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Biomedical subjects

José Meseguer

Publications and source records attributed to José Meseguer.

At least 19 recordsLinked to original sources
PubMed2006

The activation of gilthead seabream professional phagocytes by different PAMPs underlines the behavioural diversity of the main innate immune cells of bony fish.

Phagocytic cells form the cellular arm of the innate immune system. A primary role of these cells is an ability to discriminate large number of potential pathogens from self, using a restricted number of receptors. In the gilthead seabream, acidophilic granulocytes and macrophages have been described as the professional phagocytes of this species. However, no direct functional comparisons between these two phagocytic lineages exist for the seabream or for other teleost species. Therefore, purified fractions of acidophilic granulocytes and macrophages were used to characterize the ability of these cells to recognize and respond to different pathogen-associated molecular patterns (PAMPs) and the data obtained were then correlated with the expression of several pattern-recognition receptors (PRRs). The time course of the respiratory burst of acidophilic granulocytes stimulated with different PAMPs showed that muramyldipeptide (MDP), the ligand for NOD2 in mammals, induced maximal activation earlier than several ligands for toll-like receptors (TLRs), including bacterial DNA, flagellin and lipopolysaccharide (LPS). In addition, all these PAMPs strongly increased the phagocytic and bactericidal activities of acidophilic granulocytes, while other PAMPs, including poly I:C, Pam3CSK(4) and zymosan, failed to do so. The stimulation of acidophilic granulocytes and macrophages by PAMPs also resulted in the up-regulation of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), cyclooxygenase-2 (COX-2) and TLRs, although the kinetics and expression profiles observed for each cell type differed. These results suggest different roles for professional phagocytes of fish in the recognition and elimination of pathogens and in the regulation of adaptive immune responses.

Animals↗
PubMed2006

Characterization of ATP-gated P2X7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1 beta.

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 Triphosphate↗
PubMed2006

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses.

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.

Animals↗
PubMed2006

Early local and systemic innate immune responses in the teleost gilthead seabream after intraperitoneal injection of whole yeast cells.

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.

Animals↗
PubMed2006

Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3'-untranslated region of their mRNAs.

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.

3' Untranslated Regions↗
PubMed2006

Effect of heat-inactivated fish and non-fish derived probiotics on the innate immune parameters of a teleost fish (Sparus aurata L.).

The in vitro effects of four heat-inactivated bacterial species on the cellular innate immune responses of the gilthead seabream (Sparus aurata L.) were investigated. Head-kidney leucocytes were isolated and incubated for 30 min with two bacteria isolated from seabream skin (Pdp11 and 51M6; members of the Vibrionaceae and in the genus Shewanella) and two bacteria used as probiotics in humans and cattle (Lactobacillus delbrüeckii subsp. lactis and Bacillus subtilis) at 5 x 10(5), 5 x 10(6) or 5 x 10(7)CFU/ml. After incubation, different cellular innate immune parameters (leucocyte peroxidase content, phagocytosis, respiratory burst activity and cytotoxicity) were evaluated. The leucocyte peroxidase content was significantly higher after incubation with 51M6 at 5 x 10(7)CFU/ml. Head-kidney phagocytes were able to engulf the four bacterial species in all four cases, L. delbrüeckii subsp. lactis and B. subtilis being the most actively phagocytized. The incubation of seabream leucocytes with 51M6, L. delbrüeckii subsp. lactis and B. subtilis resulted in a great increase in respiratory burst activity. Cytotoxic activity was generally stimulated in a dose-dependent manner, the enhancement obtained with 5 x 10(7)CFU/ml being statistically significant. The usefulness of in vitro assays for screening and selecting candidate probiotic bacteria, as well as for optimising their effective dose, is discussed in relation with their immune-modulatory properties.

Bacillus subtilis↗
PubMed2006

Phenoloxidase activity in three commercial bivalve species. Changes due to natural infestation with Perkinsus atlanticus.

The phenoloxidase (PO) activity of the haemolymph and haemocytes from three clam species of commercial interest (Ruditapes philippinarum, Chamelea gallina and Tapes decussatus) has been compared. The activity was assayed spectrophotometrically by recording the formation of dopachrome from L-DOPA using sodium dodecyl sulphate, laminarin, trypsin or lipopolysaccharide as elicitors. Fewer PO units were observed in the haemolymph from T. decussatus than in the haemolymph from R. philippinarum, while the highest values were found in C. gallina. In all cases the activity was only significantly increased when sodium dodecyl sulphate was used as elicitor. PO activity in the haemocytes of all three clam species showed a very similar pattern to that found in the haemolymph from the same species. Furthermore, T. decussatus naturally parasitized by Perkinsus atlanticus (Protozoa, Apicomplexa) was used to study the influence of such infestation on PO activity, which was found to increase significantly in both haemolymph and haemocytes compared with non-infected (control) samples. PO activity in the haemocytes and in the haemolymph was higher when the level of parasitization was low or medium, respectively, and SDS was used as elicitor. No statistically significant differences were observed when the parasitization level was high. The present work constitutes the first report on the influence of this parasite on PO activity in haemolymph and haemocytes from T. decussatus.

Analysis of Variance↗
PubMed2006

Effect of photoperiod on the fish innate immune system: a link between fish pineal gland and the immune system.

The pineal gland via its secretory product, melatonin, influences the light-dark rhythm in most vertebrates including fish. Apart from the information concerning this circadian rhythm, the interrelation of the melatonin with other physiological processes has not been considered in fish. Thus, we evaluated the changes in the humoral innate immune system of seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) specimens exposed to a constant light-dark photoperiod (12 hr L:12 hr D). Serum was obtained from blood samples collected at 02:00, 08:00 hr (light-on), 14:00, 20:00 hr (light-off) and at 08:00 hr again. Among the humoral innate immune responses, complement, lysozyme and peroxidase activities were determined. Complement activity was higher during the day than during the night in both fish species. Seabream lysozyme activity reached its maximum at 20:00 and 02:00 hr but was hardly affected in sea bass. Finally, the peroxidase activity of seabream was significantly higher at 08:00 hr than during the rest of the cycle while, in sea bass, it showed little variation. The present results demonstrate that the humoral innate immune system has a circadian rhythm based on the light-dark cycle and that this cycle might be affected by the pineal gland.

Animals↗
PubMed2005

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics.

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.

Analysis of Variance↗
PubMed2005

Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells.

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 polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the alpha1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA+LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.

Amino Acid Sequence↗
PubMed2005

The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream.

We report the molecular cloning of the colony-stimulating factor-1 receptor gene from the bony fish gilthead seabream (sbCSF-1R). The deduced sbCSF-1R shows a predicted signal sequence, a transmembrane domain and a tyrosine kinase domain, all in conserved positions. A transcript showing a premature stop codon that predicted the removal of 84 C-terminal amino acids was also found. RT-PCR expression studies demonstrate that, although the sbCSF-1R transcripts are found in different immune tissues, including gill, liver, spleen, blood, peritoneal exudate, thymus and head-kidney (HK), their expression is confined to the monocyte/macrophage lineage. Furthermore, the expression of sbCSF-1R might be modulated by the activation stage of the macrophages, since both the infection of fish and the in vitro activation of leukocytes resulted in the down-regulation of gene expression. These data indicate that the CSF-1R may be used as a specific probe for cells of the monocyte/macrophage lineage in the gilthead seabream, an immunological tractable fish model. In addition, the functional characterisation of the CSF-1R and its ligand may shed light into the mechanisms of proliferation and the pathways of differentiation of macrophages in bony fish.

Amino Acid Sequence↗
PubMed2005

Professional phagocytic granulocytes of the bony fish gilthead seabream display functional adaptation to testicular microenvironment.

It has been shown previously that professional phagocytic granulocytes are present in the testis of the gilthead seabream, a seasonal breeding teleost that offers an excellent model for studying the testicular regression process that occurs in seasonal testicular involution and sex change. It is unexpected that testicular granulocytes produce interleukin-1beta, a regulator for spermatogonia proliferation in mammals, but are not involved in the elimination of degenerative germ cells. Here, we show that phagocytosis and reactive oxygen intermediate (ROI) production were suppressed dramatically in testicular phagocytic granulocytes, compared with their level of activity in the head-kidney, the main hematopoietic organ in fish. Furthermore, testicular-conditioned media modulated migration, phagocytosis, and ROI production of head-kidney phagocytic granulocytes, and the addition of testicular cells impaired their ROI production capacity. Until now, monocytes/macrophages were believed to be the only innate immune cells able to develop into functional subsets, whereas neutrophils only infiltrate the tissues upon infection or inflammation. Our findings demonstrate, however, that fish professional phagocytic granulocytes also display functional adaptation to different microenvironments and strongly suggest a role for these cells in the reorganization of the testis during post-spawning.

Adaptation, Physiological↗
PubMed2005

N6-Cyclohexyl-adenosine but not adenosine is a modulator of teleost fish innate immune activities.

The possible immuneregulatory effects of two purine nucleotides, adenosine and cyclohexyladenosine, on gilthead seabream (Sparus aurata L.) leucocytes were investigated. Leucocytes isolated from the head-kidney were incubated with five different concentrations (ranging from 0 to 100 microM) of adenosine or cyclohexyladenosine for 30, 180 and 360 min and the effect on leucocyte viability and some of the main innate cellular immune responses was evaluated. Adenosine did not significantly affect the viability or the innate immune parameters of seabream leucocytes at any of the assayed concentrations or incubation times. High concentrations of cyclohexyladenosine, on the other hand, caused a significant drop in the respiratory burst activity of head-kidney leucocytes at all the times assayed. In addition, phagocytosis of yeast cells was significantly rapidly depressed (30 min) after incubation with 100 microM of this analogue. The present results demonstrate that cyclohexyladenosine at high concentrations is capable of down-regulating certain innate immune responses in seabream leucocytes, suggesting that telesot fish immune cells, like their mammal counterparts, possess receptors for purine nucleotides.

Adenosine↗
PubMed2005

Early innate immune response and redistribution of inflammatory cells in the bony fish gilthead seabream experimentally infected with Vibrio anguillarum.

An obvious difference between the immune system of fish and mammals is that fish lack both bone marrow and lymph nodes; in their place, the head-kidney acts as a major haematopoietic and lymphoid organ in adult fish, whereas the thymus, spleen and mucosa-associated lymphoid tissues are common to both fish and mammals. This suggests that differences exist in antigen presentation and naive lymphocyte stimulation, a prerequisite for the initiation of adaptive immune responses. Intraperitoneal injection of the bony fish gilthead seabream (Sparus aurata L.) with intact Vibrio anguillarum, as a particulate bacterial antigen, results in the mobilisation of head-kidney leucocytes to the peritoneal cavity and priming of their respiratory burst activity. We have also observed the rapid infiltration of acidophilic granulocytes, which are leucocytes functionally equivalent to mammalian neutrophils, into the spleen. These cells may be involved in antigen capture and transport to the spleen, since an apparent association between mobilised acidophilic granulocytes, bacterial antigens and proliferating lymphocytes has been seen in this organ. Collectively, these results suggest that, in addition to being actively involved in bacterial clearance, fish phagocytic granulocytes play a role in the initiation and support of the adaptive immune response.

Animals↗
PubMed2005

In vivo effects of propolis, a honeybee product, on gilthead seabream innate immune responses.

The potential effect of the intraperitoneal or dietary administration of propolis, a honeybee product, on gilthead seabream (Sparus aurata L.) innate immune responses was evaluated. Fish were intraperitoneally injected with 5mg of water (WEP), ethanol (EEP) or both (WEP + EEP) extracts of propolis and sampled after 1, 3, 5 and 10 days. When administered in the diet, propolis was dissolved in ethanol and added to a pellet diet at a concentration of 0, 0.1 or 10 g kg(-1) diet, the fish being sampled after 1, 2, 4 and 6 weeks of feeding. Humoral (alternative complement activity and peroxidase content) and cellular (leucocyte peroxidase, phagocytosis, cytotoxicity and respiratory burst activity) immune responses were evaluated in both cases. The results suggest that propolis has limited immunostimulatory effects although intraperitoneal administration was more effective than dietary intake. The effects that were noted were at cellular level, namely, phagocytosis and cytotoxicity.

Administration, Oral↗
PubMed2005

Effects of lactoferrin on non-specific immune responses of gilthead seabream (Sparus auratus L.).

The main innate cellular immune responses of gilthead seabream (Sparus auratus L.) leucocytes were evaluated after in vitro incubation with human lactoferrin (Lf). Isolated head-kidney leucocytes were incubated with 0 (control) to 1 mg ml(-1) Lf-supplemented culture medium for 30, 120, 240 or 360 min and assayed for viability, peroxidase content, and respiratory burst, phagocytic and cytotoxic activities. Only respiratory burst activity was found to increase when using the highest Lf concentration (1 mg ml(-1)) and long incubation times (more than 120 min). Seabream were fed Lf-supplemented diets (0, control, 50, 100 or 200 mg kg(-1) diet). After 1 or 2 weeks of administration the leucocyte peroxidase content, respiratory burst, phagocytic and cytotoxic activities as a measure of cellular immune responses, as well as serum peroxidase and complement activity as a measure of humoral immune responses were evaluated. The results showed that Lf feeding at 100 mg kg(-1) diet for 1 week enhanced the cellular innate immune responses although only the cytotoxic activity did so significantly. The humoral immune response was not influenced by Lf feeding. In conclusion, Lf seems to affect innate immune cellular activity, mainly respiratory burst and natural cytotoxic activity. The possible use of Lf as an immunostimulant for farmed gilthead seabream is discussed.

Analysis of Variance↗