Mytilus galloprovincialis releases immunologically functional haemocytes to the intervalvar space in response to tissue injury and infection.

Haemocytes of Mytilus galloprovincialis represent the main component of the internal self-defence system. Although haemocytes from haemolymph are usually studied to analyse these animals' immune response, the presence of haemocytes in the intervalvar liquid, which is essentially sea water, led us to characterize them. Several functional (ROS production, phagocytosis, gene expression, travel velocity and distance) and morphological (area, size and granularity) assays were performed by applying different stimuli to the mussels (waterborne infection, shell injury and their combination). Our results revealed that intervalvar liquid haemocytes share common characteristics with haemolymph haemocytes (for instance, the cell morphology and the cell population structure divided in three main groups) but also show significant differences in size (usually smaller in the intervalvar liquid), mobility (commonly faster in the intervalvar liquid), ROS production (higher in non-stimulated intervalvar liquid cells) and gene expression (IL17, Myd88 and CathL are over expressed in liquid intervalvar cells compared to haemolymph cells). Moreover, differences were observed when mussels were subjected to the mentioned treatments. These free intervalvar haemocytes could constitute the first line of defence as external sentinels extending the immunological alert system outside of the mussel body.

Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Development and validation of a specific real-time PCR assay for the detection of the parasite Perkinsus olseni.

Perkinsus olseni is a protozoan parasite that infects a wide variety of molluscs worldwide, causing economic losses in the aquaculture sector. In the present study, a quantitative PCR (qPCR) assay was developed for the detection and quantification of P. olseni in clam gill tissue and hemolymph (Ruditapes philippinarum and R. decussatus), and the results were compared with those of the standard diagnostic methods recommended by the O.I.E. (World Organisation for Animal Health): Ray's fluid thioglycollate culture method (RFTM), a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and histopathology. The efficiency, sensitivity and reproducibility of the newly described qPCR assay were also determined. The highest prevalence was detected using the qPCR assay, and the strongest linear correlation was obtained between the RFTM infection levels and the threshold cycle (Ct) number from the gill tissue. Although better results were obtained from gill than from the hemolymph in the qPCR assays, especially with lower infection levels of the parasite, a significant linear correlation was observed between Ct values from the gill and hemolymph. The qPCR assay that was developed in this study showed high sensitivity, specificity and reproducibility for the detection and quantification of P. olseni.

Turbot (Scophthalmus maximus) Nk-lysin induces protection against the pathogenic parasite Philasterides dicentrarchi via membrane disruption.

P. dicentrarchi is one of the most threatening pathogens for turbot aquaculture. This protozoan ciliate is a causative agent of scuticociliatosis, which is a disease with important economic consequences for the sector. Neither vaccines nor therapeutic treatments are commercially available to combat this infection. Numerous antimicrobial peptides (AMPs) have demonstrated broad-spectrum activity against bacteria, viruses, fungi, parasites and even tumor cells; an example is Nk-lysin (Nkl), which is an AMP belonging to the saposin-like protein (SAPLIP) family with an ability to interact with biological membranes. Following the recent characterization of turbot Nkl, an expression plasmid encoding Nkl was constructed and an anti-Nkl polyclonal antibody was successfully tested. Using these tools, we demonstrated that although infection did not clearly affect nkl mRNA expression, it induced changes at the protein level. Turbot Nkl had the ability to inhibit proliferation of the P. dicentrarchi parasite both in vivo and in vitro. Moreover, a shortened peptide containing the active core of turbot Nkl (Nkl71-100) was synthesized and showed high antiparasitic activity with a direct effect on parasite viability that probably occurred via membrane disruption. Therefore, the nkl gene may be a good candidate for genetic breeding selection of fish, and either the encoded peptide or its shortened analog is a promising antiparasitic treatment in aquaculture.

Expression of gut-homing ß7 receptor on T cells: surrogate marker for microbial translocation in suppressed HIV-1-infected patients?

OBJECTIVES: In view of the fact that mucosal damage associated with HIV-1 infection leads to microbial translocation despite successful antiretroviral treatment, we analysed microbial translocation and expression of the gut-homing ß7 receptor on peripheral T cells in HIV-1-infected individuals. METHODS: Fifteen long-term suppressed HIV-1-infected patients, of whom seven had their treatment intensified with maraviroc and eight with raltegravir, were included in the study. Samples at baseline, at week 48 of intensification, and at weeks 12 and 24 after deintensification were analysed for soluble CD14, lipopolysaccharide (LPS), LPS-binding protein, gut-homing ß7 receptor and T-cell subsets. RESULTS: The increases in both microbial translocation and expression of the gut-homing ß7 receptor on activated CD8 T cells found during maraviroc intensification were reduced after deintensification. Moreover, the correlations between activated ß7(+) T cells and LPS levels found during intensification with maraviroc (P = 0.036 and P = 0.010, respectively) were lost during deintensification. In contrast, microbial translocation was stable during raltegravir intensification, with the exception of decreased LPS levels and activated CD4 ß7(+) T cells, which reverted to baseline values after deintensification. CONCLUSIONS: Microbial translocation is an important factor in gut immune activation and mucosa inflammation, as evidenced by the association between the dynamics of microbial translocation and activated T cells expressing the gut-homing ß7 receptor. The recruitment of activated ß7(+) T cells to the gut tract when alteration of microbial translocation is maximum may be the major mechanism for recovery of mucosal integrity.

The complexity of apoptotic cell death in mollusks: An update.

Apoptosis is a type of programmed cell death that produces changes in cell morphology and in biochemical intracellular processes without inflammatory reactions. The components of the apoptotic pathways are conserved throughout evolution. Caspases are key molecules involved in the transduction of the death signal and are responsible for many of the biochemical and morphological changes associated with apoptosis. Nowadays, It is known that caspases are activated through two major apoptotic pathways (the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway), but there are also evidences of at least other alternative pathway (the perforin/granzyme pathway). Apoptosis in mollusks seems to be similar in complexity to apoptosis in vertebrates but also has unique features maybe related to their recurrent exposure to environmental changes, pollutants, pathogens and also related to the sedentary nature of some stages in the life cycle of mollusks bivalves and gastropods. As in other animals, apoptotic process is involved in the maintenance of tissue homeostasis and also constitutes an important immune response that can be triggered by a variety of stimuli, including cytokines, hormones, toxic insults, viruses, and protozoan parasites. The main goal of this work is to present the current knowledge of the molecular mechanisms of apoptosis in mollusks and to highlight those steps that need further study.

Interpreting the reasons for the choice and changing of two drug regimens in an observational cohort: comparison of a ritonavir-boosted protease inhibitor-based versus a nonnucleoside reverse transcriptase inhibitor-based first-line regimen.

OBJECTIVES: We compared reasons for the choice of regimen, time to and reasons for third drug modification, virological response and change in CD4 T-cell counts in patients started on atazanavir/ritonavir (ATV/r)- vs. efavirenz (EFV)-based first-line regimens. METHODS: We included patients from the Cohort of the Spanish HIV Research Network (CoRIS), a multicentre cohort of HIV-positive treatment-naïve subjects, in the study. We used logistic regression to assess factors associated with choosing ATV/r vs. EFV, proportional hazards models on the subdistribution hazard to estimate subdistribution hazard ratios (sHRs) for third drug modification, logistic regression to estimate odds ratios (ORs) for virological response and linear regression to assess mean differences in CD4 T-cell count increase from baseline. RESULTS: Of 2167 patients, 10.7% started on ATV/r. ATV/r was more likely than EFV to be prescribed in injecting drug users [adjusted OR 1.85; 95% confidence interval (CI) 1.03-3.33], in 2009-2010 (adjusted OR 1.63; 95% CI 1.08-2.47) and combined with abacavir plus lamivudine (adjusted OR 1.53; 95% CI 0.98-2.43). Multivariate analyses showed no differences, comparing ATV/r vs. EFV, in the risk of third drug modification (sHR 1.04; 95% CI 0.74-1.46) or in virological response (OR 0.81; 95% CI 0.46-1.41); differences in mean CD4 T-cell count increase from baseline were at the limit of statistical significance (mean difference 29.8 cells/µL; 95% CI -4.1 to 63.6 cells/µL). In patients changing from EFV, 48% of changes were attributable to toxicity/adverse events, 16% to treatment failure/resistance, 3% to simplification, and 8 and 12%, respectively, to patients' and physicians' decisions; these percentages were 24, 6, 12, 14 and 24%, respectively, in those changing from ATV/r. CONCLUSIONS: ATV/r- and EFV-based regimens meet the requirements of both efficacy and safety for initial combination antiretroviral regimen, which relate to better durability.

The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

3D printing of PLA:CaP:GO scaffolds for bone tissue applications.

Graphene oxide (GO) has attracted increasing interest for biomedical applications owing to its outstanding properties such as high specific surface area, ability to bind functional molecules for therapeutic purposes and solubility, together with mechanical resistance and good thermal conductivity. The combination of GO with other biomaterials, such as calcium phosphate (CaP) and biodegradable polymers, presents a promising strategy for bone tissue engineering. Presently, the development of these advanced biomaterials benefits from the use of additive manufacturing techniques, such as 3D printing. In this study, we develop a 3D printed PLA:CaP:GO scaffold for bone tissue engineering. First, GO was characterised alone by XPS to determine its main bond contributions and C : O ratio. Secondly, we determined the GO dose which ensures the absence of toxicity, directly exposed in vitro (human osteoblast-like cells MG-63) and in vivo (zebrafish model). In addition, GO was microinjected in the zebrafish to evaluate its effect on immune cells, quantifying the genetic expression of the main markers. Results indicated that the GO tested (C : O of 2.14, 49.50% oxidised, main bonds: C-OH, C-O-C) in a dose ≤0.25 mg mL-1 promoted MG63 cells viability percentages above 70%, and in a dose ≤0.10 mg mL-1 resulted in the absence of toxicity in zebrafish embryos. The immune response evaluation reinforced this result. Finally, the optimised GO dose (0.10 mg mL-1) was combined with polylactic acid (PLA) and CaP to obtain a 3D printed PLA:CaP:GO scaffold. Physicochemical characterisation (SEM/EDS, XRD, FT-Raman, nano-indentation) was performed and in vivo tests confirmed its biocompatibility, enabling a novel approach for bone tissue-related applications.

A novel hepcidin-like in turbot (Scophthalmus maximus L.) highly expressed after pathogen challenge but not after iron overload.

Hepcidins are antimicrobial peptides with an important role in the host innate immunity. Moreover, it has been reported that mammalian hepcidins present a dual-function being a key regulator in the iron homeostasis. Here, we describe the coding sequence of a novel hepcidin-like peptide in turbot, Scophthalmus maximus. This molecule presents several differences with regard to the previously characterized hepcidin in this flatfish species and it has not the hypothetical iron regulatory sequence Q-S/I-H-L/I-S/A-L in the N-terminal region. Therefore we propose the existence of at least two types of hepcidin in turbot. Moreover, results revealed a higher variability in the mRNA sequences of the novel hepcidin compared with the other form. Constitutive expression of turbot hepcidins (Hepcidin-1 and Hepcidin-2) was analyzed in several tissues and as expected, both molecules were highly represented in liver. On the other hand, the effect of three different stimuli (bacterial or viral infection and iron overloading) in the level of hepcidin mRNA was also examined and a differential response to pathogens and iron was observed. Whereas both hepcidins were affected by pathogen challenge, only Hepcidin-1 was up-regulated after iron overloading. Therefore, this and other evidences suggest that these peptides could be involved in different functions covering the dual role of mammalian hepcidins.

Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation.

The morphology and functionality of Ruditapes decussatus haemocytes have been characterized by light microscopy and flow cytometry, leading to the identification of three different cellular subpopulations. Granulocytes were the largest cells, the hyalinocytes were smaller and contained fewer granules and the intermediate cells showed a size similar to hyalinocytes and a higher number of granules. The phagocytosis of different particles and the associated production of oxygen radicals were measured by flow cytometric methods. Granulocytes were the most active cells, followed by the intermediate cells and hyalinocytes. The effect of stimulation of haemocytes with lipopolysaccharide (LPS), with a heat inactivated bacterial mixture or with the infection of Vibrio splendidus on the cell viability and the expression of selected immune-related genes were studied. While significant low levels of damaged cells were registered in LPS-stimulated cells, the treatment with dead bacteria or V. splendidus reduced cell viability 1 h, 3 h and 6 h after treatment. The stimulation of haemocytes with LPS and dead bacteria induced changes in the expression of defender against cell death (DAD-1), thrombin, prosaposin, inhibitor of apoptosis (IAP), factor B and C3 complement component.

Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus).

Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV(860)) was cloned into an expression plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G(860)). In our experimental conditions, DNA immunised turbots were more than 85% protected against VHSV(860) lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report showing the efficacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the contribution of innate and adaptive immunity to the protective response elicited by the immunization.

[First case series of femoral lengthening using a Precice® intramedullary nail in our center]. / Primera serie de casos de alargamiento femoral mediante clavo intramedular Precice® en nuestro centro.

INTRODUCTION: femoral lengthening using an intramedullary nail is one of the surgical options in the treatment of severe lower limb dysmetria in routine clinical practice. MATERIAL AND METHODS: a retrospective descriptive study was carried out on a series of five patients with a mean age of 15.4 years, who underwent femoral lengthening surgery using a Precice® intramedullary nail. The etiology in all cases was idiopathic. Preoperative and definitive postoperative theoretical lengthening or dysmetry was measured, as well as lengthening accuracy, distraction rate and index (mm/day and days/cm, respectively) and consolidation index (days/cm). Intraoperative and postoperative complications were identified in all cases. RESULTS: mean follow-up was 21 months (12-42), with no loss to follow-up. The mean duration of the surgical procedure was 126 minutes (105-160). The preoperative theoretical dysmetry was 38 ± 2.7 mm. The final mean lengthening was 41 ± 7.5 mm. The mean accuracy was 108% (91-125) and the distraction rate was 0.9 ± 0.4 mm/day. The distraction rate was 13.9 ± 5.1 days/cm and the consolidation rate was 26.6 ± 9.1 days/cm. Bone consolidation was observed in all patients with a mean of 113 ± 58 days. Regarding complications, a total of four minor muscular complications were found. CONCLUSION: the Precice® intramedullary nail is a good treatment option for cases of severe femoral shortening, providing good clinical and radiological results with a low rate of complications and implant failure.

INTRODUCCIÓN: el alargamiento femoral mediante clavo intramedular es una de las opciones quirúrgicas en el tratamiento de las dismetrías severas de miembros inferiores en la práctica clínica habitual. MATERIAL Y MÉTODOS: se realizó un estudio descriptivo retrospectivo de una serie de cinco pacientes con una media de edad de 15.4 años, intervenidos de alargamiento femoral mediante clavo intramedular Precice®. La etiología en todos los casos fue idiopática. Se midió la dismetría o alargamiento teórico prequirúrgico y el definitivo postquirúrgico, así como la precisión del alargamiento, la tasa y el índice de distracción (mm/día y días/cm respectivamente) y el índice de consolidación (días/cm). Se identificaron las complicaciones intra y postoperatorias en todos los casos. RESULTADOS: la media de seguimiento fue de 21 meses (12-42), sin pérdidas en el seguimiento. La duración media del procedimiento quirúrgico fue de 126 minutos (105-160). La dismetría teórica prequirúrgica fue de 38 ± 2.7 mm. El alargamiento medio final fue de 41 ± 7.5 mm. La precisión media fue de 108% (91-125) y la tasa de distracción de 0.9 ± 0.4 mm/día. El índice de distracción fue de 13.9 ± 5.1 días/cm y el índice de consolidación, de 26.6 ± 9.1 días/cm. La consolidación ósea se observó en la totalidad de los pacientes con una media de 113 ± 58 días. Con respecto a las complicaciones, se encontraron un total de cuatro complicaciones menores de índole muscular. CONCLUSIÓN: el clavo intramedular Precice® es una buena opción de tratamiento para casos de acortamiento femoral severo aportando buenos resultados clínicos y radiológicos con una baja tasa de complicaciones y fallo del implante.

Alkane-degrading properties of Dietzia sp. H0B, a key player in the Prestige oil spill biodegradation (NW Spain).

AIMS: Investigation of the alkane-degrading properties of Dietzia sp. H0B, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. METHODS AND RESULTS: Using molecular and chemical analyses, the alkane-degrading properties of strain Dietzia sp. H0B were analysed. This Grampositive isolate was able to grow on n-alkanes ranging from C12 to C38 and branched alkanes (pristane and phytane). 8-Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia H0B, suggesting a novel alkane-degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR-amplified from Dietzia H0B and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia H0B. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. CONCLUSIONS: The results from this study suggest novel alkane-degrading pathways in Dietzia H0B and a genetic background coding for two different putative oil-degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation.

An overview of the internalization and effects of microplastics and nanoplastics as pollutants of emerging concern in bivalves.

Microplastic and nanoplastic pollution in aquatic environments is a topic of emerging concern due to the internalization, retention time and effects of these particles in aquatic biota. Bivalves are considered bioindicators due to their wide distribution, sessile behaviour, occupation of ecological niches and ability to filter a large water volume. The study of microplastics and nanoplastics in bivalves has revealed the uptake mechanisms, internalization, distribution and depuration of these particles as well as their effects on physiological parameters, morphological alterations, immunotoxicity and changes in gene expression and proteomic profiles. In this review, we examine the primary characteristics of microplastics and nanoplastics (type of material, size, coating, density, additives and shapes) involved in their possible toxicity in bivalves. Furthermore, secondary characteristics such as the suspension media, aggregation stage and adsorption of persistent pollutants were also recorded to assess the impact of these materials on bivalves. Here, we have highlighted the efforts exerted thus far and the remaining gaps in understanding the extent of microplastic and nanoplastic impacts on bivalves on the basis of laboratory experiments and mesocosm bioassays and in the field. Furthermore, further microplastic and nanoplastic toxicological studies are proposed to facilitate the realistic assessment of environmental risk.

Size matters: Zebrafish (Danio rerio) as a model to study toxicity of nanoplastics from cells to the whole organism.

The contamination of the aquatic environment by plastic nanoparticles is becoming a major concern due to their potential adverse effects in aquatic biota. Therefore, in-depth knowledge of their uptake, trafficking and effects at cellular and systemic levels is essential to understand their potential impacts for aquatic species. In this work, zebrafish (Danio rerio) was used as a model and our aims were: i) to determine the distribution, uptake, trafficking, degradation and genotoxicity of polystyrene (PS) NPs of different sizes in a zebrafish cell line; ii) to study PS NPs accumulation, migration of immune cells and genotoxicity in larvae exposed to PS NPs; and iii) to assess how PS NPs condition the survival of zebrafish larvae exposed to a pathogen and/or how they impact the resistance of an immunodeficient zebrafish. Our results revealed that the cellular distribution differed depending on the particle size: the 50 nm PS NPs were more homogeneously distributed in the cytoplasm and the 1 µM PS NPs more agglomerated. The main endocytic mechanisms for the uptake of NPs were dynamin-dependent internalization for the 50 nm NPs and phagocytosis for the 1 µm nanoparticles. In both cases, degradation in lysosomes was the main fate of the PS NPs, which generated alkalinisation and modified cathepsin genes expression. These effects at cellular level agree with the results in vivo, since lysosomal alkalization increases oxidative stress and vice versa. Nanoparticles mainly accumulated in the gut, where they triggered reactive oxygen species, decreased expression of the antioxidant gene catalase and induced migration of immune cells. Finally, although PS NPs did not induce mortality in wild-type larvae, immunodeficient and infected larvae had decreased survival upon exposure to PS NPs. This fact could be explained by the mechanical disruption and/or the oxidative damage caused by these NPs that increase their susceptibility to pathogens.

Comparison of antiviral activity of regimens containing nucleos(t)ide (NUC) Pairs in HIV-Infected Patients Initiating REScue therapy (The NUCREST Study).

BACKGROUND: recycling nucleos(t)ides (NUCs) is useful in regions where new antiretrovirals are not available. This study compares the effectiveness of NUC-containing regimens as rescue therapy in routine care. METHODS: retrospective, multicentre cohort study (January 2001 to June 2006) of patients with ≥ 1 virological failure who started therapy with 2 NUCs and 1 non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI). The primary endpoint was the rate of treatment response at 6 months (intention-to-treat [ITT] analysis). RESULTS: we included 719 patients (average of 4 prior regimens over a median 6.1 years). The most frequent NUC pairs were tenofovir plus lamivudine (TDF+3TC; 25%), tenofovir plus stavudine (TDF+d4T; 23%), and stavudine plus didanosine (d4T+ddI; 15%). A boosted PI was used in 68% of total cases. Resistance to both NUCs was more frequent in zidovudine plus lamivudine (AZT+3TC; 22.0%), abacavir plus lamivudine (ABC+3TC; 35.5%), and stavudine plus lamivudine (d4T+3TC; 31.2%). No significant differences were observed in treatment response (overall 65%, P = .67); ddI+3TC (71%) and d4T+3TC (53%) had the highest and lowest response rates, respectively. Median time to failure was shorter with d4T+3TC, d4T+ddI, and ABC+3TC (48, 51, and 58 weeks, respectively; P = .0012). Lower response rates associated with an increasing number of thymidine analog mutations (TAMs) were observed for ABC+3TC (P = .027). CONCLUSION: the clinical utility of NUCs for rescue therapy is limited and selection should be individualized. Specific combinations (d4T+3TC and d4T+ddI) might be less efficacious.

Nodavirus encephalopathy in turbot (Scophthalmus maximus): inflammation, nitric oxide production and effect of anti-inflammatory compounds.

Nodaviruses are the etiological agents of one of the most serious viral diseases affecting marine fish aquaculture. Nodavirus infection produces an abnormal swimming behaviour and causes encephalopathy and retinopathy associated to important mortalities. The expression of TNF-alpha, IRF-1 and Mx was increased in turbot after nodavirus infection. A significant increase in the production of nitrogen radicals was also observed in experimentally infected turbot. Several anti-inflammatory compounds (the antioxidant N-acetylcysteine, cortisone, dexamethasone, prednisolone and aminoguanidine) were assayed to determine the role of inflammation on nodavirus infection. Cortisone and aminoguanidine were able to accelerate the mortality onset associated to nodavirus infection, modulating the gene expression of TNF-alpha and, in addition, modifying the arrival time of nodavirus to the brain. These results suggest the importance of early inflammatory processes to overcome the infection.

Nanoplastics: From tissue accumulation to cell translocation into Mytilus galloprovincialis hemocytes. resilience of immune cells exposed to nanoplastics and nanoplastics plus Vibrio splendidus combination.

Plastic litter is an issue of global concern. In this work Mytilus galloprovincialis was used to study the distribution and effects of polystyrene nanoplastics (PS NPs) of different sizes (50 nm, 100 nm and 1 µm) on immune cells. Internalization and translocation of NPs to hemolymph were carried out by in vivo experiments, while endocytic routes and effects of PS NPs on hemocytes were studied in vitro. The smallest PS NPs tested were detected in the digestive gland and muscle. A fast and size-dependent translocation of PS NPs to the hemolymph was recorded after 3 h of exposure. The internalization rate of 50 nm PS NPs was lower when caveolae and clathrin endocytosis pathways were inhibited. On the other hand, the internalization of larger particles decreased when phagocytosis was inhibited. The hemocytes exposed to NPs had changes in motility, apoptosis, ROS and phagocytic capacity. However, they showed resilience when were infected with bacteria after PS NP exposure being able to recover their phagocytic capacity although the expression of the antimicrobial peptide Myticin C was reduced. Our findings show for the first time the translocation of PS NPs into hemocytes and how their effects trigger the loss of its functional parameters.