http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Sat, 25 May 2013 20:24:02 GMT 2013-05-25T20:24:02Z http://hdl.handle.net/2067/1357 Title: Molecular cloning, differential expression and 3D structural analysis of the MHC class-II β chain from sea bass (Dicentrarchus labrax L.) Authors: Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Costantini, Susan; Facchiano, Angelo M.; Scapigliati, Giuseppe; Stet, Rene Abstract: The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II b chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28 bp 50-UTR and a 478 bp 30-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II b chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4 h showed very little difference in the Dila-DAB expression, but after 24 h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1b (ranging from 0 to 100 ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1357 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1534 Title: Majority of TcRβ+ T-lymphocytes located in thymus and midgut of the bony fish, Dicentrarchus labrax (L.) Authors: Romano, Nicla; Rossi, Francesca; Abelli, Luigi; Caccia, Elisabetta; Piergentili, Rita; Mastrolia, Lucia; Randelli, Elisa; Buonocore, Francesco Abstract: Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence and distribution of T-lymphocytes expressing TcRβ in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology, distribution and number of T-cells and TcRβ+ cells in the various sampled lymphoid organs. The highest TcRβ expression was revealed by real-time PCR in the thymus, with high levels also being found in the gut. In the thymus, DLT15+ and TcRβ+ cell populations were concentrated in the cortex and TcRβ+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection. The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRβ+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRβ+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular immunity. The large population of TcRβ-/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRγδ phenotype fraction. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1534 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1535 Title: Interleukin-10 expression by real-time PCR and homology modelling analysis in the European sea bass (Dicentrarchus labrax L.) Authors: Buonocore, Francesco; Randelli, Elisa; Bird, Steve; Secombes, Chris J.; Facchiano, Angelo M.; Costantini, Susan; Scapigliati, Giuseppe Abstract: In this paper we investigate the interleukin-10 (IL-10) expression and 3D modelling of the European sea bass (Dicentrarchus labrax L.). IL-10 is a regulatory cytokine that has been intensively studied in mammals and has been found mainly involved in the suppression or deactivation of activated immune responses. The full-length sea bass IL-10 cDNA consists of 1015 bp and is translated in a predicted molecule of 187 amino acids. A multiple alignment of this peptide with other known sequences showed the conservation of fundamental IL-10 family characteristics. Expression analysis by real-time PCR in control fish showed a high basal expression in the head kidney (HK), followed by gut and brain. In vitro treatment of HK leucocytes with LPS showed an intense increase of IL-10 expression at 4 h and a significant decrease at 24 h, with PHA-L no differences were evidenced. A 3D model for sea bass IL-10 was obtained by accurate homology procedures and putative interaction residues with the IL-10 receptor described. The results suggested that sea bass IL-10 could be involved in anti-inflammatory responses, but further experiments are needed to assess its importance in response to pathogenic agents, vaccinations and immunostimulants. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1535 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1664 Title: Cell markers and determinants in fish immunology Authors: Randelli, Elisa; Buonocore, Francesco; Scapigliati, Giuseppe Abstract: Despite the impressive increase in the cloning and expression of genes encoding fish immunoregulatory molecules, the knowledge on ‘‘in vivo’’ and ‘‘in vitro’’ functional immunology of the corresponding peptide products is still at an initial stage. This is partly due to the lacking of specific markers for immunoregulatory peptides, that represent an indispensible tool to dissect immune reactions and to trace the fate of cellular events downstream of the activation. In this review we summarise the available information on functional immune activities of some teleost species and discuss the obtained data in an evolutionary and applied context. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1664 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1665 Title: A CD4 homologue in sea bass (Dicentrarchus labrax): Molecular characterization and structural analysis Authors: Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Guerra, Laura; Picchietti, Simona; Costantini, Susan; Facchiano, Angelo M.; Zou, Jun; Secombes, Chris J.; Scapigliati, Giuseppe Abstract: CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell coreceptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4 h and a decrease after 24 h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/1665 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1679 Title: An “immunome” gene panel for transcriptomic analysis of immune defence activities in the teleost sea bass (Dicentrarchus labrax L.): a review Authors: Randelli, Elisa; Buonocore, Francesco; Casani, Daniela; Fausto, Anna Maria; Scapigliati, Giuseppe Abstract: Teleost fish are the oldest living vertebrates displaying a morphological and functional immune system conserved until mammals, thus representing a useful model for evolutionary studies. Teleosts are increasingly employed as experimental animal species for studies on physiological activities of vertebrates, and are widely used in the aquaculture industry. Due to its importance as high-quality food, the sea bass is, at present, one of the most important farmed species in the Mediterranean sea and many studies reported on the morphological and functional organisation of immune defences with the aim of controlling its health in farming conditions. From these studies, a panel of genes coding for the main immunomodulatory peptides of innate and acquired defences is available and constituted, at present, by genes coding for innate defences, for acquired defences, and for other immune-related activities. In this review all these genes are collected and defined as the sea bass ‘‘immunome’’, that is analysed in an evolutionary context. Description: L'articolo è disponibile sul sito dell'editore http://www.tandf.co.uk/journals/default.asp Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/1679 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1680 Title: Molecular characterisation and structural analysis of an interferon homologue in sea bass (Dicentrarchus labrax L.) Authors: Casani, Daniela; Randelli, Elisa; Costantini, Susan; Facchiano, Angelo M.; Zou, Jun; Martin, Sam; Secombes, Chris J.; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047 bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an “all-alpha” domain that shows an “up-down bundle” architecture made of six helices (ABB’CDE). The two cysteine residues present in the sequence (i.e. Cys23 and Cys126) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and Vertebrates more generally. Description: L'articolo è disponibile sul site dell'editore http://www.sciencedirect.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1680 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1681 Title: Evolution of cell-mediated immune defences: cloning and structural characterisation of the T cell receptor beta chain from the icefish Chionodraco hamatus (Perciformes: Channichthyidae) Authors: Buonocore, Francesco; Randelli, Elisa; Viviana, Scala; Belardinelli, Maria Cristina; Casani, Daniela; Facchiano, Angelo M.; Costantini, Susan; Scapigliati, Giuseppe Abstract: Studies on the immune defence system of Antarctic fishes are of interest to investigate the presence of evolutionary adaptations to low temperature seawater. In the present paper, primers designed to conserved regions of TR , the antigen receptor expressed by T lymphocytes and mediator of main adaptive immune responses, were used for the homology cloning of the icefish Chionodraco hamatus TR  chain cDNA. The full length cDNA consists of 1219 nucleotides that translates in a single reading frame to give a predicted 304-amino acid molecule. The sequence shows highest nucleotide and amino acid identity with the sea bream (Sparus aurata) TR  . A multiple alignment with other known TR  molecules evidenced the presence of conserved amino acid residues involved in structural and functional domains within most teleost species. Real-time PCR analysis was used to investigate TR  basal expression, that resulted maximal in the thymus, followed by gills and spleen. Finally, the 3D structure of the icefish TR  was predicted by homology modelling and compared to the closest homologous Sparus aurata, a fish Teleost living in more temperate environmental conditions. The comparison of these two models suggests that the icefish TR  may be more flexible, as a consequence of a less compact structure and a lower number of H-bonds. Description: L'articolo è disponibile sul sito dell'editore http://www.tandfonline.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1681 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1686 Title: 3D Modelling of three pro-inflammatory molecules in selected fish species. Authors: Costantini, Susan; Facchiano, Angelo M.; Randelli, Elisa; Casani, Daniela; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The inflammatory response is the reaction of all Metazoan organisms to pathogen invasion that initiates when pathogenderived molecules are recognized by specific pattern recognition receptors expressed mainly on cells of the innate immune system. The successive expression of pro-inflammatory cytokines and chemokines limits pathogen spread, and attracts and activates immune cells to help in the elimination of the invaders. In this paper we focused on the analyses of the 3D structures of three pro-inflammatory molecules (interleukin-1􀀂, tumor necrosis factor-􀀁, interleukin-8) from selected Teleost fish species (Oncorhynchus mykiss, Dicentrarchus labrax, Chionodraco hamatus) generated using as template models those of experimental homologous proteins. These structures were discussed with the aim to investigate the differences between them and mammalian counterparts and, moreover, to verify the presence of the structural requirements for their biological activities, known mainly in mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.benthamscience.com/index.htm Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/1686 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1562 Title: Early treatment with Lactobacillus delbrueckii strain induces rise in intestinal T cells and granulocytes and modulates immune related genes of larval Dicentrarchus labrax (L.) Authors: Picchietti, Simona; Fausto, Anna Maria; Randelli, Elisa; Carnevali, Oliana; Taddei, Anna Rita; Buonocore, Francesco; Scapigliati, Giuseppe; Abelli, Luigi Abstract: Lactobacillus delbrueckii ssp. delbrueckii (AS13B), isolated from the gut of adult Dicentrarchus labrax, was administered live to developing sea bass using rotifers and Artemia as live carriers. Immune-related gene transcripts were quantified in post-larvae at day 70 post-hatch (ph) and histology, electron microscopy and immunocytochemistry of the intestinal tissue were performed at day 74 ph. Since the probiotic was orally administered the studies were focused on intestinal immunity. In treated fish gut integrity was unaffected, while the density of T-cells and acidophilic granulocytes in the intestinal mucosa was significantly higher than in controls. Probiotic-induced increases in intestinal T-cells and total body TcR-beta transcripts are first reported in fish. Significantly lower IL-1beta transcripts and a trend towards lower IL-10, Cox-2 and TGF-beta transcription were found in the treated group. Evidence is provided that early feeding with probiotic-supplemented diet stimulated the larval gut immune system and lowered transcription of key pro-inflammatory genes. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1562 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1563 Title: Lymphocyte differentiation in sea bass thymus: CD4 and CD8-α gene expression studies Authors: Picchietti, Simona; Guerra, Laura; Buonocore, Francesco; Randelli, Elisa; Fausto, Anna Maria; Abelli, Luigi Abstract: Different developmental stages (from eggs to 1-year-old juveniles) of the teleost fish Dicentrarchus labrax (L.) were assayed for CD4 gene expression. RT-PCR revealed the appearance of CD4 transcripts in postlarvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-a mRNA. Real-time PCR with specific primers quantified CD4, CD8-a and TCR-b transcripts in larvae and postlarvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-b and CD8-a transcripts were significantly higher (P < 0.001) than in previous stages, as CD4 transcripts compared with 51 dph (P < 0.01). High levels of TCR-b and CD8-a transcripts were found in the thymus, while CD4 transcripts were lower (P < 0.05 vs. TCR-b). In situ hybridization identified CD4 mRNAs at 51 dph, localized in thymocytes of the outer and lateral zones of the thymic glands. From 75 dph on the signal was mainly detected in the outer region, drawing a cortex–medulla demarcation. Developmental expression of CD4 and CD8-a almost coincided. In each adult thymic lobe CD4þ and CD8-aþ thymocytes filled the cortex. The expression patterns of CD4 and CD8-a largely overlap, except in the medulla, where CD4þ thymocytes were isolated, while CD8-aþ ones mainly arranged in cords. These results provide new information about the thymic compartmentalization and lymphocyte differentiation pathways in a teleost, almost demonstrating that double negative thymocytes fill the cortex giving rise to further selection in the medulla. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1563 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1565 Title: CD4 homologue in sea bass (Dicentrarchus labrax): molecular characterization and structural analysis Authors: Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Guerra, Laura; Picchietti, Simona; Costantini, Susan; Facchiano, Angelo M.; Zou, Jun; Secombes, Chris J.; Scapigliati, Giuseppe Abstract: CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell coreceptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4 h and a decrease after 24 h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1565 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1560 Title: Stimulation of gut immune system by early administration of probiotic strains in dicentrarchus labrax and sparus aurata Authors: Abelli, Luigi; Randelli, Elisa; Picchietti, Simona; Carnevali, Oliana Abstract: Early feeding (started during gut metamorphosis and establishment of the hypothalamic–pituitary–interrenal axis) with probiotic-supplemented diets, besides modifying the intestinal microflora, evoked profound effects on the physiology of fish larvae. Using rotifers and Artemia as living vectors, the autochthonous bacterium Lactobacillus delbrueckii delbrueckii or a multispecies probiotic formulation (autochthonous Lactobacillus fructivorans + Lactobacillus plantarum from human feces) were orally administered to sea bass and gilthead sea bream larvae, respectively. The treatments enhanced larval rearing (significantly increased body weight, decreased cortisol levels, and improved stress response compared with controls) and the immune system. In sea bass, the probiotic raised intestinal T cells, in keeping with increased total body TcR-β transcripts, and increased acidophilic granulocytes concomitant to lower transcription of pro-inflammatory genes (IL-1-β, TGF-β, IL-10, Cox-2). In sea bream, the multispecies probiotic formulation raised intestinal immunoglobulin (Ig+) cells and acidophilic granulocytesmainly belonging to theMAbG7+ phagocytic population. These results point to stimulatory actions of probiotics on the gut immune system that are devoid of harmful effects and that correlate with improvement of fry survival. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1560 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1685 Title: Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus Authors: Scapigliati, Giuseppe; Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Meloni, Sabrina; Zarletti, Gianpaolo; Tiberi, M.; Pietretti, D.; Boschi, I.; Manchado, Manuel; Martin-Antonio, B.; Jimenez-Cantizano, R.; Bovo, Giuseppe; Borghesan, Fabio; Lorenzen, Niels; Einer-Jensen, Katja; Adams, S.; Thompson, K.; Alonso, C.; Béjar, Julia; Cano, Irene; Borrego, Juan J.; Alvarez, M.Carmen Abstract: Naïve sea bass juveniles (38.4 ± 4.5 g) were intraperitoneally infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6 %. At various intervals, sampling of fish tissues were performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: IFN-1, Mx, IL-1, Cox-2; IL-10, TGF-, TCR, CD4, CD8, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable in “vitro” increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for IFN-1, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF- and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/1685 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1688 Title: Molecular and structural characterisation of a macrophage migration inhibitory factor from sea bass (Dicentrarchus labrax L.) Authors: Buonocore, Francesco; Randelli, Elisa; Facchiano, Angelo M.; Pallavicini, Alberto; Modonut, Martina; Scapigliati, Giuseppe Abstract: The macrophage migration inhibitory factor (MIF) is a cytokine produced in numerous cell types, mainly T lymphocytes and macrophages, in response to inflammatory stimuli. In this paper we report the identification of a cDNA encoding a MIF molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and its 3D structure obtained by template-based modelling. The sea bass MIF cDNA consists of 609 bp that translates in one reading frame to give the entire molecule containing 115 amino acids. The sequence contains three cysteine residues in conserved positions compared to human MIF and most Teleost fishes, with the exception of zebrafish and carp. The Cys57-Ala58-Leu59-Cys60 motif, present inside the stretch important for JAB1-interaction and mediator of the thiol-protein oxidoreductase activity of MIF, is conserved in sea bass, together with the Pro2 residue that is crucial for the tautomerase catalytic activity. Real time PCR analyses revealed that MIF is constitutively expressed in all selected tissues and organs, with the highest mRNA level observed in thymus. MIF expression was induced after 4 hours in vitro stimulation of head kidney leukocytes with LPS and decreased after 24 hours. The predicted 3D model of sea bass MIF has been used to verify the presence of structural requirements for its known biological activities. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1688 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1690 Title: Searching for immunomodulatory sequences in sea bass (Dicentrarchus labrax L.): Transcripts analysis from thymus Authors: Pallavicini, Alberto; Randelli, Elisa; Modonut, Martina; Casani, Daniela; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The thymus is a key organ of the immune system in most vertebrates and, for this reason, it has been used in this paper for the generation of a normalized cDNA library from sea bass (Dicentrarchus labrax), one of the most extensively cultured species in South Mediterranean aquaculture. A total of 1,632 ESTs from this library were initially analyzed for sequence quality and vector sequences and, after this control, 1,264 (77% of total clones sequenced) high-quality ESTs were further processed. The total collection of Dicentrarchus labrax thymus ESTs has been deposited in the EBI-GenBank-DBJ database (GenBank accession numbers from FN565576 to FN566839). The functional classification of ESTs was performed by Gene Ontology and KEGG annotation and, successively, the sequences were analysed using the ImmunomeBase software to identify potentially immuno related genes. Using this approach, we found about 100 putative genes involved in immune system responses, most new in sea bass, that were analysed most in detail. Some of the potentially interesting genes identified by these in silico analyses were studied by real-time PCR to verify their expression both at basal level and after in vitro stimulation of sea bass head kidney leukocytes. The used strategy has been confirmed as a good approach to discovery new immuno-related genes and to improve the knowledge of specific markers that could help the discrimination of T cell subpopulations in sea bass and, in general, in Teleosts. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1690 2009-12-31T23:00:00Z http://hdl.handle.net/2067/2180 Title: Analysis of the expression and modulation of selected immune-related gene transcripts in the DLEC cell line from European sea bass (Dicentrarchus labrax) Authors: Buonocore, Francesco; Randelli, Elisa; Lorenzen, Niels; Einer-Jensen, Katja; Scapigliati, Giuseppe Abstract: Cell lines have been established from different fish species especially for virus isolation and for studying cell-pathogen interactions, and therefore are of interest in aquaculture. In this paper, we have investigated the presence and the regulation of some immune genes in the DLEC (Dicentrarchus labrax embryonic cells) cell line from European sea bass (Dicentrarchus labrax L.) to preliminary elucidate their action. The basal expression of the selected genes (interleukin- 1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β), CD8-α, major histocompatibility complex II-β (MHC II-β), interferon (IFN) and Mx protein (Mx)) have been investigated and, successively, their modulation have been studied both after stimulation with different mitogen agents and after a transfection with a sequence codifying for the coat protein of a fish nervous necrosis virus (NNV). The results have evidenced that the inflammatory molecules (IL-1β, COX-2, TGF-β), constitutively expressed by the DLEC cell line, are not up-regulated by the stimulation with lipopolysaccharide (LPS) from E. coli, whether the expression of the T-cell marker transcripts (CD8-α, MHC II-β) is influenced by the action of a lectin from Phaseolus vulgaris (PHA-L). Finally, the expression of the coat NNV protein in the DLEC cell line, after the transfection, led to an high up-regulation of IFN and Mx gene transcripts. These data suggest that the DLEC cell line recognize specific pathogen-associated molecular patterns (PAMPs) and, therefore, could be useful for studying T-cell pathways and viral responses in sea bass avoiding the use of live test animals. Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/2180 2010-12-31T23:00:00Z http://hdl.handle.net/2067/2183 Title: Intestinal T cells of Dicentrarchus labrax (L.): gene expression and functional studies Authors: Picchietti, Simona; Guerra, Laura; Bertoni, Fabrizio; Randelli, Elisa; Belardinelli, Maria Cristina; Buonocore, Francesco; Rombout, Jan; Scapigliati, Giuseppe; Abelli, Luigi Abstract: Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-β, rag-1 and TCR-β gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-β and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/2183 2010-12-31T23:00:00Z http://hdl.handle.net/2067/2205 Title: Transcription of T cell-related genes in teleost fish, and the European sea bass (Dicentrarchus labrax) as a model Authors: Boschi, Ilenia; Randelli, Elisa; Buonocore, Francesco; Casani, Daniela; Bernini, Chiara; Fausto, Anna Maria; Scapigliati, Giuseppe Abstract: In recent years the cloning of genes coding for immuno-regulatory peptides, as well as the sequencing of genomes, provided fish immunologists with a growing amount of information on nucleotide sequences. Research is now also addressed in investigating the functional immunology counterpart of nucleotide sequence transcripts in various fish species. In this respect, studies on functional immunology of T cell activities are still at their beginning, and much work is needed to investigate T cell responses in teleost fish species. In this review we summarise the current knowledge on the group of genes coding for main T cell-related peptides in fish, and the expression levels of these genes in organs and tissues. Particular attention is paid to European sea bass (Dicentrarchus labrax), a marine species in which some information on functional immunology has been obtained, and we reassume here the expression of some T cell-related genes in basal conditions. In addition, we provide original data showing that T cells purified from the intestinal mucosa of sea bass with a specific mAb, express transcripts for TRgamma, TRbeta, CDalpha and RAG-1, thus showing similarities with intraepithelial leucocytes of mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/2205 2010-12-31T23:00:00Z http://hdl.handle.net/2067/2204 Title: CD3 γ/δ in sea bass (Dicentrarchus labrax): molecular characterization and expression analysis Authors: Randelli, Elisa; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The CD3 complex is the common marker on the surface of both ab and gd T cells and is essential for formation of the T-cell receptor complex and for T-cell activation. In this paper, we report the gene cloning and molecular characterization of a CD3g/d homologue in sea bass (Dicentrarchus labrax), the analysis of transcription levels in lymphoid and non-lymphoid organs and the gene regulation after in vitro stimulation with LPS and PHA. Four cysteine residues in the extracellular domain, involved in the constitution of immunoglobulinlike domain, are present in sea bass CD3g/d sequence and they are conserved both in number and position from mammals to teleost sequences. Similar to other known CD3g/ds, in sea bass CD3g/d there is also a conserved immunoreceptor tyrosine-based activation ITAM motif that could be responsible for its individual signal transduction capacity. The real time RT-PCR basal analysis shows the highest level of CD3g/d mRNA in thymus, followed by peripheral blood leucocytes, spleen, gills, gut, liver, head kidney, brain and muscle. The expression analysis under stimuli condition reveals a significant decrease of CD3g/d expression after LPS stimulation and a significant increase after PHA-L stimulation, in agreement with mammals results. In conclusion, these data allow us to affirm that sea bass CD3g/d can be used as a T cell marker and will help in adding new insight on the immune response mechanisms of sea bass. Description: L'articolo è disponibile sul sito dell'editore http://www.elsevier.com Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/2204 2010-12-31T23:00:00Z