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.

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.

Design and characterization of chionodracine-derived antimicrobial peptides with enhanced activity against drug-resistant human pathogens.

Starting from the sequence of the amphipathic α-helix of chionodracine (Cnd, 22 amino acids), we designed a series of mutants to increase Cnd's antimicrobial activity and selectivity toward prokaryotic cells and drug-resistant bacterial pathogens. We characterized these new Cnd-derived peptides using fluorescence, CD spectroscopy, and transmission electron microscopy, studying their interactions with synthetic lipid vesicles and assaying their biological function against E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter sp. Upon interaction with model membranes, these new peptides with higher net charges and hydrophobic moments adopt a helical conformation similar to Cnd. Notably, they display a low cytotoxic activity against human primary cells, a low hemolytic activity, but a significantly high bactericidal activity against drug-resistant bacterial pathogens. The low values of micromolar minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) make these Cnd-derived peptides potential templates to develop antimicrobial agents against drug-resistant human pathogens.

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.

Analysis of chromosome aberrations in somatic and germ cells of the mouse.

Chromosome aberration tests are used to evaluate the clastogenicity of chemical and physical agents, that is, the capacity of these agents to cause breaks in chromosomes and produce microscopically visible fragments or structural rearrangements. Aberrations are scored in metaphase chromosomes of dividing cells. In the mouse, bone marrow progenitors of erythrocytes and leukocytes provide abundant metaphases to study the effects on somatic cells, whereas the response of male germ cells to clastogenic agents can be visualized on metaphases of spermatogonia and primary spermatocytes. The techniques to prepare the slides for analyses are well standardized and internationally harmonized protocols for tests in bone marrow and spermatogonia provide the guidance necessary to obtain meaningful results. It is advisable to adhere as much as possible to these recommendations. Not all tests are suitable to score the same kind of aberrations. Here an overview of the application domains of these tests is provided with warnings on the scoring criteria and statistical analysis.