Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.).

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.

Vaccination and immune responses of European sea bass (Dicentrarchus labrax L.) against betanodavirus.

This review summarizes the available knowledge on the immune defences of European sea bass against antigenic preparations derived from the viral encephalopathy and retinopathy virus (betanodavirus), which represents a major threat to the health of this fish species. The nodavirus is widely present and differentiates into several strains that infect invertebrates (in insects, alphanodavirus) and teleost fish, and thus may represent a great problem for farmed fish species. Many efforts have been directed to discovering new immunizations to induce protection in sea bass, especially at young stages, and these efforts have included employing diverse betanodavirus strains, antigen preparation, vaccination routes, and the addition of adjuvants and/or immunostimulants. The obtained results showed that inactivated preparations of betanodavirus that were administered intraperitoneally may induce both immune recognition and protection. Attempts at performing mucosal immunization by immersion and/or oral administration, which is a vaccination route that is highly preferred for sea bass, have shown intriguing results, and more studies are necessary for its improvement. Overall, the objective of identifying a reliable vaccine that also cross-protects against different genotypes or reassortant viruses for use in European sea bass against betanodavirus appears to be an attainable goal in the near future.

Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.).

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.

Evolution of Th2 responses: characterization of IL-4/13 in sea bass (Dicentrarchus labrax L.) and studies of expression and biological activity.

Th2 immunity is a primary host defence against metazoan pathogens and two of the important cytokines involved in this immune response in mammals are IL-4 and IL-13. Recently the origin and evolution of Th2 immune responses have been investigated in fish where a molecule with relatedness to both IL-4 and IL-13 is present, termed IL-4/13. Different IL-4/13 paralogues (IL-4/13 A and IL-4/13B) exist in teleost fish. In this paper, we have focused on the IL-4/13 isoforms found in the European sea bass (Dicentrarchus labrax L.). Two tandem duplicated but divergent IL-4/13 A isoforms and one IL-4/13B are present, a unique situation compared to other teleosts. These genes were studied in terms of their in vitro and in vivo transcript levels after different treatments and their biological activities after production of the recombinant isoforms. The results show that the presence of these three paralogues is associated with different activities, both in terms of their expression profiles and the ability of the proteins to modulate the expression of immune genes in head kidney leukocytes. It is clear that the initiation and control of type-2 responses in seabass is complex due to the presence of multiple IL-4/13 isoforms with overlapping but distinct activities.

Immunoglobulin T from sea bass (Dicentrarchus labrax L.): molecular characterization, tissue localization and expression after nodavirus infection.

BACKGROUND: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization. RESULTS: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma. CONCLUSIONS: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.

Immune response of the Antarctic teleost Trematomus bernacchii to immunization with Psychrobacter sp. (TAD1).

Adult Trematomus bernacchii have been immunized intraperitoneally with heat-killed cells of the Antarctic marine bacterium Psychrobacter sp. (TAD1) up to 60 days. After immunizations and sampling at various times, fish sera were tested for specific IgM by ELISA, and different tissues (head kidney and spleen) were investigated for transcription of master genes of the acquired immune response (IgM, IgT, TRß, TRγ). Results from ELISA assays showed a time-dependent induction of specific serum anti-TAD1 IgM, and western blot analysis of TAD1 lysates probed with fish sera revealed enhanced immunoreactivity in immunized animals compared to controls. Quantitative PCR analysis of transcripts coding for IgM, IgT, TRß, TRγ was performed in T. bernacchii tissues to assess basal expression, and then on cDNAs of cells from head kidney and spleen of fish injected for 8, 24, and 72 h with inactivated TAD1. The results showed a differential basal expression of transcripts in the examined tissues, and a time-dependent strong up-regulation of IgT, TRß, TRγ genes upon in vivo stimulation with TAD1. These results represent a first in vivo study on the mounting of a specific immune response in an Antarctic teleost species.

Influence of titanium dioxide nanoparticles on 2,3,7,8-tetrachlorodibenzo-p-dioxin bioconcentration and toxicity in the marine fish European sea bass (Dicentrarchus labrax).

The present study investigated the influence of nano-TiO(2) (1 mg L(-1)) on 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) (46 pg L(-1)) bioconcentration and toxicity in the European sea bass (Dicentrarchus labrax) during 7 days in vivo exposure. A multimarkers approach was applied in different organs: detoxification in liver; innate immunity and pro-inflammatory response and adaptive immunity in gills and spleen; genotoxicity in peripheral erythrocytes and muscle. Bioconcentration of 2,3,7,8-TCDD in presence of nano-TiO2 was investigated in liver, skin and muscle as well as interaction between nano-TiO2 and organic pollutants in artificial sea water (ASW). Nano-TiO2 negatively influenced immune response induced by 2,3,7,8-TCDD in spleen but not in gills and reduced the DNA damage induced by 2,3,7,8-TCDD in erythrocytes. nano-TiO2 did not interfere with 2,3,7,8-TCDD detoxification and bioconcentration according to the observed no interaction of the nano-TiO2 with organic pollutants in ASW.

Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.).

In fish, the first line of defense is represented by the innate immune system and the lysozyme is one of the molecules involved in this mechanism of protection. Three types of lysozymes have been identified in metazoan, the c-type (chicken or conventional), the g-type (goose-type) and the i-type (invertebrate type). They are all involved in the hydrolysation of the bacterial cell wall. Our work has been focused on the molecular characterization, expression analysis by real-time PCR, both at basal condition and after in vivo challenges, and 3D structural studies on the g-type lysozyme from sea bass (Dicentrarchus labrax L.). Moreover, a recombinant sea bass lysozyme has been produced in Escherichia coli and used to investigate the activity of the enzyme at different pH and temperatures and to perform antibacterial assays against typical fish pathogens. The cloned sea bass cDNA for g-type lysozyme (accession number FN667957) consists of 742 bp and translates for a putative protein of 188 amino acids. The molecular weight is 20.251, 41Da with a theoretical pI of 8.53, two cysteine residues along the sequence and no putative signal peptide. These features of the enzyme are in agreement with the expected characteristics of a proper g-type lysozyme, except for the cysteine residues that in fish are quite variable in number. An alignment between known g-type lysozyme sequences evidences that the amino acid residues thought to be involved in the enzyme catalysis (Glu(71), Asp(84) and Asp(95) in sea bass) are quite well conserved between mammalian, avian and fish sequences. The sea bass g-type lysozyme gene is composed of four exons and three introns and this gene structure is more compact compared to other known fish lysozyme homologues. Modeling of 3D structure has been performed on the template structure of g-type lysozyme from Atlantic cod. The catalytic site appears well conserved when compared with known structures of fish g-type lysozymes (cod and salmon). The basal expression of lysozyme transcripts is highest in gills, followed by head kidney and peripheral blood leukocytes. The lysozyme expression is up regulated in head kidney leukocytes both after challenge with the fish bacterial pathogen Photobacterium damselae subsp. piscicida. The lytic activity, determined using as substrate Micrococcus lysodeikticus, was optimal at pH 5.5 and at a temperature of 30°C. In conclusion, these results suggest that the identified g-type lysozyme should be involved in the innate immune responses of sea bass.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) MHC class I heavy chain and ß2-microglobulin.

In this work, the gene and cDNA of sea bass (Dicentrarchus labrax) ß2-microglobulin (Dila-ß2m) and several cDNAs of MHC class I heavy chain (Dila-UA) were characterized. While Dila-ß2m is single-copy, numerous Dila-UA transcripts were identified per individual with variability at the peptide-binding domain (PBD), but also with unexpected diversity from the connective peptide (CP) through the 3' untranslated region (UTR). Phylogenetic analysis segregates Dila-ß2m and Dila-UA into each subfamily cluster, placing them in the fish class and branching Dila-MHC-I with lineage U. The α1 domains resemble those of the recently proposed L1 trans-species lineage. Although no Dila-specific α1, α2 or α3 sub-lineages could be observed, two highly distinct sub-lineages were identified at the CP/TM/CYT regions. The three-dimensional homology model of sea bass MHC-I complex is consistent with other characterized vertebrate structures. Furthermore, basal tissue-specific expression profiles were determined for both molecules, and expression of ß2m was evaluated after poly I:C stimulus. Results suggest these molecules are orthologues of other ß2m and teleost classical MHC-I and their basic structure is evolutionarily conserved, providing relevant information for further studies on antigen presentation in this fish species.

Diversity, molecular characterization and expression of T cell receptor γ in a teleost fish, the sea bass (Dicentrarchus labrax, L).

Two lineages of T cells, expressing either the αß T cell receptor (TR) or the γδ TR, exist in Gnathostomes. The latter type of T cells account for 1-10 % of T cells in blood and up to 30 % in the small intestine. They may recognize unconventional antigens (phosphorylated microbial metabolites, lipid antigens) without the need of major histocompatibility class I (MH1) or class II (MH2) presentation. In this work we have described cloning and structural characterization of TR -chain (TRG) from the teleost Dicentrarchus labrax. Further, by means of quantitative PCR analysis, we analyzed TRG expression levels both in poly I:C stimulated leukocytes in vitro, and following infection with betanodavirus in vivo. Two full length cDNAs relative to TRG, with the highest peptide and nucleotide identity with Japanese flounder, were identified. A multiple alignment analysis showed the conservation of peptides fundamental for TRG biological functions, and of the FGXG motif in the FR4 region, typical of most TR and immunoglobulin light chains. A 3D structure consisting of two domains mainly folded as beta strands with a sandwich architecture for each domain was also reported. TRG CDR3 of 8-18 AA in length and diversity in the TRG rearrangements expressed in thymus and intestine for a given V/C combination were evidenced by junction length spectratyping. TRG mRNA expression levels were high in basal conditions both in thymus and intestine, while in kidney and gut leukocytes they were up-regulated after in vitro stimulation by poly I:C. Finally, in juveniles the TRG expression levels were up-regulated in the head kidney and down-regulated in intestine after in vivo infection with betanodavirus. Overall, in this study the involvement of TRG-bearing T cells during viral stimulation was described for the first time, leading to new insights for the identification of T cell subsets in fish.

A piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus (Perciformes: Channichthyidae): molecular characterization, localization and bactericidal activity.

Antimicrobial peptides (AMPs) are considered one of the most ancient components of the innate immune system. They are able to exert their protection activity against a variety of microorganisms, and are widely distributed in both vertebrates and invertebrates. In this paper we focused on an AMP identified in the Antarctic teleost Chionodraco hamatus, an icefish species. The cDNA sequence of the AMP, named chionodracine, is comprised of 515 bp and translates for a putative protein precursor of 80 amino acids, with a signal peptide of 22 amino acids. The structural features evidenced in the primary sequence of chionodracine lead to the inclusion of the peptide in the antimicrobial family of piscidins. The analysis by real-time PCR of the basal gene transcripts of chionodracine in different icefish tissues showed that the highest expression was found in gills, followed by head kidney. The chionodracine expression levels in head kidney leukocytes were up-regulated in vitro both by LPS and poly I:C, and in vivo by LPS. A putative chionodracine mature peptide was synthesized and employed to obtain a polyclonal antiserum, which was used in immunohistochemistry of gills sections and revealed a significant positivity associated with mast cells. The bactericidal activity of the peptide was investigated and found significant against Antarctic psychrophilic bacteria strains (Psychrobacter sp. TAD1 and TA144), the Gram-positive Bacillus cereus, and at a lesser extent against the Gram-negative Escherichia coli. Interestingly, the haemolytic activity of chionodracine was tested in vitro on human erythrocytes and no significant lysis occurred until peptide concentration of 50 µM.

A CD83-like molecule in sea bass (Dicentrarchus labrax): molecular characterization and modulation by viral and bacterial infection.

The CD83 cell surface marker is an important and intriguing component of immune system. It is considered the best marker for mature human dendritic cells, but it is also important for thymic development of T cells, and it also plays a role as a regulator of peripheral B-cell function and homeostasis. A CD83-like molecule was identified in sea bass (Dicentrarchus labrax) by EST sequencing of a thymus cDNA library; the CD83 cDNA is composed of 816 bp and the mature CD83 peptide consists of 195 amino acids, with a putative signal peptide of 18 amino acids and two possible N-glycosylation sites. The comparison of sea bass CD83 sequence with its homologues in other fish species and mammals shows some differences, with two cysteine residues conserved from fish to mammals and a high variability both in the total number of cysteines and in mature CD83 sequence polypeptide length. Basal transcripts levels of CD83 mRNA are highest in liver, followed by thymus. The in vitro treatment of head kidney leukocytes with LPS resulted in a down-regulation on CD83 mRNA leves both after 4 and 24 h, whereas with poly I:C an up-regulation after 4h followed by a down-regulation at 24 h was observed. An in vivo infection of sea bass juveniles with nodavirus induced an increase of CD83 expression on head kidney leukocytes both after 6 and 24 h and a decrease after 72 h. On the other hand, an in vivo infection with Photobacterium damselae bacteria induced a decrease of CD83 transcript levels after 6 and 24 h and an increase after 72 h. These findings suggest in sea bass CD83 expression could be modulated by viral and bacterial immune response.

A monoclonal antibody for the CD45 receptor in the teleost fish Dicentrarchus labrax.

The CD45 tyrosine phosphatase plays an important role in regulating T lymphocyte activation in vertebrate species. In this study we describe some molecular and functional features of the CD45 receptor molecule from the European sea bass Dicentrarchus labrax. Following immunization with fixed sea bass thymocytes, we obtained a murine monoclonal antibody (mAb) able to stain fish leucocytes both alive, by immunofluorescence of thymus and mucosal tissues, and fixed, by in situ immunohistochemistry of tissue sections. The selected IgG(2) mAb (DLT22) was able to recognise by western blots polypeptides mainly at 180 kDa and 130 kDa in thymus, spleen, intestine and gill leucocyte. Accordingly, a 130 kDa polypeptide immunoprecipitated with DLT22 from thymocytes and analysed by nano-RP-HPLC-ESI-MS/MS, gave peptide sequences homologous to Fugu CD45, that were employed for the homology cloning of a partial sea bass CD45 cDNA sequence. This cDNA sequence was employed to measure by quantitative PCR the transcription of the CD45 gene both in unstimulated and in in vitro stimulated leucocytes, showing that the gene transcription was specifically modulated by LPS, ConA, PHA, IL-1, and poly I:C. When splenocytes were stimulated in vitro with ConA and PHA, a cell proliferation paralleled by an increase of DLT22-positive leucocytes was also observed. These data indicate that the DLT22 mAb recognizes a putative CD45 molecule in sea bass, documenting the presence of CD45-like developing lymphocytes in thymus and CD45-associated functional stages of lymphocytes in this species, thus dating back to teleost fish the functional activities of these cell populations in vertebrates.

Effects of the polycyclic ketone tonalide (AHTN) on some cell viability parameters and transcription of P450 and immunoregulatory genes in rainbow trout RTG-2 cells.

To assess the potential effects of environmental pollutants belonging to the musk fragrances group in the physiology of aquatic animal species, in this work we treated rainbow trout RTG-2 cells with the polycyclic ketone tonalide (AHTN) at dilutions ranging from 3.5 to 500 ng/ml. The following parameters were monitored: intracellular ATP concentration (energy production), mitochondrial membrane potential (early apoptosis marker), cell viability (vital staining with DFP), quantitative expression of genes coding for the cytochrome P450 detoxifying enzymes CYP1A1 and CYP3A27, and of genes coding for the immunoregulatory peptides IL-1ß, IL-8, TNFα, Cox-2 and TGF-ß. Obtained results showed that incubation with tonalide induced in RTG-2 cells no effects on cell viability, a slight increase of mitochondrial membrane potential activity, and a significant increase in intracellular ATP concentration. However, dramatic effects were observed in transcription levels of some tested genes, with upregulation levels of 300 and 600 times measured for TGF-ß and TNFα, respectively and of 150 times for the CYP3A27 gene. Our results show for the first time the potent effects exerted by tonalide on immunoregulatory genes of RTG-2 cells and also indicate that the measured sensitivity of RTG-2 towards tonalide was in the same range of that currently available using chemical methods. A possible use of the panel of genes we employed as a tool for the monitoring of musk fragrances in biological samples is discussed.

CD3γ/δ in sea bass (Dicentrarchus labrax): Molecular characterization and expression analysis.

The CD3 complex is the common marker on the surface of both αß and γδ 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 CD3γ/δ 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 immunoglobulin-like domain, are present in sea bass CD3γ/δ sequence and they are conserved both in number and position from mammals to teleost sequences. Similar to other known CD3γ/δs, in sea bass CD3γ/δ 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 CD3γ/δ 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 CD3γ/δ 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 CD3γ/δ can be used as a T cell marker and will help in adding new insight on the immune response mechanisms of sea bass.

3D modelling of three pro-inflammatory molecules in selected fish species.

The inflammatory response is the reaction of all Metazoan organisms to pathogen invasion that initiates when pathogen-derived 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-1ß, tumor necrosis factor-α, 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.

Searching for immunomodulatory sequences in sea bass (Dicentrarchus labrax L.): transcripts analysis from thymus.

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 1632 ESTs from this library were initially analysed for sequence quality and vector sequences and, after this control, 1264 (77% of total clones sequenced) high-quality ESTs were further processed. The total collection of D. 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 more 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 discover 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.

Molecular and structural characterisation of a macrophage migration inhibitory factor from sea bass (Dicentrarchus labrax L.).

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 609bp 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 Cys(57)-Ala(58)-Leu(59)-Cys(60) 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 Pro(2) 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 4h in vitro stimulation of head kidney leukocytes with LPS and decreased after 24h. The predicted 3D model of sea bass MIF has been used to verify the presence of structural requirements for its known biological activities.

Lymphocyte differentiation in sea bass thymus: CD4 and CD8-alpha gene expression studies.

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 post-larvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-alpha mRNA. Real-time PCR with specific primers quantified CD4, CD8-alpha and TCR-beta transcripts in larvae and post-larvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-beta and CD8-alpha 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-beta and CD8-alpha transcripts were found in the thymus, while CD4 transcripts were lower (P < 0.05 vs. TCR-beta). 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-alpha almost coincided. In each adult thymic lobe CD4(+) and CD8-alpha(+) thymocytes filled the cortex. The expression patterns of CD4 and CD8-alpha largely overlap, except in the medulla, where CD4(+) thymocytes were isolated, while CD8-alpha(+) 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.

Stimulation of gut immune system by early administration of probiotic strains in Dicentrarchus labrax and Sparus aurata.

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-beta transcripts, and increased acidophilic granulocytes concomitant to lower transcription of pro-inflammatory genes (IL-1-beta, TGF-beta, IL-10, Cox-2). In sea bream, the multispecies probiotic formulation raised intestinal immunoglobulin (Ig(+)) cells and acidophilic granulocytes mainly belonging to the MAb G7(+) 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.