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    M. Esteban

    Fish cytotoxic effectors form a cell population whose ultrastructure and properties of conjugation with target cells have not been completely established. We report the ultrastructure of the nonspecific cytotoxic cells in a seawater... more
    Fish cytotoxic effectors form a cell population whose ultrastructure and properties of conjugation with target cells have not been completely established. We report the ultrastructure of the nonspecific cytotoxic cells in a seawater teleost (Sparus aurata L.) and compare it to a freshwater species (Cyprinus carpio L.). Blood leucocytes were incubated with HeLa or B16 melanoma cells. Samples were processed for transmission electron microscopic study. Conjugates consisting of leucocytes binding targets were regularly observed after 30 min, 1 hr, or 2 hr of incubation. In both species leucocytes binding to targets showed ultrastructural features of either monocyte-like or lymphocyte-like cells. Monocyte-like cells usually appeared flattened against the targets and seemed to enclose fragments of the target to form cytoplasmic vesicles and the content of their scarce cytoplasmic granules seemed to be delivered into these vesicles. In the seabream lymphocyte-like cells, dense cytoplasmic granules occurred only occasionally, and neither microvilli nor cell processes were present at the contact areas with the targets. In the carp, the contacts were more numerous and formed regularly interdigitating contact areas and the lymphocytes showed granules with characteristic dense and fibrillar contents. We conclude that seabream and carp have a leucocyte cell population with ultrastructural features of either monocytes or lymphocytes showing nonspecific cytotoxic ability.
    Thrombocytes and platelets, beyond their primary function in hemostasis, seem to play an active role in inflammation. As regards their phagocytic ability, the results to date are confusing, incomplete, and somewhat contradictory. Whereas... more
    Thrombocytes and platelets, beyond their primary function in hemostasis, seem to play an active role in inflammation. As regards their phagocytic ability, the results to date are confusing, incomplete, and somewhat contradictory. Whereas the interaction of avian thrombocytes or mammalian platelets with bacteria both in vitro and in vivo has received wide attention, almost no information exists on the topic in "lower" vertebrates. The aim of this work is to review the available information on the phagocytic properties of thrombocytes and platelets. Particular attention is payed to the ontogeny of these cells, the soluble factors involved in the inflammatory process derived from them, and their interaction with particulate material, mainly with bacteria.
    One of the most powerful innate immune responses against virus is mediated by the type I interferon (IFN). In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes but the viral RNA... more
    One of the most powerful innate immune responses against virus is mediated by the type I interferon (IFN). In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes but the viral RNA sensors and mediators leading to the IFN production are scarcely known. Thus, we have searched the presence of these genes in gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In seabream, a resistant fish species to the VNNV strain used, we found an up-regulation of the genes encoding MDA5, TBK1, IRF3, IFN, Mx and PKR proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we identified, in addition, transcripts cod...
    This paper describes an easy and sensitive flow cytometric assay for assessing the non-specific cytotoxic activity of gilthead seabream (Sparus aurata L.) head-kidney leucocytes against tumor target cells. Concomitantly, the cytotoxic... more
    This paper describes an easy and sensitive flow cytometric assay for assessing the non-specific cytotoxic activity of gilthead seabream (Sparus aurata L.) head-kidney leucocytes against tumor target cells. Concomitantly, the cytotoxic process and the cell types involved were microscopically studied. The assay was based on the consecutive use of two fluorochromes. The targets were preincubated with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and then mixed with effectors. At the end of incubation time propidium iodide (PI) was added. While live effectors were non-fluorescent, live and dead targets retained the DiO (green) fluorescence and non-viable targets and effectors showed PI (red) fluorescence staining. The kinetics of the cytotoxic activity was studied from 10 to 240 min. Lymphocytes, monocyte-macrophages and granulocytes showed non-specific cytotoxic activity, as demonstrated by light and electron microscopy. In conclusion, the technique presented validates the effectiveness of a dual-color flow cytometric assay for assessing the activity of non-specific cytotoxic cells in fish.
    The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream (Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its... more
    The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream (Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5'-UTR region of 84 bp, an ORF of 729 bp and a 3'-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, alpha1, alpha2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is ...
    The early cellular innate immune responses of the teleost gilthead seabream (Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte... more
    The early cellular innate immune responses of the teleost gilthead seabream (Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte mobilization, degranulation, peroxidase content, respiratory burst, phagocytic and cytotoxic activities were assayed in both head-kidney leukocytes (HKLs) and peritoneal exudate leukocytes (PELs). The total number of PELs significantly increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis revealed variations in the proportion of cell-types in the PE. Thus, PE acidophilic granulocytes increased to a significant extent 4 h post-injection and were restored thereafter. Moreover, PE monocyte-macrophages started to increase from 24 h, the enhancement being statistically significant after 48 and 72 h. Degranulation was greater in PELs throughout the assay. The peroxidase content of the leukocytes was...
    The possible expression of FasL in gilthead seabream leucocytes was studied by flow cytometry, immunoblotting and immunocytochemistry, using an anti-mouse FasL monoclonal antibody. The results pointed to a cytosolic FasL-like, but not a... more
    The possible expression of FasL in gilthead seabream leucocytes was studied by flow cytometry, immunoblotting and immunocytochemistry, using an anti-mouse FasL monoclonal antibody. The results pointed to a cytosolic FasL-like, but not a membrane-like form, in resting leucocytes from head-kidney, thymus, spleen, blood and peritoneal exudate. Immunoblotting revealed a 19kDa band in resting leucocytes, while activated leucocytes showed the same band and another of 39kDa. The FasL-like molecule is identified in lymphocytes, monocyte-macrophages and acidophilic granulocytes. Phylogenetical and functional implications are suggested.
    Teleost fish NCCs (nonspecific cytotoxic cells) are thought to be the evolutionary precursors of the mammalian NK cells. A novel mechanism mediating the NCC-mediated cytotoxicity has been described in teleosts. Now, this NCC receptor... more
    Teleost fish NCCs (nonspecific cytotoxic cells) are thought to be the evolutionary precursors of the mammalian NK cells. A novel mechanism mediating the NCC-mediated cytotoxicity has been described in teleosts. Now, this NCC receptor protein-1 (NCCRP-1) was characterized in gilthead seabream. The NCCRP-1 full-length sequence contains 1036 bp with an open reading frame of 702 bp. A comparison of the predicted 233-amino acid protein with several fish orthologues indicates a highly conserved sequence containing the F-box associated (FBA) domain and proline-rich motifs (PRM) characteristics of this family. The phylogenetical tree shows that seabream NCCRP-1 belongs to the NCCRP subfamily within the FBA family of proteins. This is a single copy gene with a constitutive and ubiquitous expression as determined by RT-PCR and flow cytometry. The results show that lymphocytes, monocyte/macrophages and acidophilic granulocytes from lymphoid tissues express the receptor, both at gene and protei...
    COPHES/DEMOCOPHES has its origins in the European Environment and Health Action Plan of 2004 to... more
    COPHES/DEMOCOPHES has its origins in the European Environment and Health Action Plan of 2004 to "develop a coherent approach on human biomonitoring (HBM) in Europe". Within this twin-project it was targeted to collect specimens from 120 mother-child-pairs in each of the 17 participating European countries. These specimens were investigated for six biomarkers (mercury in hair; creatinine, cotinine, cadmium, phthalate metabolites and bisphenol A in urine). The results for mercury in hair are described in a separate paper. Each participating member state was requested to contract laboratories, for capacity building reasons ideally within its borders, carrying out the chemical analyses. To ensure comparability of analytical data a Quality Assurance Unit (QAU) was established…
    Fish skin mucus contains several immune substances that provide the first line of defence against a broad spectrum of pathogens although they are poorly studied to date. Terminal carbohydrate composition and levels of total IgM... more
    Fish skin mucus contains several immune substances that provide the first line of defence against a broad spectrum of pathogens although they are poorly studied to date. Terminal carbohydrate composition and levels of total IgM antibodies, several immune-related enzymes (lysozyme, peroxidase, alkaline phosphatase, esterases, proteases and antiproteases) as well as the bactericidal activity (against fish pathogenic Vibrio harveyi, Vibrio angillarum, Photobacterium damselae and non-pathogenic bacteria Escherichia coli, Bacillus subtilis, Shewanella putrefaciens) were identified and measured in the skin mucus of five marine teleosts: gilthead seabream (Sparus aurata), European sea bass (Dicentrarchus labrax), shi drum (Umbrina cirrosa), common dentex (Dentex dentex) and dusky grouper (Epinephelus marginatus). First, lectin binding results suggests that skin mucus contain, in order of abundance, N-acetylneuraminic acid, glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, galactose an...