Cell mediated immune response of the Mediterranean sea urchin Paracentrotus lividus after PAMPs stimulation

9 páginas, 5 figuras, 1 tablaThe Mediterranean sea urchin (Paracentrotus lividus) is of great ecological and economic importance for the European aquaculture. Yet, most of the studies regarding echinoderm's immunological defense mechanisms reported so far have used the sea urchin Strongylocentrotus purpuratus as a model, and information on the immunological defense mechanisms of Paracentrotus lividus and other sea urchins, is scarce. To remedy this gap in information, in this study, flow cytometry was used to evaluate several cellular immune mechanisms, such as phagocytosis, cell cooperation, and ROS production in P. lividus coelomocytes after PAMP stimulation. Two cell populations were described. Of the two, the amoeboid-phagocytes were responsible for the phagocytosis and ROS production. Cooperation between amoeboid-phagocytes and non-adherent cells resulted in an increased phagocytic response. Stimulation with several PAMPs modified the phagocytic activity and the production of ROS. The premise that the coelomocytes were activated by the bacterial components was confirmed by the expression levels of two cell mediated immune genes: LPS-Induced TNF-alpha Factor (LITAF) and macrophage migration inhibitory factor (MIF). These results have helped us understand the cellular immune mechanisms in P. lividus and their modulation after PAMP stimulationThis work has been funded by the National Project A/026000/09AECID, 2010–2012 “Respuesta inmune de invertebrados marinos”Peer reviewe

Cell mediated immune response of the Mediterranean urchin Paracentrotus lividus to PAMPs stimulation

Póster presentado en el 13th International Society of Developmental and Comparative Immunology Congress, Murcia, 28 junio - 3 julio 2015The Mediterranean sea urchin (Paracentrotus lividus) is of great ecological and economic importance for the European aquaculture. Few studies explain how this animal interacts with pathogens and which immune mechanisms are induced to overcome the diseases. The immune system involves humoral and cellular components. The immune cells are coelomocytes and move in the coelomic spaces. There is not a single standard classification of coelomocytes for all echinoderms since they are heterogeneous in morphology and size. The immune functions of each type of coelomocyte are still not totally understood, but it is postulated that amoeboid-phagocytic cells and spherule cells are the only cellular components of the immune systemPeer reviewe

Establishment of Infection Models in Zebrafish Larvae (Danio rerio) to Study the Pathogenesis of Aeromonas hydrophila

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CCBY)Aeromonas hydrophila is a Gram-negative opportunistic pathogen of fish and terrestrial animals. In humans, A. hydrophila mainly causes gastroenteritis, septicaemia, and tissue infections. The mechanisms of infection, the main virulence factors and the host immune response triggered by A. hydrophila have been studied in detail using murine models and adult fish. However, the great limitation of studying adult animals is that the animal must be sacrificed and its tissues/organs extracted, which prevents the study of the infectious processes in the whole living animal. Zebrafish larvae are being used for the analysis of several infectious diseases, but their use for studying the pathogenesis of A. hydrophila has never been explored. The great advantage of zebrafish larvae is their transparency during the first week after fertilization, which allows detailed descriptions of the infectious processes using in vivo imaging techniques such as differential interferential contrast (DIC) and fluorescence microscopy. Moreover, the availability of fluorescent pathogens and transgenic reporter zebrafish lines expressing fluorescent immune cells, immune marker genes or cytokines/chemokines allows the host–pathogen interactions to be characterized. The present study explores the suitability of zebrafish larvae to study the pathogenesis of A. hydrophila and the interaction mechanisms between the bacterium and the innate immune responses through an infection model using different routes for infection. We used an early-embryo infection model at 3 days post-fertilization (dpf) through the microinjection of A. hydrophila into the duct of Cuvier, caudal vein, notochord, or muscle and two bath infection models using 4 dpf healthy and injured larvae. The latter resembled the natural conditions under which A. hydrophila produces infectious diseases in animals. We compared the cellular processes after infection in each anatomical site by confocal fluorescence imaging and determined the implication of inflammatory immune genes by measuring gene expression by qPCR14 páginas, 5 figuras.-- This work was supported by the European Union Seventh Framework Programme (FP7PEOPLE-2011-ITN) under the Marie-Curie Initial Training Network FishForPharma (PITN-GA-2011-289209) and by Project AGL2014-51773-C3 and Project 201230E057 (CSIC) from the Spanish Ministerio de Economía y Competitividad.Peer reviewe

Determination of Exosome Mitochondrial DNA as a Biomarker of Renal Cancer Aggressiveness

This study is partially supported by ID#53169721 project funded by Pfizer and the exosomes characterization was financed thanks to the grant PY20_00241 (FEDER/Junta de Andalucia-Regional Ministry of Economic Transformation, Industry, Knowledge and Universities).Here, the role of non-invasive biomarkers in liquid biopsy was evaluated, mainly in exosomes and mitochondrial DNA (mtDNA) as promising, novel, and stable biomarkers for renal cell carcinoma (RCC). A total of 140 fractions (named from B to F) obtained by ultracentrifugations of whole blood samples from 28 individuals (13 patients and 15 controls) were included. Nanoparticle Tracking Analysis (NTA) was conducted to characterized exosomal fraction. Subsequently, an analysis of digital PCR (dPCR) using the QuantStudio (TM) 3D Digital PCR platform was performed and the quantification of mtDNA copy number by QuantStudioTM 12K Flex Real-Time PCR System (qPCR) was developed. Moreover, Next Generation Sequencing (NGS) analyses were included using MiSeq system (Illumina, San Diego, CA, USA). An F fraction, which contains all exosome data and all mitochondrial markers, was identified in dPCR and qPCR with statistically significant power (adjusted p values <= 0.03) when comparing cases and controls. Moreover, present analysis in mtDNA showed a relevant significance in RCC aggressiveness. To sum up, this is the first time a relation between exosomal mtDNA markers and clinical management of RCC is analyzed. We suggest a promising strategy for future liquid biopsy RCC analysis, although more analysis should be performed prior to application in routine clinical practice.Pfizer 53169721FEDER/Junta de Andalucia-Regional Ministry of Economic Transformation, Industry, Knowledge and Universities PY20_0024

Rag1 immunodeficiency‐induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress [Poster]

12th European Zebrafish Meeting, Krakow, 9-13 July 2023In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1−/− zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity- and apoptosis-related genes was increased in the rag1−/− fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1−/− zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1−/− zebrafish compared to their wild-type (wt) siblings, rag1−/− showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence-related genes and senescence-associated β-galactosidase, respectively, diminished telomere length, and abnormal self-renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1−/− fish, as was the deficiency of the antioxidant metabolite L-acetylcarnitine (ALCAR) in rag1−/− fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT-263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature agingN

Is the invasive X. securis a risk for the production of M. galloprovincialis?

Poster.-- Encontros IIM Primeira fase, 12 de decembro de 2019Peer reviewe

Genes of the mitochondrial apoptotic pathway in Mytilus galloprovincialis

13 páginas, 6 figuras, 2 tablasBivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress.This work was partially funded through the EU Projects BIVALIFE (266157) and REPROSEED (245119).Peer reviewe

Interaction of the attenuated recombinant rIHNV-Gvhsv GFP virus with macrophages from rainbow trout (Oncorhynchus mykiss)

7 figurasOne of the most important threats to the salmonid aquaculture industry is infection caused by novirhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV). Using reverse genetics, an avirulent recombinant rIHNV-Gvhsv GFP strain was generated, which was able to replicate as effectively as wild type IHNV in a fish cell line and in macrophages. Although this recombinant virus induced protective responses against IHNV and VHSV, the response did not involve the production of antibodies or modulate the expression of some antiviral genes. To determine the immune mechanisms underlying the protection conferred by the rIHNV-Gvhsv GFP virus, different immune parameters (NO production, respiratory burst activity and the induction of apoptosis) were assessed in the macrophage population. The results obtained in the present work may indicate that the Nv protein could be important in the modulation of NO and ROS production. rIHNV-Gvhsv GFP did not appear to have a clear effect on nitric oxide production or apoptosis. However, an increased respiratory burst activity (with levels induced by the recombinant virus significantly higher than the levels induced by the wild type virus), suggests a stimulation of the macrophage population, which could be related to the protection against virulent viruses.This work was supported by the projects FAIRCT 98-4398 from the European Union and BIO 2000-0906 from the Spanish Ministerio de Ciencia y Tecnología. European structural funds were used for confocal microscopy. Fondo Europeo de Desarrollo Regional (FEDER) y Ministerio de Ciencia e Innovación. A. Romero acknowledges the CSIC for the I3P fellowship.Peer reviewe

Effect of the temperature during antiviral immune response ontogeny in teleosts

1 tabla, 6 figurasZebrafish were used to investigate the expression levels of several antiviral and inflammatory genes (IL-1β, iNOS, TNF-α, TLR3, IFN-I, IFNγ, IRF3, MDA-5, Mx) constitutively and after viral stimulation during early development. We also determined how their expression was affected by changes in the temperature. The antiviral genes were almost completely inhibited at 15 °C with the exception of TLR3. In contrast, IL-1β, iNOS and TNF-α expression was not obviously different between the two temperatures. At 15 °C, most of the genes examined did not differ following stimulation with poly I:C or viral hemorrhagic septicemia virus (VHSV). However, at 28 °C, all of the genes showed significant differences in at least some of the sampling points after poly I:C treatment with the largest differences observed for Mx. Mx expression in adult zebrafish was not significantly altered by temperature and poly I:C treatment led to a smaller increase in gene expression when compared to larval Mx levels. Thus, Mx seems to play an important role in viral immunity in larvae, when the adaptive immune response is not fully functionalThis research was supported by the project CSD2007-00002 Aquagenomics funded by the program Consolider-Ingenio 2010 from the Spanish Ministerio de Educación y Ciencia (MEC).Peer reviewe

Genomic and transcriptomic identification of the cathepsin superfamily in the Mediterranean mussel Mytilus galloprovincialis

Poster.-- 4th Congress of the International Society of Fish & Shellfish Immunology, December 12-15, 2022, Bode, NorwayCathepsins are lysosomal enzymes that participate in important physiological processes, such as development, tissue remodelling, senescence and innate and adaptive immunity. The description of these proteins in molluscs is fragmented and incomplete. In the present work, we identified most of the cathepsin family members in the bivalve Mytilus galloprovincialis by screening published genomic and transcriptomic information. In this specie, the cathepsin family is composed of 41 proteins showing a high diversification of cathepsins D, L and F, not previously observed in other taxonomic groups. Specific set of cathepsins are constitutively expressed in the different mussel tissues. Transcriptomic analyses suggested coordinated activity of the different cathepsins and their sequential activation during larval development. Cathepsins also play an important role in the immune response of bivalves, and different immune pathways seem to be activated in response to Vibrio splendidus infectionN