Invertebrate Models in Innate Immunity and Tissue Remodeling Research

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Bio-plastic recognition by mussels hemocytes

The growing use of bio-polymers derivatives poses an increasingly pressing problem regarding their environmental sustainability. In particular, it should be still ascertained the claimed absence of direct and indirect influence on ecosystems and the health of living organisms, including humans. Our goal was about assessing the potential effects of poly-lactates and polyhydroxyalkanoates, the most widely used bio polymers classes with promising different applications for replacing conventional plastics on natural aquatic environments. We chose M. galloprovincialis as sentinel species since their extensive filter-feeding activity. When it is exposed to microparticles can bioaccumulate them in soft tissues and organs. In the immunobiological investigation, to highlight if bio-polymers can influence the marine ecosystems, in vitro exposure assays on bivalve mussel have been carried out, and their impacts have been explored, by evaluating the cellular response of hemocytes referred to their phagocytic and/or encapsulation activity. Preliminary evidences have shown that bioplastic particles behave in a very similar way to fossil plastic triggering the immuno-system and activating the elimination of non-self particles via cellular response. As future perspectives, although it is widely recognized that in vitro testing is an effective method for defining the effects of emerging pollutants, the in vitro test will be further deepened with in vivo experiments

The multiple potentialities of anthozoans: analyses and comparisons between animal models

Anthozoans are the richest class of species of the phylum Cnidaria. They are a candidate group for studying the evolution of mutualisms and immunity and despite their morphological simplicity exhibit a repertoire of immunological components with large genomes and gene families similar to those of the Bilateria. Like other invertebrates, anthozoans immunity is based on self/non-self recognition mechanisms and allorecognition responses, therefore, maintaining their integrity and responding actively to selection pressures. Highlight and investigate the link between innate immunity, homeostasis maintenance, inflammation, tissue remodelling and regeneration in Anthozoa could be useful to elucidate the adaptive capability features to different stress factors. We have carried out studies demonstrating that all these processes are highly conserved among the anthozoans species. We have compared the inflammatory responses and the morpho-functional aspects related to regeneration in different species of Mediterranean anthozoans using histological, cellular and molecular technical approaches on organisms, maintained in aquaria under environmental and pathogenic stressful conditions. This approach appears to be a useful tool from baseline studies in immunology and anthozoans result valid models able to respond to environmental stress conditions. Important results have been obtained with potential biotechnological transferability in pharmacology

Actinins as novel broad-spectrum AMP isolated from the tentacle of Anthozoan Actinia equina (Linnaeus, 1758)

Capturing activities and defense mechanisms of Cnidarian are strongly associated with toxins and peptide with antimicrobial properties. AMP are an important component of many organisms’ innate immune system with a good inhibitory or killing effect against invaders pathogens. We investigated the AMP activity of acid extracts obtained from tentacle and body of Actinia equina (Cnidaria, Anthozoa) against Gram positive (Micrococcus lysodeikticus) and Gram negative (Escherichia coli, Vibrio alginolyticus) bacteria. The peptide fractions showed interesting minimum inhibitory concentrations (MIC) values (concentrations up to 0.125 μg/ml) against tested pathogens. Tentacle acid extracts exhibiting a good antimicrobial activity, were further investigated, characterized and the peptides purified by reverse phase chromatography on solid phase Sep-Pak C8 column followed by several HPLC runs on C18 column. A broad-spectrum antibacterial peptides activity was detected in 40 % acetonitrile fractions. The Peptide 6.2 has a molecular weight of 2612.91 Da and is composed of 27 amino acids (Actinin A); while peptide 7.3 has a molecular weight of 4323.07 Da and is composed of 35 amino acids (Actinin B). The two peptides were completely sequenced and their aa sequence revealed similarity with the already described AMPs identified in amphibians and fish, with anti-Gram+ & Gram-, antifungal, candidacidal, anti- Methicillin-resistant Staphylococcus aureus (MRSA) activity Actinins A and B were chemically synthesized and tested in vitro against the above-mentioned bacterial pathogens. The analysis identified the peptide Actinin B which showed an interesting antibacterial and can be considered good candidates for new therapeutic applications

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Evolution, adaptation and immune functions of fish F-type lectins. The novelty of FBL from Trematomus bernacchii (Boulenger, 1902)

Lectins are a protein family, present in almost all living organisms and involved in different biological pathways, such as immune responses. The Fucose Binding Lectin (FBL), constitute the latest lectin family identified and characterized in fishes. The FBL family is constituted by a large number of proteins exhibiting multiples of the F-type motif, either tandemly arrayed or in mosaic combinations with other domains. In an early step a FBL has been isolated and characterized from serum of the Antarctic fish Trematomus bernacchii by affinity chromatography on fucose-agarose column. A clear Bacterial agglutinating activity (BA) towards different bacteria strains (Escherichia coli, Kokuria rhizophyla and Bacillus subtilis) and Hemagglutinating activity (HA) toward rabbit erythrocytes was induced from the serum as well from the purified protein and thus confirm its involvement in host pathogen interactions. In SDS-PAGE analysis, the FBL exhibited an apparent molecular weight of 30 kDa. This data is confirmed from the sequence of the F lectin recognised on the T. bernacchii transcriptome. The sequence shows a similar and coherent structure with a supposed Mw 32.16 kDa and an isoelectropoint of 5.21. Furthermore, sequencing the N-terminus confirmed the identity of the sequence runned on SDS PAGE and blotted on PVDF membrane. The HA activity was analyzed at different temperatures and it was maintained also at the physiological living low temperatures of this fish habitat (close to 0 °C). Therefore, in order to identify trends linked to cold adaptation in Antarctic fish, we present our hypothesis on the conformational change determined by the aminoacidic substitutions respect the others fish fucolectins living in warmer water on the light of the general phylogenetic scenario including the new preliminary data on sharks FB

Inflammation events occurring upon bacterial infection in Mytilus galloprovincialis

Bivalves, and in particular the Mediterranean Mytilus galloprovincialis are important sources of food in several countries in the world. Because of that, mussels farming has a strong economic impact. Due to their status as sessile and filter-feeding animals, bivalves accumulate in their tissues environmental pollutants and a larger amount of microorganisms and between these, a multitude of infective bacteria for higher vertebrates and humans, such as Vibrio species. Several immunological responses of M. galloprovincialis were investigated and described after Vibrio infection both, in vitro and in vivo conditions, such as hemocytes count and different cellular subpopulations. Particularly, intracellular signaling pathways are activated to trigger the synthesis of antimicrobial effectors Here, were investigated the modulation of immunological cellular markers of the Mediterranean bivalve M. galloprovincialis in response to in vivo exposure with Vibrio splendidus. The activation of inflammatory cascade was examined through immunolabeling with antibodies involved in the pathway: Toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MYD88), Allograft inflammatory factor-1 (AIF1) and ribonucleases RNASET2 (T2 family), that trigger the recruitment and activation of macrophages in vertebrates. Results confirmed the activation of TRL4 during bacterial infection and MYD88 adapter suggesting a role in recognition and intracellular signaling. Moreover, Gram-negative bacteria determine the recruitment by the ribonuclease RNASET2 of haemocytes and a huge migration of AIF-1+ cells. This approach is suitable to understand the molecular defense mechanisms in invertebrates during the exposure to possible pathogens, also in order to develop new technics and tools to evaluate mussel immunity response used in aquaculture to prevent mass mortality of these mollusks, economic loss and potential risks for consumers of seafood

METHYLMERCURY EFFECTS ON Mytilus galloprovincialis HAEMOCYTES ACTIVITY

Bivalves, filter-feeding organisms, due to their wide distri-bution, are used by many countries in biomonitoring, particu-larly in the assessment of xenobiotics in the marine ecosystem.These sentinel organisms are able to accumulate trace metalsand other substances in their tissues. Haemocytes are effectorsof cellular immunity in bivalves and are capable of respond-ing to stressors through activities such as phagocytosis or cyto-toxicity. In order to identify cellular markers to investigate pol-lution, the effect of different concentrations of organic mercury(CH3HgCl) on the morphology and responses of Mytilus gal-loprovincialishaemocytes was studied in this work. Sublethalconcentrations of methylmercury, as evidenced by the Trypanblue exclusion test, were used to investigate its effect on mor-phology, the efficiency of phagocytosis towards yeast cells,the maintenance of the lysosomal membrane and the ability torelease cytotoxic molecules. Alterations in haemocyte viabili-ty, morphological changes and alterations in the cytoskeletonwere observed. The spreading ability, a cell morphometricparameter, was also used as an additional method. Exposureto CH3HgCl influenced the percentage and index of phagocy-tosis. Finally, cytoskeletal and morphological modificationslead to a reduction in the ability to adhere to the substrate andincorporate the target. The cytotoxic activity of M. gallo-provincialis haemocytes towards erythrocytes and the activityrevealed from lysis plaque assay has not been modified byadequate concentrations of methylmercury in the medium. Inaddition, membrane permeability could be affected bymethylmercury due to the reduced retention capacity of neu-tral red by the cells. This evidence confirms that theMediterranean mussel M. galloprovincialisis a suitable modelorganism in the study of the state of health of the marine envi-ronment and in particular for investigation of pollution causedby xenobiotics