Nicolò Parrinello

Confinement stress in sea bass (Dicentrarchus labrax) depresses peritoneal leukocyte cytotoxicity

Fish respond to stressful conditions via neuroendocrine responses (primary response) which result in increased levels of plasma cortisol which is considered immunosuppressive. Sea bass were confined at low (10 kg/m3) and high (60 kg/m3) density for 3–48 h. Plasma cortisol and glucose were evaluated and two principal cellular immune responses were assayed. A significant increase in plasma cortisol and glucose levels, as well as osmolarity, was found following stress. In addition, phagocytic activity, as shown by reactive oxygen species (ROS) production by challenged head kidney phagocytes and cytotoxic activity of eosinophilic granule cells from peritoneal cavity against K562 tumour cell lines appeared to be suppressed. Plasma levels of cortisol, glucose, osmolarity were correlated with cellular immunity, by the linear regression method. The suppression of cytotoxic activity was found to be significantly correlated with high plasma cortisol and glucose levels. These parameters could affect the eosinophilic granule cells of the peritoneal cavity.

Structural and functional diversity of the lectin repertoire in teleost fish: Relevance to innate and adaptive immunity

Protein-carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly diversified, and include not only representatives of the lectin families described in mammals, but also members of lectin families described for the first time in fish species; (b) the tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; (c) although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; (d) in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as complement activation and regulation of immune functions; (e) some lectins that recognize exogenous ligands mediate processes unrelated to immunity: they may act as anti-freeze proteins or prevent polyspermia during fertilization.

CiC3-1a-Mediated Chemotaxis in the Deuterostome Invertebrate Ciona intestinalis (Urochordata)

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a59–76) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a59–75) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is Gi protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3′-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.

INFLAMMATORY-LIKE REACTION IN THE TUNIC OF CIONA INTESTINALIS (TUNICATA). I. ENCAPSULATION AND TISSUE INJURY

Particulate (sheep erythrocytes, ascidian oocytes, stromata, colloidal carbon) or soluble agents (bovine serum albumin or hemoglobin, hemocyanin) were injected in varying doses into the tunic of Ciona intestinalis. This ascidian reacted by producing a capsule and/or tissue injury. Statistical analysis suggests that the two phenomena are independent, probably related to the nature and dose of the irritant.Light histological observations showed granulocyte degranulation in the damaged tissue, suggesting that an acute inflammatory-like process is involved in the tunic reaction.

Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria.

Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.

Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (~50%), granulocytes (~35%), prohemocytes (~8%), oenocytes (~4%) and spherulocytes (~3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (~12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system.

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzymatic assay following electrophoretic analysis on polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE indicated that hemocyte lysate presented orthodiphenoloxidase (catecholase) activity. The enzymes from the three species differed in molecular size, activating substances and trypsin sensitivity.

HEMAGGLUTININ AND HEMOLYSIN LEVELS IN THE COELOMIC FLUID FROM HOLOTHURIA POLII (ECHINODERMATA) FOLLOWING SHEEP ERYTHROCYTE INJECTION

After injection of formalinized sheep erythrocytes into the coelomic cavity of Holothuria polii the activity of the naturally occurring hemagglutinins remained constant, while the hemolysin level rose over an eight day period. The kinetics of the response were the same after a further injection, although the hemolytic titers reached higher levels over a longer period. Results obtained using rabbit erythrocytes indicate that this response can be considered a secondary one: higher titers were demonstrated over a 24 h period. Some properties of both naturally occurring and induced hemolysins are discussed.

Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus

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.

Naturally occurring hemolysins in the coelomic fluid of Holothuriapolii Delle Chiaie (echinodermata)

Abstract The coelomic fluid of Holothuria polii D.Ch contains hemolytic activity against erythrocytes of several vertebrate species. The hemolytic potency depends upon calcium ion concentration and varies according to erythrocyte source and cell number in the reaction mixture. Absorption experiments with formalinized rabbit erythrocytes suggest that hemolytic activity is not specific. Its heat lability, water insolubility at low pH values, and sensitivity to proteolytic enzymes suggest that hemolytic activity resides in protein molecules. The activity, maximal in alkaline media, appears to depend up time and temperature.