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Background Oral immunotherapy is a frequent treatment for the management of food allergies, but adverse events (AE) are common. This study assessed the outcome of cow's milk oral immunotherapy (MOIT) in severe cow`s milk-allergic patients treated with omalizumab in a real-life setting. Methods OmaBASE was a national, multicenter, open, and observational registry that collected clinical, immunologic, and treatment from patients with food allergy receiving omalizumab. Results Data derived from 58 patients aged 10.3 years (IQR 6.3-13.2) and median milk-specific IgE 100 kU(A)/L at the start of omalizumab treatment. Most had experienced anaphylaxis by accidental exposures (70.7%) and had asthma (81.0%). Omalizumab in monotherapy induced tolerance to >= 6000 mg of cow's milk protein (CMP) to 34.8% of patients tested by oral food challenge. Omalizumab combined with MOIT conferred desensitization to >= 6000 mg of CMP to 83.0% of patients. Omalizumab withdrawal triggered more AE (P = .013) and anaphylaxis (P = .001) than no discontinuation. Anaphylaxis was observed in 36.4% of patients who discontinued omalizumab, and more in those with sudden (50.0%) rather than progressive (12.5%) discontinuation. At database closure, 40.5% of patients who had completed follow-up tolerated CMP without omalizumab (7.2% 1500-4500 mg; 33.3% >= 6000 mg). Conclusion Milk oral immunotherapy initiated under omalizumab allows the desensitization of subjects with severe cow's milk allergy even after omalizumab discontinuation. However, discontinuation of omalizumab can lead to severe AE and should be carefully monitored.

Background: The role of estrogens in immune functioning is relatively well known under both physiological and pathological conditions. Neutrophils are the most abundant circulating leukocytes in humans, and their abundance and function are regulated by estrogens, since they express estrogen receptors (ERs). Traditionally, estrogens were thought to act via classical nuclear ERs, namely ERα and ERβ. However, it was observed that some estrogens induced biological effects only minutes after their application. This rapid, “nongenomic” effect of estrogens is mediated by a membrane-anchored receptor called G protein-coupled estrogen receptor 1 (GPER1). Nevertheless, the expression and role of GPER1 in the immune system has not been exhaustively studied, and its relevance in neutrophil functions remains unknown. Methods: Human neutrophils were incubated in vitro with 10-100 µM of the GPER1-specific agonist G1 alone or in combination with lipopolysaccharide. GPER1 expression and subcellular localization, respiratory burst, life span, gene expression profile, and cell signaling pathways involved were then analyzed in stimulated neutrophils. Results: Human neutrophils express a functional GPER1 which regulates their functions through cAMP/protein kinase A/cAMP response element-binding protein, p38 mitogen-activated protein kinase, and extracellular regulated MAPK signaling pathways. Thus, GPER1 activation in vitro increases the respiratory burst of neutrophils, extends their life span, and drastically alters their gene expression profile. Conclusions: Our results demonstrate that GPER1 activation promotes the polarization of human neutrophils towards a proinflammatory phenotype and point to GPER1 as a potential therapeutic target in immune diseases where neutrophils play a key role.

Skin mucus is increasingly used as a source for determining immunity-related proteins and enzymes. However, the ability to accurately measure some activities may be modified by inadequate handling and storage of the samples. This study aims to measure the effect of freezing and lyophilization at the time of collection on such activities. Fresh, frozen (immediately after collection at -20 °C and -80 °C) and lyophilized skin mucus samples obtained from the same groups of fish specimens of gilthead seabream (Sparus aurata L.) were analysed in the assays. The amount of total proteins and sugar residues (determined by lectin binding) present in skin mucus samples fell after both freezing and lyophilization of the samples. While no significant differences were exhibited in the levels of some proteins or enzymes (immunoglobulin M, antiprotease, peroxidase, esterase and alkaline phosphatase) determined in fresh or frozen mucus samples, protease and lysozyme activities were lower in frozen mucus samples than in fresh samples. Lyophilization of the mucus samples drastically decreased the total level of proteins obtained, as well as of protease, peroxidase, lysozyme and alkaline phosphatase activities. The results suggest that freezing skin mucus samples is more suitable than lyophilization if samples are stored before determining enzymatic activities.

The in vitro use of fish erythrocytes to test the toxicity of aquatic pollutants could be a valuable alternative to fish bioassays but has received little attention. In this study, erythrocytes from marine gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) specimens were exposed for 24 h to Cd, Hg, Pb and As and the resulting cytotoxicity was evaluated. Exposure to metals produced a dose-dependent reduction in the viability, and mercury showed the highest toxicity followed by MeHg, Cd, As and Pb. Moreover, fish erythrocytes incubated with each one of the metals exhibited alteration in gene expression profile of metallothionein, superoxide dismutase, catalase, peroxiredoxin, glutathione reductase, heat shock proteins 70 and 90, Bcl2-associated X protein and calpain1 indicating cellular protection, stress and apoptosis death as well as oxidative stress. This study points to the benefits for evaluating the toxicological mechanisms of marine pollution using fish erythrocytes in vitro.

There is increasing concern about the possible effect of pharmaceutical compounds may have on the fish immune system. Bath exposition of 17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives, altered the immune response of the gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. Tamoxifen (Tmx) is a selective estrogen-receptor modulator used in hormone replacement therapy, the effects of which are unknown in fish immunity. This study aims to investigate the effects of dietary administration of EE2 (5 μg/g food) and Tmx (100 μg/g food) on the immune response of gilthead seabream, and the capacity of the immune system to recover its functionality after a recovery period. The results show for the first time the reversibility of the effect of EE2 and Tmx on the fish immune response. Tmx promoted a transient alteration in hepatic vitellogenin gene expression of a different magnitude to that produced by EE2. Both, EE2 and Tmx inhibited the induction of interleukin-1β gene expression while reversed the inhibition of ROI production in leukocytes following vaccination. However, none of these effects were observed after ceasing EE2 and Tmx exposure. EE2 and Tmx stimulated the antibody response of vaccinated fish although Tmx, but not EE2, altered the antibody response and modulated the percentage of IgM(+) B lymphocytes of vaccinated fish during the recovery phase. Taken together, our results suggest that EE2 and Tmx might alter the capacity of fish to appropriately respond to infection and show that Tmx has a long-lasting effect on humoral adaptive immunity.

Heavy metals provoke toxicological effects on aquatic animal species, including fish, though their effects on fish leucocytes and immunotoxicology are still limited. In the present work the effects of heavy metals (Cd, Hg, Pb or As) on viability, oxidative stress and innate immune parameters of isolated head-kidney leucocytes from gilthead seabream (Sparus aurata) are studied. Cytotoxicity results indicated that short exposures (30 min or 2h) to Hg promoted both apoptosis and necrosis cell death of leucocytes whilst Cd, Pb and As did only by apoptosis, in all cases in a concentration- and time-dependent manner. In addition, production of free oxygen radicals was induced by Cd, Hg and As heavy metals. Cd failed to change phagocytosis but Hg and As increased the percentage of phagocytic cells but decreased the number of ingested particles per cell whilst Pb increased both phagocytic parameters. On the other hand, respiratory burst activity was significantly reduced by incubation with Cd, Hg and As but increased with Pb. Furthermore, the gene expression profiles partly support the functional finding of this work. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

The knowledge about the direct effects of heavy metals on fish leucocytes is still limited. We investigate the in vitro effects of heavy metals (Cd, Hg, Pb or As) on oxidative stress, viability and innate immune parameters of head-kidney leucocytes (HKLs) from European sea bass (Dicentrarchus labrax). Production of free oxygen radicals was induced by Cd, Hg and As, mainly after 30 min of exposure. Cd and Hg promoted both apoptosis and necrosis cell death while Pb and As did only apoptosis, in all cases in a concentration-dependent manner. Moreover, expression of genes related to oxidative stress and apoptosis was significantly induced by Hg and Pb but down-regulated by As. In addition, the expression of the metallothionein A gene was up-regulated by Cd and Pb exposure though this transcript, as well as the heat shock protein 70, was down-regulated by Hg. Cd, methylmercury (MeHg) and As reduced the phagocytic ability, whereas Hg and Pb increased it. Interestingly, all the heavy metals decreased the phagocytic capacity (the number of ingested particles per cell). Leucocyte respiratory burst changed depending on the metal exposure, usually in a time- and dose-manner. Interestingly, the expression of immune-related genes was slightly affected by Cd, MeHg, As or Pb being Hg the form producing the greatest alterations, which included down-regulation of immunoglobulin M and hepcidin, as well as the up-regulation of interleukin-1 beta mRNA levels. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

Although aluminum salts (Alum) have been extensively used in human vaccination for decades, its mechanism of action is controversial. In fish, the use of Alum as a vaccine adjuvant is scarce and there are no studies aimed at identifying its mechanism of action. In the present study we report that Alum is a powerful adjuvant in the gilthead seabream (Sparus aurata L., Sparidae) and the European seabass (Dicentrarchus labrax L. Moronidae). Thus, Alum increased the specific antibody titers to the model antigen keyhole limpet hemocyanin as the commonly used Freund's adjuvant did in both species. In addition, both adjuvants were able to increase the transcript levels of the gene encoding the major pro-inflammatory mediator interleukin-1β (Il1b). Strikingly, however, Alum failed to promote Il1b release by seabream leukocytes and even impaired Il1b induction, processing and release in macrophages. However, it increased NADPH oxidase-dependent reactive oxygen species (ROS) production in gilthead seabream leukocytes and purified granulocytes. In addition, Alum promoted gilthead seabream leukocyte death independently of ROS production and caspases, suggesting that damage-associated molecular patterns release from dying cells mediate Alum adjuvant activity. Our results pave the way for future studies aimed at investigating the relevance of danger signals generated by Alum in vivo on its adjuvant activity in order to increase our understanding of the mechanisms of action of Alum in fish vaccines and to help in the design of new adjuvants for aquaculture.

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the central nervous system, is considered one of the most serious viral diseases in marine aquaculture. Although some studies have localized NNV in the retina cells, none has dealt with immunity in the retina. Thus, for the first time, we intravitreally infected healthy specimens of European sea bass (Dicentrarchus labrax) with NNV with the aim of characterizing the immune response in the retina. Ultrastructural analysis detected important retinal injuries and structure degradation, including pycnosis, hydropic degeneration and vacuolization in some cell layers as well as myelin sheaths in the optic nerve fibres. Immunohistochemistry demonstrated that NNV re- plicated in the eyes. Regarding retinal immunity, NNV infection elicited the transcription of genes encoding proteins involved in the interferon (IFN) and cell-mediated cytotoxicity (CMC) responses as well as B and T cell markers, demonstrating that viral replication influences innate and adaptive responses. Further studies are needed to understand the retina immunity and whether the main retinal function, vision, is affected by noda- virus., Detección de nodavirus en el mar Mediterráneo. Implicación y riesgo para la acuicultura, NODAMED, SI

The potential benefits of probiotics when administering to fish could improve aquaculture production. The objective of this study was to examine the modulation of immune status and gut microbiota of gilthead seabream ( Sparus aurata L.) specimens by a probiotic when administered encapsulated. Commercial diet was enriched with Shewanella putrefaciens Pdp11 (SpPdp11, at a concentration of 10 8 cfu g −1 ) before being encapsulated in calcium alginate beads. Fish were fed non-supplemented (control) or supplemented diet for 4 weeks. After 1, 2 and 4 weeks the main humoral and cellular immune parameters were determined. Furthermore, gene expression profile of five immune relevant genes ( il1β , bd , mhcIIα , ighm and tcrβ ) was studied by qPCR in head kidney. On the other hand, intestinal microbiota of fish was analysed at 7 and 30 days by DGGE. Results demonstrated that administration of alginate encapsulated SpPdp11 has immunostimulant properties on humoral parameters (IgM level and serum peroxidase activity). Although no immunostimulant effects were detected on leucocyte activities, significant increases were detected in the level of mRNA of head-kidney leucocytes for mhcIIα and tcrβ after 4 weeks of feeding the encapsulated-probiotic diet. The administration of SpPdp11 encapsulated in alginate beads produced important changes in the DGGE patterns corresponding to the intestinal microbiota. Predominant bands related to lactic acid bacteria, such as Lactococcus and Lactobacillus strains, were sequenced from the DGGE patterns of fish fed the probiotic diet, whereas they were not sequenced from fish receiving the control diet. The convenience or not of probiotic encapsulation is discussed.

Summary Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNFα activates the Notch and NF-κB signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNFα, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system.

A key goal of a successful vaccine formulation is the strong induction of persistent protective immune responses without producing side-effects. Adjuvants have been proved to be successful in several species at inducing increased immune responses against poorly immunogenic antigens. Fish are not the exception and promising results of adjuvanted vaccine formulations in many species are needed. In this study, over a period of 300 days, we characterized the apparent damage and immune response in gilthead seabream immunized by intraperitoneal injection with the model antigen keyhole limpet hemocyanin (KLH) alone or formulated with Montanide ISA water-in-oil (761 or 763), or Imject™ aluminum hydroxide (aluminium), as adjuvants. Throughout the trial, external tissue damage was examined visually, but no change was observed. Internally, severe adhesions, increased fat tissue, and hepatomegaly were recorded, but, without impairing animal health. At 120 days post priming (dpp), histopathological evaluations of head-kidney, spleen and liver revealed the presence of altered melanomacrophage centers (MMC) in HK and spleen, but not in liver. Surprisingly, in all aluminium treated fish, classical stains unmasked a toxic effect on splenic-MMC, unequivocally characterized by a strong cell depletion. Furthermore, at 170 dpp transmission electron microscopy confirmed this data. Paradoxically, at the same time powerful immune responses were recorded in most vaccinated groups, including the aluminium treatment. Whatever the case, despite the observed adhesions and MMC depletion, fish physiology was not affected, and most side-effects were resolved after 300 dpp. Therefore, our data support adjuvant inclusion, but strongly suggest that use of aluminium must be further explored in detail before it might benefit the rational design of new vaccination strategies in aquaculture.

Natural antibodies play crucial roles in pathogen elimination, B-cell survival and homeostasis, and inflammatory and autoimmune diseases. Although estrogens are able to regulate both innate and adaptive immune responses, their role in the production of natural antibodies is unknown. Here, we show that the dietary intake of the synthetic estradiol analog, 17α-ethinylestradiol (EE2), one of the most potent pharmaceutical estrogens and intensively used in human therapeutics as a component of most oral contraceptives, regulates the abundance and proliferation of T and IgM+ B lymphocytes in the teleost fish gilthead seabream (Sparus aurata L.). Furthermore, for the first time in vertebrates, it is shown that estrogen signaling through G protein-coupled estrogen receptor 1 (GPER1) induces the production of polyreactive natural antibodies, which are able to crossreact with unrelated antigens and commensal and pathogenic bacteria. In addition, the serum from fish treated with EE2 or the GPER1 agonist G1 shows higher complement-dependent bactericidal activity than that from non-treated specimens. These results demonstrate that estrogens and GPER1 are the key regulators of natural antibody production and pathogen clearance in fish, paving the way for future studies in other vertebrate classes., SI

Histamine is stored inside hemocytes of the tunicate Styela plicata (Chordata, Tunicata, Ascidiacea), but no evidence on its role in the regulation of the immune response of this species has been reported. We examined whether histamine participated in the regulation of inflammation and host defense in S. plicata. The presence of histamine inside S. plicata hemocytes was confirmed by flow cytometry, and histamine release was detected by ELISA, after in vitro hemocyte stimulation with different PAMPs. In vitro hemocyte treatment with histamine, or specific histamine-receptor agonists, reduced their phagocytic ability. Injection of histamine into the tunic recruited hemocytes to the site of injection. Systemic injection of histamine, or the histamine-releasing agent compound 48/80, decreased the phagocytic ability of hemocytes. Histamine promoted the constriction of tunic hemolymph vessels in vivo, having a direct effect on vasoconstriction in tunic explants. These results provide for the first time clear evidence for the involvement of histamine in the regulation of inflammation and host defense in tunicates.

Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1β and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms., SI

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 and fucose residues. Second, results showed that while some immune activities were very similar in the studied fish (e.g. IgM and lysozyme activity) other such as protease, antiprotease, alkaline phosphatase, esterase and peroxidase activities varied depending on the fish species. High levels of peroxidase and protease activity were found in U. cirrosa respect to the values obtained in the other species while E. marginatus and S. aurata showed the highest levels of alkaline phosphatase and esterase activities, respectively. Moreover, skin mucus of S. aurata revealed higher bactericidal activity against pathogenic bacteria, contrarily, to what happened with non-pathogenic bacteria (E. coli, B. subtilis). Thus, study of the variations in the carbohydrate profile and immune-related components of the fish skin mucus could help to understand the fish resistance as well as the presence and distribution of pathogens and magnitude of infections, aspects that are of major importance for the aquaculture industry.

TLR22 is a fish-specific TLR that recognizes dsRNAs. In the present study, a TLR22 homologue gene from gilthead seabream (sbTLR22) was identified and characterized. The full coding sequence contained a single open-reading frame of 2895 nucleotides encoding a predicted protein of 964 amino acids in length. Its 3′-UTR was relatively long, 1380 nucleotides, and contained three AU-rich sequences frequently associated with mRNA instability. Functional studies showed that the sbTLR22 transcript had a short half-life, although the three AU-rich sequences in its 3′-UTR did not seem to be related with this fact. The sbTLR22 was highly expressed in the spleen, thymus and gills of healthy fish. After Vibrio anguillarum infection, the mRNA levels of sbTLR22 increased greatly in head kidney, blood and peritoneal exudate, but were only moderately induced in spleen and liver, suggesting the involvement of sbTLR22 in the immune response against bacterial infections. In addition, acidophilic granulocytes and macrophages, both considered professional phagocytes in seabream, displayed cell-type-specific sbTLR22 expression profiles when stimulated with different pathogen-associated molecular patterns (PAMPs). Although acidophilic granulocytes expressed sbTLR22, polyinosinic:polycytidylic acid (poly I:C) was unable to up-regulate the expression of this receptor. In contrast, poly I:C induced the expression of sbTLR22 in macrophages, in a process that was partially endosome-dependent. Taken together, our results suggest that sbTLR22 is involved in bacterial infection and might sense bacterial PAMPs.

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered.

Neutrophils are major participants in innate host responses. It is well known that estrogens have an immune-modulatory role, and some evidence exists that neutrophil physiology can be altered by these molecules. Traditionally, estrogens act via classical nuclear estrogen receptors, but the identification of a G protein–coupled estrogen receptor (GPER), a membrane estrogen receptor that binds estradiol and other estrogens, has opened up the possibility of exploring additional estrogen-mediated effects. However, information on the importance of GPER for immunity, especially, in neutrophils is scant. In this study, we report that gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils, express GPER at both mRNA and protein levels. By using a GPER selective agonist, G1, it was found that GPER activation in vitro slightly reduced the respiratory burst of acidophilic granulocytes and drastically altered the expression profile of several genes encoding major pro- and anti-inflammatory mediators. In addition, GPER signaling in vivo modulated adaptive immunity. Finally, a cAMP analog mimicked the effects of G1 in the induction of the gene coding for PG-endoperoxide synthase 2 and in the induction of CREB phosphorylation, whereas pharmacological inhibition of protein kinase A superinduced PG-endoperoxide synthase 2. Taken together, our results demonstrate for the first time, to our knowledge, that estrogens are able to modulate vertebrate granulocyte functions through a GPER/cAMP/protein kinase A/CREB signaling pathway and could establish therapeutic targets for several immune disorders in which estrogens play a prominent role. The Journal of Immunology, 2013, 191: 4628–4639., SI

Studies in fish have demonstrated that Cd-exposure produce skeletal deformities and alterations in tissue morphology, enzyme activities, stress response, ion regulation and immune response. In the present work, gilthead seabream (Sparus aurata) specimens were exposed to waterborne Cd (5 μM CdCl2 or 1 mg L(-1)) for 2, 10 or 30 days. Organo-somatic changes, Cd accumulation, liver histology and humoral and cellular immune responses were determined. Results showed that exposure of seabream specimens to Cd induced no alterations on spleen and liver organo-somatic indexes whilst produced progressive deleterious morphological alterations in liver and exocrine pancreas that correlated with the hepatic Cd-accumulation. Regarding the immunotoxicological potential, strikingly, Cd-exposure produced a reduction in the serum complement activity and leucocyte respiratory burst to a significant extent after 10 and 30 days whilst the serum peroxidase activity and leucocyte phagocytosis were increased at different sampling times. On the other hand, serum IgM levels and leucocyte peroxidase activity resulted unaltered. The present results seem to indicate that seabream exposed to Cd in the present conditions suffer toxicity.

Vaccines for fish need to be improved for the aquaculture sector, with DNA vaccines and the oral administration route providing the most promising improvements. In this study, we have created an oral chitosan-encapsulated DNA vaccine (CP-pNNV) for the nodavirus (NNV) in order to protect the very susceptible European sea bass (Dicentrarchus labrax). Our data show that the oral CP-pNNV vaccine failed to induce serum circulating or neutralizing specific antibodies (immunoglobulin M) or to up-regulate their gene expression in the posterior gut. However, the vaccine up-regulated the expression of genes related to the cell-mediated cytotoxicity (CMC; tcrb and cd8a) and the interferon pathway (IFN; ifn, mx and ifng). In addition, 3 months after vaccination, challenged fish showed a retarded onset of fish death and lower cumulative mortality with a relative survival of 45%. Thus,we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and up-regulates the transcription of genes related to CMC and IFN. However, further studies are needed to improve the anti- NNV vaccine and to understand its mechanisms., SI

Combined or individual effects of two microalgae (Phaeodactylum tricornutum and Tetraselmis chuii) and Bacillus subtilis on immune response, gene expression, and survival to challenge with Photobacterium damselae subsp. piscicida of gilthead sea bream were investigated. To test the capacity of B. subtilis to grow employing the microalgae polysaccharides as energy and carbon source, an in vitro assay was defined, and demonstrated that the digestion product of microalgae, mainly P. tricornutum, supported the growth of B. subtilis much better than glucose. For the in vivo study, fish were distributed in six equal groups (each of two replicates) and received one of the following experimental diets: C) control, non-supplemented diet; T) T. chuii 100 g kg(-1); P) P. tricornutum 100 g kg(-1); B) B. subtilis (10(7) cfu g(-1)); BT) B. subtilis (10(7) cfu g(-1))+T. chuii (100 g kg(-1)); and BP) B. subtilis (10(7) cfu g(-1))+P. tricornutum (100 g kg(-1)). The complement activity, serum IgM level, respiratory burst, phagocytic activity, and expression of seven selected immune-related genes in head-kidney were evaluated following two and four weeks of treatment. At the end of the feeding trial, fish were challenged by intraperitoneal injection of LD(50) concentration of P. damselae subsp. piscicida and mortality was recorded. This is the first study testing the immunomodulatory capacity of the microalgae used in the present work. The dietary applications of B. subtilis, T. chuii, and P. tricornutum, singly or in combination, may exhibit up-regulating effects on gilthead sea bream immune parameters. P. tricornutum demonstrated the highest immunostimulant activity. There were no significant differences between combination feeding and feeding ingredients separately. Our results demonstrated the potential of microalgae as immunostimulants for fish, although further studies regarding the implications and effects of a stimulated immune system against pathogens, especially the protective capacity against specific diseases, are necessary.

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabream's immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.

The oxytetracycline (OTC) is an antibiotic widely used in the aquaculture, principally in the treatment of infections caused by the pathogenic Gram-negative bacteria. In the present study, the consequences of the oral administration of OTC on the main humoral (natural haemolytic complement activity, IgM levels and seric peroxidase level) and cellular (respiratory burst, intracellular peroxidase level and phagocytosis) immune parameters of gilthead seabream ( Sparus aurata , L) were evaluated after 7, 14 and 21 days of treatment. Furthermore, the expression levels of immune-associated genes (MHC-IIα, C3, IL-1β, COX-2 and β-defensin) were analysed by real-time polymerase chain reaction in head-kidney and gut. Three experimental groups were established: control group (fed with non-supplemented OTC food) and experimental groups (4 and 8 mg OTC g − 1 feed, respectively) ( N = 30). The results reveal a decrease of the natural haemolytic complement activity to the higher dose of OTC administered. By contrast, after 7 days of treatment, fish fed the lowest concentration of OTC were observed to have a statistically significant increase in the percentage of phagocytic cells, while the phagocytic capacity and respiratory burst increased after 14 days of treatment for both concentrations of OTC. No significant effects were observed after 21 days of treatment for both concentrations of OTC with regard to the values obtained in the control group. In head-kidney and gut, the dietary administration of OTC not caused statistically significant variation, only in C3 gene expression at 7 days in gut. The present results suggest that after dietary administration of OTC, the humoral and cellular innate immune parameters of gilthead seabream are depressed and stimulated respectively, and induce up- or down-regulation of the immune-relevant genes analysed depending on the concentration of OTC supplied in diet.

In the present study, a feeding trial was conducted to evaluate the effect of inulin and heat-inactivated Bacillus subtilis, single or combined, on several innate immune activities of gilthead seabream (Sparus aurata). Forty-eight specimens were randomly assigned to four dietary treatments: 0 (control), inulin (10 g/kg, prebiotic group), B. subtilis (107 cfu/g, probiotic group), or B. subtilis + inulin (107 cfu/g + 10 g/kg, synbiotic group). After two and four weeks, six fish of each group were sampled, with the main innate immune parameters (natural haemolytic complement activity, serum and leucocyte peroxidase, phagocytosis, respiratory burst, and cytotoxic activities) being determined. Inulin or heat-inactivated B. subtilis failed to significantly stimulate the innate immune parameters assayed, although some activities showed no significant increase through these treatments. A combination of inulin and B. subtilis resulted in an increase of such parameters, with the haemolytic complement activity being the only one significantly stimulated. To conclude, inulin and B. subtilis, when administered as a synbiotic, have a synergistic effect and enhance some innate immune parameters of gilthead seabream.

Phagocytosis is the process by which cells engulf some solid particles to form internal vesicles known as phagosomes. Phagocytosis is in fact a specific form of endocytosis involving the vesicular interiorization of particles. Phagocytosis is essentially a defensive reaction against infection and invasion of the body by foreign substances and, in the immune system, phagocytosis is a major mechanism used to remove pathogens and/or cell debris. For these reasons, phagocytosis in vertebrates has been recognized as a critical component of the innate and adaptive immune responses to pathogens. Furthermore, more recent studies have revealed that phagocytosis is also crucial for tissue homeostasis and remodeling. Professional phagocytes in teleosts are monocyte/macrophages, granulocytes and dendritic cells. Nevertheless, in recent years phagocytic properties have also been attributed to teleost lymphocytes and thrombocytes. The possible implications of such cells on this important biological process, new factors affecting phagocytosis, evasion of phagocytosis or new forms of phagocytosis will be considered and discussed., SI

Interferons (IFNs) and their receptors exist in all classes of vertebrates, where they represent early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish and type I IFN has recently been classified into two groups based on their primary protein sequences and biological activities. Thus, although groups I and II zebrafish IFN show powerful antiviral activities, only group I (IFNphi1) is able to protect the fish against bacterial infection. In addition, group II IFNs (IFNphi2 and IFNphi3) induce a rapid and transient expression of antiviral genes, while group I IFN exerts a slow but more powerful induction of several antiviral and pro-inflammatory genes. To gain further insight into the IFN system of fish, we have developed a waterborne infection model of zebrafish larvae with the spring viremia of carp virus (SVCV). Larvae were challenged 3 days post-fertilization by immersion, which considerably reduces the manipulation of fish and represents a more natural route of infection. Using this infection model, we unexpectedly found an inability on the part of zebrafish larvae to mount a protecting antiviral response to waterborne SVCV. Nevertheless, zebrafish larvae showed a functional antiviral system since ectopic expression of the cDNA of both groups I and II IFN was able to protect them against SVCV via the induction of IFN-stimulated genes (ISGs). Interestingly, group II IFNs also induced group I IFN, suggesting crosstalk between these two kinds of antiviral IFN. These results further confirm the antiviral activities of type I IFN in the zebrafish and provide the first viral infection model for zebrafish larvae using a natural route of infection. This model, in combination with the powerful gene overexpression and morpholino-mediated knockdown techniques, will help to illuminate the IFN system of teleost fish.

The IFNs and their receptors have existed in early chordates for ∼500 million years and represent the early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish, and type I has recently been classified into two groups based on their primary protein sequences. However, the biological activities of fish IFNs and their roles in infection are largely unknown. Using the zebrafish and manageable bacterial (Streptococcus iniae) and viral (spring viremia of carp virus) infection models, we are reporting in this study that zebrafish IFN (zfIFN) γ failed to induce antiviral and proinflammatory genes when administered in vivo, which correlates with its inability to protect the fish against bacterial and viral infections. We also found that, although both group I (i.e., zfIFN1) and group II zfIFNs (i.e., zfIFN2 and zfIFN3) displayed strong in vivo antiviral activities, only group I zfIFN was able to protect the fish against bacterial infection, which may reflect the different patterns and kinetics of immune-related genes elicited by these two groups of IFNs. Thus, group II zfIFNs induced a rapid and transient expression of antiviral genes, whereas group I zfIFN exerted a slow but more powerful induction of several antiviral and proinflammatory genes. Collectively, our results suggest nonredundant, complementary roles of type I zfIFNs in viral infections and provide evidence for a pivotal role of the recently identified group II IFN of fish in the early stages of viral infections.

Melatonin, a molecule produced in the pineal gland and retina of vertebrates, plays a major role in day-night circadian rhythms and other physiological processes, including the immune responses. Because little is known in this respect in fish, we have evaluated the in vivo role of melatonin in the main innate immune activities and the expression of immune-relevant genes in a teleost fish, the gilthead seabream (Sparus aurata L.). An intraperitoneal injection of 1 or 10 mg melatonin/kg of body weight produced a dose-time dependent increase in circulating melatonin serum levels. Several innate immune responses such as peroxidase, phagocytic, reactive oxygen intermediates and cell-mediated cytotoxic activities were significantly enhanced by the administration of melatonin at different sampling times. The expression of immune-relevant genes such as interleukin-1beta, major histocompatibility complex, virus-related response (interferon-regulatory factor-1 and Mx) and lymphocyte markers (immunoglobulin M and T-cell receptor for B and T lymphocytes, respectively), as analysed by semi-quantitative reverse transcription-polymerase chain reaction, was up-regulated in the head-kidney of melatonin-injected fish 1 and 3 days postinjection and decreased after 7 days. These data, together with our previous observations describing how photoperiod and in vitro melatonin treatment may affect seabream and sea bass immunology, confirm melatonin as a regulator of fish immunology. However, further studies are still needed to reveal the mechanisms underlying the direct or indirect interactions of melatonin with the fish immune system.

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the two first reproductive cycles (RCs), in which the specimens develops as males. During the first RC (RC1), 11-ketotestosterone (11-KT) and testosterone (T), the main androgens in fish, play different and specific roles in the testicular physiology as they peak at different stages of RC1. Moreover, the profiles of T serum levels during the second RC (RC2) demonstrated that this androgen is not essential in the testicular regression process that occurs during this cycle. However, changes in serum levels suggest that 17beta-estradiol (E2) orchestrates this process during both RCs. Moreover, the E2 serum levels recorded during RC2 indicate that there is a threshold level that determines the initiation of ovarian development during this cycle without promoting complete feminization. We analysed triglyceride, protein, glucose and lactate serum levels in order to establish a relationship between the mobilization and transfer of nutrients, the hormonal changes that take place during the RC and body composition, finding that in vivo serum levels of metabolites change significantly throughout the first two RCs, although the physiological relevance of such changes is still unknown. Triglyceride levels seem to be affected by the beginning of ovary development during RC2 but not by testicular recrudescence during RC1. Moreover, glucose and lactate might be important sources of energy during the resting and testicular involution stages.

Rewrite theories and their associated Kripke structures constitute a flexible and executable framework in which a wide range of systems can be studied. We present a general notion of simulation between Kripke structures, study its categorical aspects, and propose rewriting logic as a framework in which these simulations can be represented. Several representability results showing that rewriting logic is indeed a suitable framework for this purpose are given, and we illustrate its use with two examples.

Summary Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr + ) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr + cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Control of intestinal homeostasis, inflammation suppression and a reduction in the incidence of cancer all rely on the antiproliferative potential of probiotics. In this paper, we assess the antiproliferative activity of probiotics in two teleost fish cell lines SAF-1, a fibroblast cell line and EPC, an epithelioma from carp. Cells were grown in the presence of cytoplasmic extracts obtained from two bacterial strains, Lactobacillus delbrueckii subsp. lactis (LDL) and 51M6. Proliferation and apoptosis were measured after 4, 24, 48 or 72 h in culture by the crystal violet or by double staining flow cytometry assays, respectively. Generally, LDL had stronger effects on cell growth than 51M6. Moreover, SAF-1 cells were more susceptible to growth inhibition than EPC cells. Apoptosis took place following growth inhibition, especially when LDL extracts were used. The results are discussed in terms of the biological significance of probiotic bacteria that naturally occur on the fish mucosal surfaces with an emphasis on how dose and species specificity may be determinant factors.

Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens. The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, fish, and insects. In this study, we report that the AMPs called piscidins were primarily present in the mast cells (MCs) of fish and were only identified in fish belonging to the Order Perciformes. It is striking that histamine was seen to have a similar evolutionary history, since the only piscine MCs endowed with this molecule are in the Perciformes. We also show that both MCs and professional phagocytic granulocytes were armed with different piscidin molecules. In contrast, macrophages were devoid of these AMPs. More importantly, we found by immunoelectron microscopy that piscidins were delivered to the bacteria-containing phagosome of granulocytes upon phagocytosis, suggesting a role for these AMPs in the killing of both extracellular and intracellular pathogenic bacteria.

Mast cells are important as initiators and effectors of innate immunity and regulate the adaptive immune responses. They have been described in all classes of vertebrates and seem to be morphologically and functionally similar. However, early studies had shown that fish and amphibian mast cells were devoid of histamine. In this study, we take a fresh look at the evolution of histamine and find that the mast cells of fish belonging to the Perciformes order, the largest and most evolutionarily advanced order of teleosts, are armed with histamine. More importantly, histamine is biologically active in these fish where it is able to regulate the inflammatory response by acting on professional phagocytes. In addition, the actions of histamine in these immune cells seem to be mediated through the engagement of H1and H2receptors, which, together with the H3receptor, are well conserved in bony fish. We propose that the storage of histamine in vertebrate mast cells and its use as an inflammatory messenger was established in primitive reptiles (Lepidosauria) ≈276 million years ago. This same feature seems to have developed independently in Perciform fish much more recently in the Lower Eocene, between 55 and 45 million years ago, a short period during which the great majority of Percomorph families appeared.

The humoral innate immune response of sharpsnout seabream Diplodus puntazzo against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by cohabitation and a group of uninfected fish served as control. At 5, 12, 19, 26, 40 and 55 days post-exposure (p.e.), control and recipient fish were sampled to determine the prevalence of infection and some humoral innate immune parameters (antiprotease, antitumoral and peroxidase activities). Prevalence of infection was high from day 12 p.e. and reached 100% at days 40 and 55, when intensity of infection was medium to severe. The antiprotease activity was significantly increased in E. leei-exposed fish with respect to control fish at days 12 and 19 p.e. The serum antitumoral activity was slightly lower in recipient than in control fish at all sampling times, except at 40 days p.e., though no statistically significant differences were observed. Serum peroxidases were higher in all recipient fish than in control ones, with the highest stimulation index at 40 days p.e. Within recipient fish, no differences were detected between sampling times in any of the measured activities. The possible implication of these immune factors in the high susceptibility of D. puntazzo to this enteromyxosis is discussed., This work has been funded by the EU Project MyxFishControl (QLRT-2001-00722). A. Cuesta is recipient of a fellowship from Fundación CajaMurcia.

Interleukin-1beta (IL-1beta) is the prototypic pro-inflammatory cytokine. All the biological effects of IL-1beta are mediated through interaction with type 1 IL-1 receptor (IL-1RI), whereas another receptor, called type 2 IL-1R (IL-1RII), lacks an intracellular signalling domain and acts as a decoy receptor that down-regulates responses to IL-1beta. Although both receptors are present in bony fish, their expression and biological role in the regulation of IL-1beta activity in non-mammalian vertebrates remain to be established. In this study, a homologue of mammalian IL-1RII was isolated and characterized in the gilthead seabream (Sparus aurata). The seabream IL-1RII harboured two Ig-like domains in its extracellular region and a short cytoplasmic tail lacking a signalling domain. The seabream IL-1RII cDNA showed an unexpectedly long 3'UTR compared with that from other species and contained three ATTTA instability motifs, which seem to be responsible for its relatively short half-life (less than 2h). The expression of seabream IL-1RII was dramatically up-regulated after infection with Vibrio anguillarum in all the immune tissues examined and was even more strongly induced than the IL-1beta gene in the head kidney, spleen and liver. Strikingly, the mRNA levels of IL-1RII were 15-fold higher than those of IL-1beta in the liver, suggesting a role for this organ in the neutralization of IL-1beta leaking into the systemic circulation from the sites of inflammation. In vitro, bacterial DNA and flagellin increased the mRNA levels of IL-1RII in macrophages, while only flagellin was able to weakly induce its expression in acidophilic granulocytes. Finally, the seabream IL-1RII was localized in the plasma membrane when expressed in HEK293 cells and was able to bind IL-1beta.

A flow-injection pulse amperometric method for determining imipramine, based on ion transfer across a plasticized poly(vinyl chloride) (PVC) membrane, was developed. A four-electrode potentiostat with ohmic drop compensation was used, while a flow-through cell incorporated the four electrodes and the membrane, which contained tetrabutylammonium tetraphenylborate. The influence of the applied potential, the flow-injection variables and of the pH on the determination of imipramine was studied. In the selected conditions, a linear relationship between current peak height and imipramine concentration over a range of 1 × 10 −6 to 8 × 10 −5 M imipramine was found. Good repeatability and between-day reproducibility were obtained. No interference was observed on the part of some common ions and pharmaceutical excipients. The method proposed was applied satisfactorily to the determination of imipramine in pharmaceuticals and human serum.

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 affected differently in HKLs and PELs. The respiratory burst activity was not affected in HKLs but significantly increased in PELs from 4 to 48 h post-injection with yeast cells. On the other hand, HKL phagocytosis had decreased 72 h post-injection with yeast cells while it increased after 4 and 24 h post-injection in the PELs. Conversely, the cytotoxic activity was significantly enhanced in HKLs from 24 to 72 h post-injection but slightly decreased in PELs. Finally, our data demonstrate that seabream injected with the yeast Saccharomyces cerevisiae show leukocyte mobilization and cellular innate immune response activation at the site of invasion and also in the head-kidney. The implications of the leukocyte-types and the immune responses observed, as well as analogies with other particulated antigens, will be discussed as possible models for investigating the effect of potential pathogens.

Mammalian interleukin-1beta (IL-1beta) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1beta-converting enzyme (ICE) after the activation of P2X(7) receptor by extracellular ATP. The mechanism of IL-1beta release in non-mammalian vertebrates is largely unknown, although most of the IL-1beta gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X(7) receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X(7) receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X(7) receptors. We used this information to further investigate the mechanisms of IL-1beta release induced by mammalian and fish P2X(7) receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1beta remained unprocessed within the cell. However, activation of rat P2X(7) receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1beta. In contrast, neither seabream nor zebrafish P2X(7) receptors induced the secretion of mammalian or fish IL-1beta when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X(7) and the intracellular region of its rat counterpart did so. These findings indicate that P2X(7) receptor-mediated activation of ICE and release of IL-1beta result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1beta secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.

Adenosine (A) and its derivatives have important biological functions, including the inhibition of immune responses and apoptosis induction. The aim of this work was to investigate whether A and N 6 -cyclohexyladenosine (CHA) regulate apoptosis in leukocyte cultures from the teleost, gilthead seabream ( Sparus aurata L.) by flow cytometry using a double fluorescence staining method. Head kidney leukocytes (HKLs) were cultured for 0, 24 or 48 h. The kinetics of FDA + /PI − (viable), FDA − /PI − (apoptotic) and FDA − /PI + (necrotic) leukocyte subpopulations were followed during the culture process. Apoptosis was induced by addition of resveratrol or staurosporine to the culture media and the study was validated by transmission electron microscopy. A and CHA did not induce but decreased apoptosis of seabream HKLs, in particular HK lymphocytes (L), after 24 h in culture. Results raise the question whether apoptosis is differently modulated by purine nucleosides in fish L and phagocytes and also in fish compared to mammalian cells.

The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E2) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E2 modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E2 would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E2 are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6–14 wk) to high doses of E2 result in feminization of the males. Taking all this into account, we sharply increased E2 levels d...

The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

Although neuroendocrine-immune system interaction has been shown in teleost fish, no study has evaluated the role of melatonin (Mel) on fish immune response even considering that it is affected by the photoperiod. Gilthead seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) head-kidney leucocytes were incubated with Mel (0-control-, 20 pM-400 microM) and leucocyte viability and main innate cellular immune parameters were evaluated. Overall, seabream and sea bass head-kidney leucocytes incubated with low (similar to physiological) doses of Mel unchanged the innate immune response, whereas very high (pharmacological) dosages did. Phagocytosis was not affected by any Mel treatment while the peroxidase activity was significantly inhibited with the highest Mel concentration. In contrast, the sea bass respiratory burst activity was increased in a dose-dependent manner with 400 nM Mel or higher. Further studies are needed to clarify whether there are interactions between the fish pineal gland, and its hormone Mel, and the fish immune system.

Although it is believed that fish fry depend fundamentally on their innate defence mechanisms, the ontogeny of fish innate immune cells is poorly understood. In the present study, we have used a specific monoclonal antibody against acidophilic granulocytes (AGs), the main professional phagocytic cell type of the bony fish gilthead seabream, to study their localization during the development of the main lymphomyeloid organs, namely the head kidney, spleen and thymus, and of the two major portals for pathogen entry, namely the gills and intestine. AGs were observed in the posterior intestine and in the blood earlier than in the haematopoietic kidney (21 vs. 27 days post-hatching, dph). AGs were observed scattered between other cells of the haematopoietic lineage in the head kidney of larvae, but were grouped around the blood vessels of this organ in juveniles and adults, where they were also much more numerous. In the spleen and in the thymus, AGs were observed much later (62 dph) and appeared scattered. AGs were also observed in the gill lamella and the posterior intestine near the anus throughout development.

17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives and hormone replacement therapy, tamoxifen (Tmx), a selective estrogen-receptor modulator used in hormone replacement therapy, and G1, a G protein-coupled estrogen receptor (GPER) selective agonist, differentially increased the hepatic vitellogenin (vtg) gene expression and altered the immune response in adult gilthead seabream (Sparus aurata L.) males. However, no information exists on the effects of these compounds on the immune response of juveniles. This study aims, for the first time, to investigate the effects of the dietary intake of EE2, Tmx or G1 on the immune response of gilthead seabream juveniles and the capacity of the immune system of the specimens to recover its functionality after ceasing exposures (recovery period). The specimens were immunized with hemocyanin in the presence of aluminium adjuvant 1 (group A) or 120 (group B) days after the treatments ceased (dpt). The results indicate that EE2 and Tmx, but not G1, differentially promoted a transient alteration in hepatic vtg gene expression. Although all three compounds did not affect the production of reactive oxygen intermediates, they inhibited the induction of interleukin-1β (il1b) gene expression after priming. Interestingly, although Tmx increased the percentage of IgM-positive cells in both head kidney and spleen during the recovery period, the antibody response of vaccinated fish varied depending on the compound used and when the immunization was administered. Taken together, our results suggest that these compounds differentially alter the capacity of fish to respond to infection during ontogeny and, more interestingly, that the adaptive immune response remained altered to an extent that depends on the compound.

Aquatic animals are continuously exposed to chemical pollutants but the effects evoked in skin surfaces, which receive the most direct contact with them, are poorly investigated. Terminal carbohydrate composition and immunological components present in skin mucus of gilthead seabream (Sparus aurata L.) specimens exposed to waterborne sublethal dosages of heavy metals [arsenic (As2O3), cadmium (CdCl2) and mercury (CH3HgCl) at 5, 5 and 0.04 μM, respectively for 2, 10 and 30 days were analysed. Moreover, the presence of a fucose binding lectin (FBL) was evaluated by western blot and the protein profiles were by SDS-PAGE and HPLC. Results showed little effects of heavy metals in the presence of several terminal carbohydrates with few increments or decrements. Most of the enzyme activities related to immune responses were increased upon heavy metal exposure in the skin mucus including bactericidal activity. Methylmercury produced the most dramatic changes increasing all the activities. Moreover, the FBL was undetected in any of the control fish skin mucus but was evident in all the heavy metal exposed fish. In addition, As and Cd produced a clear change in the protein profile as evidenced by the lack of a protein band of around 12 kDa which is absent. These protein changes were more evident with the HPLC study showing the presence of different peaks and differences in intensity. The present results could be useful for better understanding the role and their behaviour of the mucosal immunity in skin as a key component of the innate immune system against pollutants.

TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections., Author Summary Tumor necrosis factor alpha (TNFα) is one of the main pro-inflammatory cytokines produced in response to a broad type of infections [1]. Although TNFα has a crucial role in protecting the host organism from pathogens, its deregulation can promote susceptibility to pathogens by impairing pathogen clearance and, ultimately, promoting maintenance of infection and death. In addition, some viruses might utilize the host produced TNFα to their benefit. Thus, anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor certain virus replication have not been identified. Here, we have used a viral infection model in zebrafish to identify the mechanism of action by which TNFα has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa does not affect viral ability to infect host cells or to antagonize the main host antiviral pathway, namely the interferon pathway. However, Tnfa impairs viral clearance by blocking the host autophagy response, which is usually used by host cells to degrade unnecessary or dysfunctional cellular components, and that we found to be critical to eliminate intracellular viral particles. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish in aquaculture and probably to other viral infection affecting cattle industry and human.

Although several studies have demonstrated the ability of some endocrine disruptive chemicals (EDCs) to alter the physiology of zebrafish, the immune-reproductive interaction has received little attention in this species. In this study, we used a homozygous line carrying an insertion of 8 amino acids in the ligand-binding domain of the estrogen receptor 2b gene (esr2b) to further understand the role of estrogen signaling on innate immunity. Adult mutant fish showed distorted sexual ratios related with alterations in testicular morphology and supraphysiological testosterone and 17β-estradiol (E2) levels. Immunity-wise, although esr2b mutant fish showed unaltered antibacterial responses, they were unable to mount an effective antiviral response upon viral challenge. RT-qPCR analysis demonstrated that mutant fish were able to induce the genes encoding major antiviral molecules, including Ifnphi1, Ifnphi2, Infphi3, Mxb and Mxc, and the negative feedback regulator of cytokine signaling Socs1. Notably, although esr2b mutant larvae showed a similar resistance to SVCV infection to their wild type siblings, waterborne E2 increased their viral susceptibility. Similarly, the exposure of adult wild type zebrafish to E2 also resulted in increased susceptibility to SVCV infection. Finally, the administration of recombinant Ifnphi1 hardly reversed the higher viral susceptibility of esr2b mutant zebrafish, suggesting that elevated socs1 levels impair Ifn signaling. All together, these results uncover an important role for E2 and Esr signaling in the fine-tuning of sexual hormone balance and the antiviral response of vertebrates.