Viral pathogen-induced mechanisms to antagonize mammalian interferon (IFN) signaling pathway.

Antiviral responses of interferons (IFNs) are crucial in the host immune response, playing a relevant role in controlling viralw infections. Three types of IFNs, type I (IFN-α, IFN-ß), II (IFN-γ) and III (IFN-λ), are classified according to their receptor usage, mode of induction, biological activity and amino acid sequence. Here, we provide a comprehensive review of type I IFN responses and different mechanisms that viruses employ to circumvent this response. In the first part, we will give an overview of the different induction and signaling cascades induced in the cell by IFN-I after virus encounter. Next, highlights of some of the mechanisms used by viruses to counteract the IFN induction will be described. And finally, we will address different mechanism used by viruses to interference with the IFN signaling cascade and the blockade of IFN induced antiviral activities.

Virus-induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade.

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response, and viral pathogens have evolved multiple mechanisms to antagonize this pathway and to facilitate infection. Bluetongue virus (BTV), an orbivirus of the Reoviridae family, is transmitted by midges to ruminants and causes a disease that produces important economic losses and restriction to animal trade and is of compulsory notification to the World Organization for Animal Health (OIE). Here, we show that BTV interferes with IFN-I and IFN-II responses in two ways, by blocking STAT1 phosphorylation and by degrading STAT2. BTV-NS3 protein, which is involved in virion egress, interacts with STAT2, and induces its degradation by an autophagy-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 polyubiquitination. Taken together, our study identifies a new mechanism by which a virus degrades STAT2 for IFN signaling blockade, highlighting the diversity of mechanisms employed by viruses to subvert the IFN response.

T cells loaded with magnetic nanoparticles are retained in peripheral lymph nodes by the application of a magnetic field.

BACKGROUND: T lymphocytes are highly dynamic elements of the immune system with a tightly regulated migration. T cell-based transfer therapies are promising therapeutic approaches which in vivo efficacy is often limited by the small proportion of administered cells that reaches the region of interest. Manipulating T cell localisation to improve specific targeting will increase the effectiveness of these therapies. Nanotechnology has been successfully used for localized release of drugs and biomolecules. In particular, magnetic nanoparticles (MNPs) loaded with biomolecules can be specifically targeted to a location by an external magnetic field (EMF). The present work studies whether MNP-loaded T cells could be targeted and retained in vitro and in vivo at a site of interest with an EMF. RESULTS: T cells were unable to internalize the different MNPs used in this study, which remained in close association with the cell membrane. T cells loaded with an appropriate MNP concentration were attracted to an EMF and retained in an in vitro capillary flow-system. MNP-loaded T cells were also magnetically retained in the lymph nodes after adoptive transfer in in vivo models. This enhanced in vivo retention was in part due to the EMF application and to a reduced circulating cell speed within the organ. This combined use of MNPs and EMFs did not alter T cell viability or function. CONCLUSIONS: These studies reveal a promising approach to favour cell retention that could be implemented to improve cell-based therapy.

Effect of a grapevine-shoot waste extract on red wine aromatic properties.

BACKGROUND: The use of a grapevine-shoot extract (VIN) is being studied as an alternative to sulfur dioxide (SO2 ). VIN stabilizes anthocyanins and preserves polyphenolic compounds, and thus improves chromatic wine properties. In this study, selected aroma compounds (esters, C13 -norisoprenoids, oxidation and vine-shoot-related compounds), sensory analysis and the olfactometric profile were determined in the wines treated with VIN at two concentrations. RESULTS: Treatment with VIN hardly modified the content of esters and oxidation-related compounds in the wines. However, the high ß-damascenone and isoeugenol contents and the increase in astringency at tasting in VIN wines were noteworthy, as were some odorant zones. All these were established as VIN markers after the chemometric data analysis. CONCLUSION: These data revealed that only the lowest dose tested may be recommended as a suitable alternative to SO2 . Although some aromatic properties of these wines may change, these changes are not considered to affect the quality of the wines negatively. These results are useful for wineries, which face having to discover the aroma-related processes in the challenge of producing SO2 -free wines without detriment to their sensory properties. © 2018 Society of Chemical Industry.

Inhibition of PI3Kδ reduces kidney infiltration by macrophages and ameliorates systemic lupus in the mouse.

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease generated and maintained throughout life by autoreactive T and B cells. Class I phosphoinositide 3-kinases (PI3K) are heterodimers composed of a regulatory and a catalytic subunit that catalyze phosphoinositide-3,4,5-P3 formation and regulate cell survival, migration, and division. Activity of the PI3Kδ isoform is enhanced in human SLE patient PBLs. In this study, we analyzed the effect of inhibiting PI3Kδ in MRL/lpr mice, a model of human SLE. We found that PI3Kδ inhibition ameliorated lupus progression. Treatment of these mice with a PI3Kδ inhibitor reduced the excessive numbers of CD4(+) effector/memory cells and B cells. In addition, this treatment reduced serum TNF-α levels and the number of macrophages infiltrating the kidney. Expression of inactive PI3Kδ, but not deletion of the other hematopoietic isoform PI3Kγ, reduced the ability of macrophages to cross the basement membrane, a process required to infiltrate the kidney, explaining MRL/lpr mice improvement by pharmacologic inhibition of PI3Kδ. The observations that p110δ inhibitor prolonged mouse life span, reduced disease symptoms, and showed no obvious secondary effects indicates that PI3Kδ is a promising target for SLE.

Superparamagnetic iron oxide nanoparticle uptake alters M2 macrophage phenotype, iron metabolism, migration and invasion.

Superparamagnetic iron oxide nanoparticles (SPIONs) have shown promise as contrast agents and nanocarriers for drug delivery. Their impact on M2-polarised macrophages has nonetheless not been well studied. Here we explored the effects of SPIONs coated with dimercaptosuccinic acid, aminopropyl silane or aminodextran in two M2 macrophage models (murine primary IL-4-activated bone marrow-derived macrophages and human M2-like differentiated THP-1 cells). All SPIONs were internalised and no cell toxicity was observed. SPION treatment produced reactive oxygen species and activated the extracellular signal-regulated kinase and AKT pathways. After 24-h SPION incubation, M2 macrophages switched their iron metabolism towards an iron-replete state. SPION treatment in both M2 macrophage models altered their M2 activation profiles, promoted IL-10 production, and stimulated protease-dependent invasion. These results highlight the need to evaluate the interactions between SPIONs and cells to take full advantage of the intrinsic properties of these nanoparticles in biological systems. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxide nanoparticles (SPIONs) have been used as an MRI contrast agent in many experimental studies. The authors here investigated the effects of these nanoparticles on M2 macrophages after cellular uptake. The findings of cell activation further enhanced our current knowledge on the interaction of SPIONS with macrophages.

Variety and Harvesting Season Effects on Antioxidant Activity and Vitamins Content of Citrus sinensis Macfad.

Five sweet orange (Citrus sinensis Osbeck) varieties cultivated in Huelva (Spain) and picked at two seasons during two consecutive years, were characterized for their antioxidant activity (free radicals scavenging activity, reducing power and lipid peroxidation inhibition) and vitamin content (vitamin E and vitamin C). The effects induced by sweet orange variety and stage of maturity were comprehensively compared by applying 2-way ANOVA and linear discriminant analysis. The results indicated higher differences in antioxidant activity and vitamin contents in response to the effect of the harvesting season, when compared to the effect of sweet orange variety. Nevertheless, the results observed in 2012 showed less marked differences among the assayed sweet orange varieties. Either way, it might be concluded that oranges sampled in January show the highest antioxidant activity and vitamin contents. Furthermore, concerning the properties evaluated in this work, all sweet orange varieties represent good alternatives, except for Rhode Summer, which would not be the preferable choice as a target to enhance sweet orange overall characteristics.

Effects of salinity and nitrogen supply on the quality and health-related compounds of strawberry fruits (Fragaria × ananassa cv. Primoris).

BACKGROUND: Different nitrogen inputs and/or development under adverse water conditions (water stress/low quality and/or high salinity/electrical conductivity), such as those prevailing in Almeria (Mediterranean coast, south-east Spain), may affect overall fruit and vegetable quality. This study evaluated the influence of salinity and nitrogen reduction in hydroponic nutrient solution on strawberry fruit quality and nutritional compounds (Fragaria × ananassa Duch., cv. Primoris). RESULTS: Strawberries obtained under salinity treatments recorded the highest values for soluble solids content (SSC; all samplings); fruit taste was thus enhanced. Additionally, salinity improved fruit nutritional value, with higher contents of antioxidants compounds (first sampling). During first and second samplings, strawberries grown under N reduction and non-saline conditions showed higher values for firmness compared to fruits developed under other treatments. Regarding health-related compounds, few differences were found except for total polyphenols concentration and antioxidant activity for the first sampling, where strawberries grown under saline treatments obtained the highest values for both parameters. CONCLUSION: The use of low-quality waters, such as those found in Almeria (salinity, N9S and N5S) and low nitrogen inputs (N5, avoid environmental impact) for strawberry cultivation does not exert a negative impact on overall quality. Positive differences could be found in SSC, firmness and health-related compounds when compared against the control treatment (N9).

Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes.

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4+ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4+ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.

Comprehensive immune profiling reveals that Orbivirus infection activates immune checkpoints during acute T cell immunosuppression.

Bluetongue virus (BTV) is an arbovirus transmitted by the bite of infected Culicoides midges that affects domestic and wild ruminants producing great economic losses. The infection induces an IFN response, followed by an adaptive immune response that is essential in disease clearance. BTV can nonetheless impair IFN and humoral responses. The main goal of this study was to gain a more detailed understanding of BTV pathogenesis and its effects on immune cell populations. To this end, we combined flow cytometry and transcriptomic analyses of several immune cells at different times post-infection (pi). Four sheep were infected with BTV serotype 8 and blood samples collected at days 0, 3, 7 and 15pi to perform transcriptomic analysis of B-cell marker+, CD4+, CD8+, and CD14+ sorted peripheral mononuclear cells. The maximum number of differentially expressed genes occurred at day 7pi, which coincided with the peak of infection. KEGG pathway enrichment analysis indicated that genes belonging to virus sensing and immune response initiation pathways were enriched at day 3 and 7 pi in all 4 cell population analyzed. Transcriptomic analysis also showed that at day 7pi T cell exhaustion pathway was enriched in CD4+ cells, while CD8+ cells downregulated immune response initiation pathways. T cell functional studies demonstrated that BTV produced an acute inhibition of CD4+ and CD8+ T cell activation at the peak of replication. This coincided with PD-L1 upregulation on the surface of CD4+ and CD8+ T cells as well as monocytes. Taken together, these data indicate that BTV could exploit the PD1/PD-L1 immune checkpoint to impair T cell responses. These findings identify several mechanisms in the interaction between host and BTV, which could help develop better tools to combat the disease.

Effects of Different Heating Treatments on the Antioxidant Activity and Phenolic Compounds of Ecuadorian Red Dacca Banana.

The banana is a tropical fruit characterized by its composition of healthy and nutritional compounds. This fruit is part of traditional Ecuadorian gastronomy, being consumed in a wide variety of ways. In this context, unripe Red Dacca banana samples and those submitted to different traditional Ecuadorian heating treatments (boiling, roasting, and baking) were evaluated to profile their phenolic content by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) and the antioxidant activity by ORAC, ABTS, and DPPH assays. A total of sixty-eight phenolic compounds were identified or tentatively identified in raw banana and treated samples, highlighting the content in flavonoids (flavan-3-ols with 88.33% and flavonols with 3.24%) followed by the hydroxybenzoic acid family (5.44%) in raw banana samples. The total phenolic compound content significantly decreased for all the elaborations evaluated, specifically from 442.12 mg/100 g DW in fresh bananas to 338.60 mg/100 g DW in boiled (23.41%), 243.63 mg/100 g DW in roasted (44.90%), and 109.85 mg/100 g DW in baked samples (75.15%). Flavan-3-ols and flavonols were the phenolic groups most affected by the heating treatments, while flavanones and hydroxybenzoic acids showed higher stability against the heating treatments, especially the boiled and roasted samples. In general, the decrease in phenolic compounds corresponded with a decline in antioxidant activity, evaluated by different methods, especially in baked samples. The results obtained from PCA studies confirmed that the impact of heating on the composition of some phenolic compounds was different depending on the technique used. In general, the heating processes applied to the banana samples induced phytochemical modifications. Even so, they remain an important source of bioactive compounds for consumers.

Adenoviral delivery of soluble ovine OX40L or CD70 costimulatory molecules improves adaptive immune responses to a model antigen in sheep.

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

Non-replicative antibiotic resistance-free DNA vaccine encoding S and N proteins induces full protection in mice against SARS-CoV-2.

SARS-CoV-2 vaccines currently in use have contributed to controlling the COVID-19 pandemic. Notwithstanding, the high mutation rate, fundamentally in the spike glycoprotein (S), is causing the emergence of new variants. Solely utilizing this antigen is a drawback that may reduce the efficacy of these vaccines. Herein we present a DNA vaccine candidate that contains the genes encoding the S and the nucleocapsid (N) proteins implemented into the non-replicative mammalian expression plasmid vector, pPAL. This plasmid lacks antibiotic resistance genes and contains an alternative selectable marker for production. The S gene sequence was modified to avoid furin cleavage (Sfs). Potent humoral and cellular immune responses were observed in C57BL/6J mice vaccinated with pPAL-Sfs + pPAL-N following a prime/boost regimen by the intramuscular route applying in vivo electroporation. The immunogen fully protected K18-hACE2 mice against a lethal dose (105 PFU) of SARS-CoV-2. Viral replication was completely controlled in the lungs, brain, and heart of vaccinated mice. Therefore, pPAL-Sfs + pPAL-N is a promising DNA vaccine candidate for protection from COVID-19.

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.

An experimental test of the role of environmental temperature variability on ectotherm molecular, physiological and life-history traits: implications for global warming.

Global climate change is one of the greatest threats to biodiversity; one of the most important effects is the increase in the mean earth surface temperature. However, another but poorly studied main characteristic of global change appears to be an increase in temperature variability. Most of the current analyses of global change have focused on mean values, paying less attention to the role of the fluctuations of environmental variables. We experimentally tested the effects of environmental temperature variability on characteristics associated to the fitness (body mass balance, growth rate, and survival), metabolic rate (VCO(2)) and molecular traits (heat shock protein expression, Hsp70), in an ectotherm, the terrestrial woodlouse Porcellio laevis. Our general hypotheses are that higher values of thermal amplitude may directly affect life-history traits, increasing metabolic cost and stress responses. At first, results supported our hypotheses showing a diversity of responses among characters to the experimental thermal treatments. We emphasize that knowledge about the cellular and physiological mechanisms by which animals cope with environmental changes is essential to understand the impact of mean climatic change and variability. Also, we consider that the studies that only incorporate only mean temperatures to predict the life-history, ecological and evolutionary impact of global temperature changes present important problems to predict the diversity of responses of the organism. This is because the analysis ignores the complexity and details of the molecular and physiological processes by which animals cope with environmental variability, as well as the life-history and demographic consequences of such variability.

A New Look at Vaccine Strategies Against PPRV Focused on Adenoviral Candidates.

Peste des petits ruminants virus (PPRV) is a virus that mainly infects goats and sheep causing significant economic loss in Africa and Asia, but also posing a serious threat to Europe, as recent outbreaks in Georgia (2016) and Bulgaria (2018) have been reported. In order to carry out the eradication of PPRV, an objective set for 2030 by the Office International des Epizooties (OIE) and the Food and Agriculture Organization of the United Nations (FAO), close collaboration between governments, pharmaceutical companies, farmers and researchers, among others, is needed. Today, more than ever, as seen in the response to the SARS-CoV2 pandemic that we are currently experiencing, these goals are feasible. We summarize in this review the current vaccination approaches against PPRV in the field, discussing their advantages and shortfalls, as well as the development and generation of new vaccination strategies, focusing on the potential use of adenovirus as vaccine platform against PPRV and more broadly against other ruminant pathogens.

Vaccination as a Strategy to Prevent Bluetongue Virus Vertical Transmission.

Bluetongue virus (BTV) produces an economically important disease in ruminants of compulsory notification to the OIE. BTV is typically transmitted by the bite of Culicoides spp., however, some BTV strains can be transmitted vertically, and this is associated with fetus malformations and abortions. The viral factors associated with the virus potency to cross the placental barrier are not well defined. The potency of vertical transmission is retained and sometimes even increased in live attenuated BTV vaccine strains. Because BTV possesses a segmented genome, the possibility of reassortment of vaccination strains with wild-type virus could even favor the transmission of this phenotype. In the present review, we will describe the non-vector-based BTV infection routes and discuss the experimental vaccination strategies that offer advantages over this drawback of some live attenuated BTV vaccines.

The Interplay between Bluetongue Virus Infections and Adaptive Immunity.

Viral infections have long provided a platform to understand the workings of immunity. For instance, great strides towards defining basic immunology concepts, such as MHC restriction of antigen presentation or T-cell memory development and maintenance, have been achieved thanks to the study of lymphocytic choriomeningitis virus (LCMV) infections. These studies have also shaped our understanding of antiviral immunity, and in particular T-cell responses. In the present review, we discuss how bluetongue virus (BTV), an economically important arbovirus from the Reoviridae family that affects ruminants, affects adaptive immunity in the natural hosts. During the initial stages of infection, BTV triggers leucopenia in the hosts. The host then mounts an adaptive immune response that controls the disease. In this work, we discuss how BTV triggers CD8+ T-cell expansion and neutralizing antibody responses, yet in some individuals viremia remains detectable after these adaptive immune mechanisms are active. We present some unpublished data showing that BTV infection also affects other T cell populations such as CD4+ T-cells or γδ T-cells, as well as B-cell numbers in the periphery. This review also discusses how BTV evades these adaptive immune mechanisms so that it can be transmitted back to the arthropod host. Understanding the interaction of BTV with immunity could ultimately define the correlates of protection with immune mechanisms that would improve our knowledge of ruminant immunology.