases in 1 marker. The microsatellite instability-positive and stable cases did not differ with respect to age, tumour stage, lymph node or distant metastases. Conclusions Our data showed that a proportion of laryngeal squamous cell carcinomas are microsatellite instability positive. Knowledge of microsatellite instability patient status will allow adjusting anticancer therapy at an individual level.

Solitary schwannoma is a benign, encapsulated tumour of Schwann cell origin, therefore the olfactory and optic nerves are never affected. About 25%-45% of all schwannomas occur in the head and neck but schwannoma of the external auditory canal is a rare finding, and we have found only 6 previous cases reported in the literature world-wide. Our patient was discovered by chance during a stapedectomy because the tumour was sited in the external auditory canal without distorting it.

A survey of routes for aminal cleavage of N-sulfinylimidazolidines has been carried out, and selective conditions to cleave the aminal moiety while preserving the sulfinamide group unaltered have b...

Coinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log10 units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48-72 h post infection, in contrast to the 50-80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference; the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections.

Background Paratuberculosis vaccination has been in use in some regions for many decades, but results have not been widely spread. A new Mycobacterium avium subsp. paratuberculosis (MAP) killed vaccine was studied in relationship with its effects on fecal shedding and milk production in four farms while other two were kept as controls submitted to a test and cull scheme. Findings Fecal detection (n = 1829) and milking records (n = 2413) have been analyzed after two (5 herds) and four (1 herd) years of the beginning of the intervention. Shedder prevalence was reduced by 100% in three of the four vaccinated farms, 68% in the total of vaccinated animals and 46% in the two control farms. Total amount of MAP shed was reduced 77% in the vaccinated farms and 94% in the control farms. Overall milk production increased up to 3.9% after vaccination, while there was no significant difference in production after intervention in the non-vaccinated farms. Conclusion MAP shedding reduction can be quickly accomplished both by vaccination and by testing and culling. However, vaccination appears to be a less expensive and more sustainable strategy since it required one single intervention and was also associated with an increase in milk production.

AIM: To investigate if the clinical efficacy of granulocytes and monocytes by adsorption (GMA) is associated with an increased frequency of peripheral regulatory T cells (Tregs), as these cells have proven to be successful in suppressing inflammatory bowel disease (IBD) in animal models. METHODS: We report four cases of corticosteroid-dependent ulcerative colitis (UC) and two Crohn’s disease (CD) cases with severe cutaneous lesions who received GMA therapy. The frequency of CD4+ CD25high (Tregs) in peripheral blood was analyzed by flow cytometry and the expression of FoxP3 and TGF beta in purified CD4+ T cells was determined by real time PCR prior to and one month after the last apheresis session, and at the time of endoscopic and clinical assessing. RESULTS: Increased expression of Fox P3 mRNA was found in all five patients who responded to cytapheresis with remission of clinical symptoms, mucosal inflammation and cutaneous lesions, and an increased frequency of circulating Tregs was found in four patients. These changes were not observed in the patient with UC who did no respond to GMA. Variations in TGF-β (mRNA) did not parallel that of FoxP3 mRNA. CONCLUSION: The clinical efficacy of GMA on IBD and related extra intestinal manifestations was associated with an expansion of circulating CD4+ CD25+ Tregs and higher expression of FoxP3 in CD4+ T cells. Accordingly, an elevated CD4+ CD25+ FoxP3 may be a valuable index of remission in patients with IBD and other chronic relapsing-remitting inflammatory conditions during treatment with GMA.