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Inflammation is a complex reaction of host defence mechanisms aiming at neutralization of an insult and restoring normal tissue structure and function. In human IL-17 is T-cell derived cytokine plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. Here, we report on three Ciona intestinalis IL-17 homologues (CiIL17-1, CiIL17-2, CiIL17-3). The gene organization, phylogenetic tree and modeling supported the close relationship with the mammalian IL-17A and IL-17F suggesting that the C. intestinalis IL-17 genes share a common ancestor in the chordate lineages. Real time PCR analysis showed a prompt expression induced by LPS inoculation showing that they are involved in the first steps of inflammatory response. In situ hybridization assays disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels. As in human, we can assume that CiIL-17-like stimulates the release of CiTNFalpha, which synergizes with CiIL-17 in its effects on cells and molecules of Ciona intestinalis immunity system. In addition, a comparative evaluation with others molecules upregulated by LPS challenge as CiTNF-alpha, phenoloxisases, peroxinectin, galectins and mannan binding lectin, have been evaluated in terms of temporal and quantitative gene expression.

The MD2 (Myeloid Differentiation factor-2) protein belongs to the ML superfamily. This group of proteins contain a specific lipid binding domain (ML domain) that plays an important role in lipid recognition and metabolism. In vertebrates, MD-2 is involved in innate immune response as co-receptor in the LPS/TLR4 signaling pathway; MD2 recognizes and binds the bacterial lipid A and drives the TLR4 activation. Two TLR isoforms, CiTLR-1 and CiTLR-2, were identified in Ciona intestinalis with a TIR domain most similar to human TLR4 and TLR 6 respectively. Using a PCR-based subtractive hybridization strategy for isolation of differentially expressed genes between LPS-challenged and naïve C. intestinalis, we identified a full-length cDNA (855 bp) encoding for a 150 a.a. protein (CiMD-2-like). In silico analysis showed that the deduced protein contains a signal peptide (1-19 a.a.) and an E1/Der p2/Der f2/ML domain-MD2 related lipid recognition domain (21-148 a.a) with similarities to ML(MD-2 related Lipid-recognition) domain identified in MD-2 and NPC2 (Niemann-Pick disease type C2). Phylogenetic and structural analysis supported the close relationship with MD-2 and NPC2 suggesting that CiMD-2-like originated from a common ancestor gene. Furthermore, gene expression studies by Real-time PCR demonstrated that this cDNA is up-regulated after LPS injection in the body wall. In situ hybridization performed in controls and LPS-induced animals has shown that this gene is expressed in granular amebocytes, large granules hemocytes and URG (univacuolar refractile granulocyte) in pharynx, the main organ of the ascidian immune system

Several diagnostic genetic markers were identified in Pomatoschistus marmoratus and P. tortonesei using polyacrylamide gel electrophoresis (PAGE) of allozymes. Twenty-one loci were resolved, including the electrophoretic pattern of muscle proteins. The MDH*, PGM1,2*, EST-1,2*, FUM* and PGI-2* loci exhibited different alleles which were fixed for the two species being analysed. Genetic distance, as calculated by Nei's index, showed a value of 0.413. Environmental hypersalinity, could have influenced the geographical distribution of P. tortonesei.

Phylogenetic analysis recognizes echinoderms as a key group of deuterostomes, therefore the species in this group are useful for the study of the evolution of innate immunity responses. In addition, this marine invertebrate lives in coastal and estuarine waters that are directly exposed to potentially pathogenic microorganisms and stressful anthropogenic factors. Coelomocyte populations seem to be essential to immune-defence functions such as phagocytosis, ROI production, cytotoxicity, synthesis and release of antimicrobial substances including lectin, cytokine, C3-like expression, prophenoloxidase activity and capsule formation. Holothurians’ coelomocyte populations contain several coelomocyte types, including phagocytes, and can form brown bodies in response to multicellular parasites. Holothuria tubulosa coelomic fluid contains three main coelomocyte categories: amoebocytes, spherulocytes and progenitor cells. The amoebocytes represent about 30 % of the total population, and in living cell preparations exhibit two distinct forms: petaloid and philopodial; spherule cells represent the numerically dominant cell type (about 60 %). Progenitor cells are present in a lesser amount (about 20 %); they are similar to lymphocytes and show a nucleus that is typically prominent with a thin rim of cytoplasm. In the present report we show that coelomocytes of Holothuria tubulosa are able to exert cytotoxic activity against different cellular targets such as rabbit or sheep erythrocytes and the human erythromyeloid leukaemia-derived cell line K562. Moreover, the unseparated coelomocyte supernatant lysate (CLS) exerts a lytic activity, even in the absence of calcium, against the same targets. Analysis of the coelomocyte lysate by overlay assays with sheep and rabbit erythrocytes on PAGE without SDS showed a protein pattern composed by two main hemolytic bands with different electrophoretic mobility. The one with low mobility (I) showed calcium independent haemolytic activity while high mobility band (II) showed calcium dependent activity. The two bands were eluted from the gel and analyzed by SDS-PAGE and stained with silver stain; band I could be separated in three components of different sizes (52, 42 and 41 kDa), and band II had a size of 43 kDa. Spherule cells seemed to be the effector cells, as shown by a plaque lysis assay. Further studies are in progress to identify the lytic proteins and the lytic mechanism at work on both a molecular and cellular level.

Antimicrobial peptides (AMPs) represent the most universal immune effectors. Molluscs constitute the second largest animal phylum, after Arthropods, in term of number of species. Only a negligible number has been investigated regarding AMPs. The choice of the species to be studied relied on their economical importance and availability. First studies on molluscan AMPs dated from 1996 and were based on biological activities of biochemical-purified fractions. Such approach released all the original structures we know, with biological activity sometimes different from one isoform to another. Then, molecular biology techniques were applied to molluscan AMPs starting in 1999. Complete screening of genome expression in various situations became available, as well as some exotic Molluscs, the ones collected from deep-sea hydrothermal vent, for instance. Full sequences of active peptides and precursors, and gene organizations were established. A breakthrough consisted in the discovery of numerous AMP variants, even within the same animal. In addition, computer homology revealed the existence of already known AMPs in new studied molluscan species.

Aims: Staphylococcal biofilm-associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system. Methods and Results: We tested the activity of the 5-kDa peptide fraction of the cytosol from coelomocytes (5-CC) against a group of Gram-positive, Gram-negative bacteria and fungi. We determined minimal inhibitory concentrations (MICs) ranging from 253.7 to 15.8 mg ml(-1). We observed an inhibitory activity and antibiofilm properties of 5-CC against staphylococcal biofilms of reference strains Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. The antimicrobial efficacy of 5-CC against the biofilms of clinical strain Staph. epidermidis 1457 was also tested using live/dead staining in combination with confocal laser scanning microscopy. At a sub-MIC concentration (31.7 mg ml(-1)) of 5-CC the formation of young (6-h old) and mature (24-h old) staphylococcal biofilms was inhibited. Conclusions: The biological activity of 5-CC could be attributed to three peptides belonging to the sequence segment 9-41 of a beta-thymosin of P. lividus. Significance and Impact of the Study: The effector cells of P. lividus represent an interesting source of marine invertebrates-derived antimicrobial agents in the development of new strategies to treat staphylococcal biofilms

Phenoloxidases/tyrosinases initiate melanin synthesis in almost all organisms, and are involved in different biological activities such as the colour change of human hair and the browning or blackening of fruit skin etc. In many invertebrates, defence reactions are linked to phenoloxidase activity and/or melanization. Contacts with foreign molecules are able to trigger the prophenoloxidase (proPO) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (PO). It is generally accepted that the proPO system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect proPO activation steps have been achieved. After cells were stimulated by components of pathogen associated molecular pattern (PAMP), proPO activation takes place via zimogenic serine proteinase in turn activated by PAMPs followed by cascade, spatial and temporal control. The proPO activating system plays a defensive role in arthropods, molluscs, annelids, ascidians and the cephalochordate Branchiostoma belcheri. In the present paper, we report on ascidian proPO system and related molecules, with particular focus on the biochemical, cellular and molecular aspects of the Ciona intestinalis, proPO system of circulating hemocytes from naïve ascidians as well as of body wall following LPS inflammatory challenge. Phenoloxidases/tyrosinases initiate melanin synthesis in almost all organisms, and are involved in different biological activities such as the colour change of human hair and the browning or blackening of fruit skin etc. In many invertebrates, defence reactions are linked to phenoloxidase activity and/or melanization. Contacts with foreign molecules are able to trigger the prophenoloxidase (proPO) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (PO). It is generally accepted that the proPO system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect proPO activation steps have been achieved. After cells were stimulated by components of pathogen associated molecular pattern (PAMP), proPO activation takes place via zimogenic serine proteinase in turn activated by PAMPs followed by cascade, spatial and temporal control.

Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish beta-1,3-glucan-binding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units. The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via ionic interaction with its C-terminal region. This interaction with a peroxidase seems to be a novel function for an SOD. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances.