[Show abstract][Hide abstract] ABSTRACT: In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 12/2012; · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca²+-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1-B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by D-galactose, α-lactose, lactulose, LacNAc, thiodigalactoside (TDG), L-fucose, D-mannose, D-glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca²+-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.
Fish & Shellfish Immunology 02/2011; 30(4-5):1014-23. · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In invertebrates, cellular and humoral components are evolved to maintain their homeostais and integrity. Both these components respond to different non-self such as microorganisms, vertebrate erythrocytes and foreign proteins. The aim of this research was to study the possible modulating effects of Bacillus thuringiensis (Bt) in a commercial and vegetative form on the immune response of the echinoderm, sea cucumber Holothuria tubulosa. Bt is one of the most used biopesticides worldwide, covering the 90% of the organic market. Sicily is a region vocated to biological control and agricultural area are often closed to the coastal area. Investigate the effect of biopesticides on other non target organisms is important in a more exhaustive environmental risk assessment. Moreover correlative studies indicate that the immune surveillance is affected by both Bt forms in vivo challenge. In particularly we have evaluated the phagocytosis vs Saccharomyces cerevisiae, hemagglutination using rabbit erythrocytes and coelomocyte cytotoxic activity against rabbit erythrocytes and K562 tumor target cells. These results can contribute to improve the knowledge about the use of Bt like biological insecticidal on non target organisms.
[Show abstract][Hide abstract] ABSTRACT: The coelomic fluid from the echinoderm contains several coelomocyte types involved in immune defences. In this article we report a study of some activity involved in immune response of the sea urchin Paracentrotus lividus. Correlative studies indicate that in this species a cytotoxic and hemoagglutinating activity is present in a manner sex-dependent. Introduzione -Alcuni studi effettuati sul sistema immunitario dei vertebrati fanno emergere una relazione tra sesso ed immunità, in generale le femmine sembrano possedere una maggiore reattività immunitaria rispetto ai maschi (Stoehr e Kokko, 2006). Tra gli invertebrati la situazione è molto meno chiara. Nel presente lavoro abbiamo studiato l'immuno-reattività di esemplari di entrambi i sessi dell'echinoderma Paracentrotus lividus esaminando alcune reazioni immunitarie di tipo umorale e cellulare. In particolare nella specie in esame recentemente è stata mostrata un'attività citotossica calcio–dipendente in grado di lisare gli eritrociti di coniglio e le cellule tumorali della linea K562 in seguito al rilascio di sostanze litiche ad opera degli sferulociti non colorati (S-CLS) (Arizza et al., 2007). Materiali e metodi – I saggi biologici sono stati condotti analizzando in totale campioni preparati singolarmente da 100 individui maschili e 100 femminili. È stata analizzata l'attività citotossica ed emoagglutinante del fluido celomatico (CF) e del supernatante del lisato degli S-CLS. Saggio di citotossicità: 200 µl di campione sono stati diluiti serialmente e miscelati con 200 µl di sospensione di eritrociti di coniglio (8×10 6 cell) in ISO-Ca 2+ (20 mM Tris, 0.5 M NaCl, 10 mM CaCl 2 pH 7.5) e incubati per 1 ora a 37 °C. Il rilascio dell'emoglobina è stato determinato attraverso la lettura dell'assorbanza a 541 nm. Saggio di emoagglutinazione: 25 µl di campione sono stati diluiti serialmente in ISO-Ca 2+ e miscelati con 25 µl di sospensione eritrocitaria all'1% in ISO-Ca 2+ contenente gelatina allo 0,1% e incubati per 1 ora a 37 °C in piastre a 96 pozzetti con fondo a U (Microtiter-SIGMA).
[Show abstract][Hide abstract] ABSTRACT: Gli studi eff ettuati sul sistema immunitario dei vertebrati, fanno emergere una relazione tra sesso ed immunità. In generale, le femmine sembrano possedere una maggiore reattività immunitaria rispetto i maschi. Tra gli invertebrati, la situazione è molto meno chiara. Nel presente lavoro abbiamo esaminato l'immuno-reattività di esemplari di entrambi i sessi dell'echinoderma Paracentrotus lividus. Preliminarmente è stato determinato per gli individui di ciascun sesso, il numero totale dei celomociti. In seguito, sono state valutate alcune reazioni immunitarie di tipo umorale e cellulare. In particolare è stata valutata: l'attività emagglutinante del fl uido celomatico (CF) e del supernatante del lisato dei celomociti (CLS); l'attività citotossicità del CF, del CLS e dei celomociti; l'attività di formazione di placche di lisi e di fagocitosi. Dai dati fi nora ottenuti si è potuto apprezzare, per la prima volta, che gli individui di P. lividus hanno una diff erente capacità immunitaria dipendente dal sesso. In generale si è notato, per ciascun saggio sperimentale, un certo grado di variabilità individuale, ma in ogni caso si sono ottenuti valori migliori per gli individui di sesso femminile. In particolare il numero dei celomociti è stato mediamente maggiore nelle femmine (~ 2.0 × 10 8) rispetto ai maschi (~1.0 × 10 8). Anche l'attività citotossica del CF, CLS e degli emociti ha raggiunto valori signifi cativamente superiori negli individui femminili. Lo stesso risultato è stato trovato anche nei test di formazione di placche e di fagocitosi. I risultati suggeriscono che gli individui femminili del P. lividus sono più immunoreattivi dei maschi. Poiché i sistemi di difesa degli invertebrati si basano soprattutto sui meccanismi cellulo-mediati, il minor numero di celomociti trovati nei maschi, potrebbe spiegare questa minore capacità di difesa. Ulteriori studi sono necessari per meglio comprendere il signifi cato di questi diff erenti valori di attività immunitaria e la relazione con la riproduzione.
[Show abstract][Hide abstract] ABSTRACT: Negli invertebrati marini il sistema di difesa contro i microbi è basato su meccanismi immunitari di tipo cellulare e umorale. Tra questi ultimi sono compresi i peptide antimicrobici (AMP). Gli AMP sono di norma peptidi di piccole dimensioni con un numero di amminoacidi che va da 10 a 50, con carica positiva o anfi patica. Quelli che derivano da invertebrati marini, sono specifi ci contro le cellule procariotiche e possiedono un ampio spettro antimicrobico che comprende i patogeni umani patogeni umani. In questo lavoro, dimostriamo che la frazione peptidica con massa inferiore o uguale a 5kDa estratta dal supernatante del lisato dei celomociti del riccio di mare Paracentrotus lividus (5-CC) possiede un ampio spettro di attività antimicrobica contro tutti i ceppi batterici patogeni umani saggiati in vitro, come Staphylococcus. aureus ATCC 29213, ATCC 25923, ATCC 43866, Staphylococcus epidermidis DSM3269, 1457, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231 e Candida tropicalis ATCC 13813. La concentrazione minima inibente (MIC) del 5-CC varia da 253,7 a 15,8 mg ml-1. Inoltre, come dimostrato da osservazioni al microscopio confocale, la 5-CC è anche capace inibire la formazione dei biofi lm di stafi lococco. Per caratterizzare gli AMP presenti nella frazione peptidica, il 5-CC è stato analizzato con un RP-HPLC/nESI-MSMS. Le analisi hanno individuato tre principali peptidi (Parcentrina I, II e III) con una taglia molecolare rispettivamente di 1251.7, 2088.1, e 2292.2 daltons. L’analisi MSMS ha evidenziato che i pepditi comprendono rispettivamente le sequenze peptidiche 9-19, 12-31 e 24-41 della �- timosina di P. lividus (AN AJ439718). La �-timosina è uno dei peptidi antibatterici presenti nelle piastrine dei vertebrati. Ulteriori studi sono in corso per valutare, attraverso tecniche di biologia molecolare la sua localizzazione sia a livello tissutale, sia a livello cellulare e la modulazione della sua espressione in presenza di microrganismi. In conclusione, le paracentrine potrebbero essere candidate come agenti anti-biofilm di patogeni umani e/o come sostanze antifouling.
[Show abstract][Hide abstract] ABSTRACT: In the last few years, interest in the mutual
relationships between ascidians hemocytes and
products of innate immunity gene repertoire has led
to a more clear-cut knowledge of multifunctional role
of hemocytes in Ciona intestinalis immunity. This is
a suitable approach that may include morphofunctional
screening methods allowing us to
disclose differentiation of the hemocytes, their
activities in immune responses leading to a more
precise and reasonable classification taking in
account a multi-parameter approach. The genome
sequence provided new insight in studying the
innate immunity. Bioinformatic approach and
extensive in silico search have concerned
immunorelevant molecules, gene expression
patterns and some specific immune properties that
contribute to clarify morpho-functional aspects.
The involvement in immunity of hemoblasts,
lymphocyte-like cells, hyaline amebocytes, and
various types of granulocytes have been described.
[Show abstract][Hide abstract] ABSTRACT: A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFalpha-like cytokine was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFalpha epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNF alpha-like protein therefore appeared to be modulated by the LPS challenge.
Cell and Tissue Research 11/2008; 334(2):305-17. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tunicate immunocytes can be cytotoxic toward foreign cells, and cytolytic molecules (“lysins”) have been revealed in vitro by using erythrocyte targets. In Ciona intestinalis the hemocyte cytotoxic activity has been examined towards mammalian erythrocytes in a medium isosmotic to the hemolymph containing 10 mM
Ca2+ (TBS). Unilocular refractile hemocytes (URGs) release cytotoxic factors inhibited by sphingomyelin in a plaque-forming assay. To separate the lysin- releasing cells from the hemolymph and characterize lysins, a discontinuous Percoll gradient was performed and hemocyte populations were separated in 6 bands. URGs cytotoxic for RE were enriched (~40 %) in the band 5 (B5) and then lysed to obtain the supernatant. The B5-lysate
supernatant (B5-HLS) showed lytic activity (~84 %) specific for RE and K562 cells whereas a
lower level of cytotoxicity was found against sheep erythrocytes (SE). Such an activity was Ca2+- dependent and thermostable at 56 °C, B5-HLS also showed a Ca2+-independent hemagglutinating activity against trypsinized RE (HT 4.3) but not toward trypsinized SE. Inhibition experiments displayed that lysins and lectins could be inhibited by carbohydrates (galactose, thio-digalactoside, lactose, lactulose) whereas sphingomyelin (2.5 µg/ml) only inhibited the
lytic activity, suggesting that lectins may be involved in membrane sphingomielyn-lysin
interactions. To check
for the enzymatic nature of the lysins, phospholipase A2 inhibitors such as dibucain and quinacrine were assayed. The experiments showed that both the molecules were inhibitors of B5-HLS cytotoxic activity suggesting the involvement of a Ca-dependent phospholypase A2 activity. A cytotoxic mechanism based on phospholypase A2 activation due to lectin- sugar interactions is discussed as a model. Lysins against RE and K562 cells with the same properties including sugar and phospholypase A2 inhibition, can be promptly (within 3 h) released
in vitro by URGs in a culture medium suggesting that activated cells could participate in the defence response exerting a cytotoxic role.
II scientific meeting of the Italian Ascidiologists, Palermo; 06/2008
[Show abstract][Hide abstract] ABSTRACT: The immune-system of invertebrates recognizes and then reacts to foreign particles potentially pathogen by means of cytotoxicity, phagocytosis, encapsulation, and humoral effector
mechanisms (agglutination, lysis). Recent studies have shown the presence in invertebrates,
including tunicates, of hemocyte/coelomocyte cytolytic molecules (lysins) that can be released into
the hemolymph or coelomic fluid, and their lytic activity depends on their integration inside
target cell membranes. In some cases membrane lesions appear as circular pores as shown by electron
microscopy observations. There are few data on ascidian lysins and their lytic mechanisms. In Ciona
intestinalis a cytotoxic activity towards mammalian erythrocytes has been reported. The lytic
activity, examined using a Tris-buffered saline containing calcium ions and isosmotic to the
hemolymph, was inhibited by sphingomyelin (25 µg/ml). To identify the lysin-releasing cells,
hemolymph was separated through a discontinuous Percoll gradient. The hemocyte populations were
separated in 6 bands, each of them appeared to be enriched with a particular hemocyte
population. The hemocytes from band 5 (B5) mainly composed with unilocular refractile
granulocytes were responsible for the lysis of rabbit erythrocytes (RE). To characterize the
activity, the supernatant of the B5 hemocyte lysate supernatant (B5HLS) or B5 hemocyte culture
supernatant (B5HCS) was assayed with different mammal targets. Both B5HLS and B5HCS contain at
various extent lytic activity against RE and K562 tumor cell line. A lower level of
cytotoxicity was found against sheep erythrocytes. Such an activity was calcium-dependent,
thermo-stable (56 °C), inhibited by sphingomyelin (25 µg/ml), phospholypase A2 inhibitors e.g.
dibucain and quinacrine, as well as by D-galactose and cell-free hemolymph. Present results suggest
that a complex lytic mechanism dependent on sugar-CRD interaction may be involved in innate
immune response of Ciona intestinalis.
[Show abstract][Hide abstract] ABSTRACT: In invertebrates immune system, cell proliferation, phagocytosis and chemotaxis are regulated by cytophilic humoral molecules with functional similarities to vertebrate cytokines. These molecules modulate defense responses to exogenous and endogenous insults, tissue repair and recovery of homeostasis by ligand-specific receptor interactions required to initiate and regulate immune responses. tumor necrosis factor
(TNF) is a pro-inflammatory cytokine produced as part of the innate response. In invertebrates
TNF-like molecules have been identified by using various methods. Since in Ciona intestinalis
genome (Ensembl) TNF gene has been identified (CinTNF), Real-time PCR analysis was carried out. Results
showed a prompt (2-4 hrs) enhanced CinTNF gene expression in the inflamed body wall after intratunic LPS injection. In situ hybridization assays supported the involvement of pharynx hemocytes in the inflammatory response, and transcript was mainly found in morula cells and in unidentified cells associated with epidermis. Similar results were found by examining hemocytes from the hemolymph. Immunoblotting assay with anti-CinTNF specific antibodies revealed that a 17 kDa CinTNF is released in the hemolymph.
[Show abstract][Hide abstract] ABSTRACT: Heul siwek f!l'Olein.1 (HSP70 UI/d 9IJj IUII'e nl!OI II/I!ilsurl!ll in sf!C'Cill/l!ns or hil'IIIVI! Brachidonles phmaonis eXI)Us(,d (1/ l'IIITing lell/jierullll'e (5. iIJ, i5. 2IJ. 25 III/d 32°(: iI/ U clill/illic roOIl/. earrml lI/oleculuI' !IIetlloc!ologies 'dOI-hlol) highlighted U lelilj!l!I'ulal'e-depend('/71 I'1!Sf!onse 0/ 1I'00uied him/ I'C-': Ihe highel' II'e 10011I1)I!I'(IIUI'0', Ihe higllCl' Ihe NSPs cX/Il'l'Ssioll, il/llIl'ljrOIl1 II III/COITl'loll!d pl!uk III 5 °C Key-II'ords: hml sllOck /)l'iJlciIlS, Brachidonles phat'aonis, /elil/h'I'IIII1I'(' I'j1<'el, MediteITlllu'ul1. Introduzione -Gli organismi intertidali atTrontano un ambiente estremamente variabile dal punto di vista fisico poiche regime idrologico, aridita, disponibilita di cibo e molti altri f'attori variano su scala giornaliera durante tutto il lora cicio vitale, L'auJ11ento della temperatura, in particolare, dovuto a possibili fattori umani, ha un effello diretto sugli organismi durante la bassa marea, quando I'umidita ambientale e drammalicamente diminuita. Le possibili alteraziol1l dovute all'incremento tcrmico
[Show abstract][Hide abstract] ABSTRACT: Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1alpha-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca(2+)-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (alpha-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules contain interleukin-1alpha (IL1alpha) epitopes, and their activities are specifically inhibited by anti-human recombinant IL1alpha antibody. Analysis by SDS-polyacrylamide gel electrophoresis has revealed that the density of the bands of several serum proteins increases within 4 h after LPS injection, correlated with the enhanced serum activity. Moreover, Western blot patterns demonstrate that several serum proteins (59, 37, 30, 23, 15 kDa) cross-react with the antibody as early as 4 h post-injection. Although we have not been able to establish whether, in adition to galectins, various types of D-galactose-specific lectins are contained in the serum, we show, for the first time in invertebrates, that galectin molecules with opsonic properties can be enhanced in response to a non-specific inflammatory stimulus, and that their release can be further stimulated by LPS. Finally, we reveal that multiple galectins share human IL1alpha epitopes, probably because of steric configuration and the oligomerization process.
Cell and Tissue Research 09/2007; 329(2):379-90. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The coelomic fluid from the sea urchin Paracentrotus lividus contains several coelomocyte types including amoebocytes and uncoloured spherulocytes involved in immune defences. In the present paper, we show a Ca(2+)-dependent cytotoxic activity for the unfractionated coelomocytes assayed in vitro, with rabbit erythrocytes and the K562 tumour cell line. In a plaque-forming assay, whole coelomocyte preparations as well as density gradient separated coelomocyte populations revealed that cell populations enriched in uncoloured spherulocytes, exerted high cytotoxic activity by releasing lysins in the presence of amoebocytes. This cooperative effect could be dependent on soluble factors released by amoebocytes. With regard to this, we show that an enhanced cytotoxic activity was found by adding the supernatant from sonicated amoebocytes or hemocyte culture medium into spherulocyte preparations.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 07/2007; 147(2):389-94. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin and evolution of the innate immunity, including cell-cell, cell-cytokines, cell-lectins, cellmatrix interactions, appear to be product of cells and genetic markers for self recognition that grow with molecules and mechanisms to identify and destroy non-self. Lectins are components of a wellconserved protein-carbohydrate recognition system, the activity of most of them resides in a carbohydrate-recognition domain (CRD). They present an ample repertoire and have been proposed to mediate cell-cell or cell-extracellular matrix interactions in developmental processes, cell adhesion, inflammation and metastasis. Several immunomodulatory functions have been reported, among them mitogenic properties, opsonic properties, complement pathway activation and several immune responses. Cytokines are the major regulators of the host defence processes and are involved in responses to exogenous and endogenous insults, tissue repair and recovery of homeostasis. Many cytokines are bifunctional molecules having, beside a receptorbinding domain, a CRD. The expression of the biological activity relies on the association between both domains. Several reports have also shown that cytokine-CRD can interact with various pathogens and presents the recognition site that contributes to pathogen elimination via opsonization and/or leukocyte activation. Lectin-like activities of several cytokines, including IL-1, have been described. Our understanding of invertebrate cytokine-lectin biological functions and evolution are lacking. In some studies, similarities at the physicochemical level of vertebrate cytokines and functional invertebrate analogues have also been described. Among experimental approaches to identify cytokine-like molecules, antibodies neutralizing the activity of mammalian cytokines, have been used to screen for cross-reactivity with invertebrate factors in hemolymph. Tunicates are a key group in chordate phylogenesis. In ascidian species, lectins are responsible for the in vitro opsonization, modulate cell proliferation activity, phagocytosis and complement activation, stimulate proliferation of mouse thymocytes and L-929 fibroblasts. Recently we have shown that, in the serum from the LPSchallenged Ciona intestinalis, IL1-like inducible components cross-reacted with anti-humanrIL1a antibodies. CiIL1 with hemagglutinating properties, appears to be involved in sugar specific opsonization of yeast in an in vitro phagocytosis assay. Therefore a putative structural model of the opsonin include both CRD and IL1 epitopes. We propose an evolutionary model in which multifunctional costituive/inducible lectins can express cytokine activity with CRD responsible for pleiotropy and redundance, evolutionary conserved to guaranty a basic recogniton mechanism. Several inflammatory factors have been hypothesized as responsible for this process including components known in mammal innate immunity (cytokines, complement components, collagens) and phenoloxidase activity. A putative evolutionary model in which lectins are represented as multifunctional inducible molecules (cytokine-like function) involved in adult defence mechanisms, could be hypothesized. The lectin CRD, conserved in the evolution to guaranty a basic recognition mechanism, could explain lectin pleiotropy and redundancy as already suggested for mammalian cytokines.
[Show abstract][Hide abstract] ABSTRACT: Phagocytosis and plaque lysis activity (PLA) of coelomocyte from Paracentrotus lividus were
examined after exposure to cadmium chloride (CdCl2·H2O), a potentially toxic metal salt, widely
used in industry. P. lividus specimens were exposed at different Cd concentration (100, 200, and 400 μg
l-1) for 24 hours (sampled at 0, 6, 12 and 24 hrs) at 15°C, in tanks containing artificial sea water (ASW)
or injected with ASW containing the metal at 50, 100 and 200 μg l-1 in to the coelomic cavity. The
treatment without Cd did not affect phagocytosis and PLA, whereas treatment with Cd, significantly
lowered. This effect was dose and time dependent, presumably dependent on the cytotoxic effect of
cadmium on coelomocyte as indicated by neutral uptake assay.
[Show abstract][Hide abstract] ABSTRACT: Cytosolic lectins, Ca2+-independent and β-galactoside-specific, were determined to be containedin hemocyte and pharynx lysate supernatants of Ciona intestinalis, as revealed by hemagglutinationassay with trypsinized rabbit erythrocytes. Ca2+-independence and decreasing β-galactosidesinhibitory capacity (TDG > LacNAc ≥ Lactose > Galactose) have been considered properties typical ofgalectins. These lectins can be promptly released by hemocytes maintained in vitro suggesting theirinvolvement in defense responses including inflammatory reactions. Both cell lysate supernatants andhemocyte culture medium presented β-galactoside-inhibitable opsonizing activity versus yeast.Although a Percoll density gradient separation method showed that several hemocyte types containand release β-galactoside-specific molecules, results suggest that hyaline and granular amoebocytesare the primary source of these molecules.
[Show abstract][Hide abstract] ABSTRACT: There are several reasons for analyzing tunicate immune systems and phylogenesis. First, they can be established as primitive models, ancestors of vertebrates, for understanding fundamental immunological
mechanisms by analyzing their cells and molecular products; inflammation is the first defence system that
includes tissue response to foreign agents locally injected. Second, molecular and morpho functional study
coupled with molecular approach are requested to characterize molecules (e.g. cytokines) in a phylogenetic
contest. Third, tunicates are protochordates, pivotal in phylogenetic reconstruction. The body wall of Ciona
intestinalis mounts a defence inflammatory reaction to LPS. The kinetics of the response, cell components and
histochemical aspects have already been described. In Botryllus schlosseri the non fusion reaction shows inflammatory characters. The comparative study is of interest because these ascidians belong to distinct clade
and present different life cycle. Cytokine –like molecules have a basic role in inflammatory responses. We will
examine cells and molecules, by using several approaches including immunochemistry and immunohistochemistry, protein purification methods, specific antibodies production, functional assay of purified proteins. Protein and gene sequence data will contribute to prepare primers, cDNA sequences and riboprobes to evaluate hemocyte and tissue expression and modulation (in situ hibridization, real time
PCR). With the aim of verifying the cytokine-like role, functional approaches will be carried out to modulate
immune responses as phagocytosis and cytotoxicity. Here we show an opsonizing activity of C. intestinalis hemolymph that can be significantly decreased by absorption with antihuman-IL1.
(Research granted by MIUR PRIN Cofin 2004)
[Show abstract][Hide abstract] ABSTRACT: Paracentrotus lividus coelomic fluid contains several coelomocyte types that have been identified as amoebocytes, uncoloured spherulocytes (US), red cells and vibratile cells. Previous studies on coelomocytes, revealed that amoebocytes show phagocytic activity and are able to encapsulate foreign p articles, in addition they release haemoagglutinins; USs show in vitro cytotoxic activity against rabbit erythrocytes (RE) as revealed by plaque forming assay. In this study, the coelomic fluid was fractionated through a discontinuous Iodixinol gradient, a nd four cell bands(B1-B4) were obtained. Each of them appeared to be enriched in a particular coelomocyte type: B1 mainly contained amoebocytes; in B2 are present vibratile cells and little amount of amoebocytes; B3 was enriched with USs, and B4 contained red cells. The reported results support the phagocytic activity of amoebocytes assayed with yeast cells, whereas the separated US population were not able to show their in vitro cytotoxic activity (plaque forming assay) against RE. When amoebocytes (1 x 10 6ml-1) from the enriched fraction were added to USs (1:1), this activity was restored revealing a coelomocyte interaction. Since supernatant from amoebocyte cultures, added to the reaction medium, was able in inducing US cytotoxicity, the interaction seems to be based on substances released from amoebocytes. Western–blot assay with anti -human IL1 á antibodies (Sigma) showed that, in a short time, cultured amoebocytes release interleukin -like molecules. On the other side, protein preparations from US cell me mbranes cross-reacted with anti -human IL1-R antibodies. Further studies are requested to characterize the molecules that contain human IL1 and IL1-R epitopes.