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Biomedical subjects

J Meseguer

Publications and source records attributed to J Meseguer.

At least 19 recordsLinked to original sources

17Beta-estradiol triggers postspawning in spermatogenically active gilthead seabream (Sparus aurata L.) males.

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 during short times by i.p. injecting E2 diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E2 had on spermatogenesis. It was observed that E2 levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E2 accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.

Animals↗

Turbot TNFalpha gene: molecular characterization and biological activity of the recombinant protein.

The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNFalpha), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNFalpha family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNFalpha cluster. Therefore, the turbot TNF here studied was identified as TNFalpha. The complete TNFalpha gene was obtained by gene walking, and, similarly to the other known fish TNFalpha genes, presented three introns and four exons. A PCR was designed to study the turbot TNFalpha expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNFalpha expression, but this response was shorter in time than that induced by bacteria. In addition, TNFalpha expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNFalpha (rTNFalpha) was obtained by IPTG induction of bacteria transformed with the pET15b-TNFalpha construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNFalpha neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNFalpha was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNFalpha.

Amino Acid Sequence↗

Gilthead seabream ( Sparus aurata L.) innate defence against the parasite Enteromyxum leei (Myxozoa).

The humoral innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei has been studied. At 10, 22, 38, 52 and 108 days of cohabitation fish were sampled to examine gut histology and to determine serum innate immune parameters and the mRNA expression of pro-inflammatory cytokines (IL-1beta and TNFalpha) in head-kidney. The parasite was successfully transmitted to 45% of the recipient fish and prevalence reached a maximum (62.5%) at the last sampling time (108 days). Recipient fish started to die after 74 days of cohabitation. In general, alternative complement activity was higher whereas the peroxidase level was lower in recipient fish than in controls. Moreover, IL-1beta mRNA expression increased while the TNFalpha gene expression decreased in recipient fish. These data demonstrate the involvement of complement activity in the defence mechanisms of the gilthead seabream against the myxosporean E. leei. Within the recipient fish group, few differences were observed in the studied immune parameters between E. leei-parasitized and non-parasitized recipient fish. Parasitological and immunological implications of E. leei infections in Mediterranean fish farms are discussed.

Animals↗

Cell-mediated cytotoxicity is the main innate immune mechanism involved in the cellular defence of gilthead seabream (Teleostei: Sparidae) against Enteromyxum leei (Myxozoa).

The cellular innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by maintaining uninfected fish (recipients) together with infected animals. A group of fish not exposed to the infection served as controls. After 10, 22, 38, 52 and 108 days, control and recipient fish were sampled and leucocyte subpopulations and cellular immune responses (leucocyte peroxidases, phagocytosis, respiratory burst and cytotoxicity) of the head-kidney leucocytes were determined. The percentage of acidophilic granulocytes was significantly lower in non-parasitized and parasitized recipient fish than in control fish after 22 days but no significant differences were seen between non-parasitized and parasitized recipient animals. The leucocyte peroxidase content, phagocytosis and respiratory burst activity were seen to have decreased significantly at different sampling times in both non-parasitized and parasitized recipient fish with respect to the controls, whereas cytotoxic activity was up to 2.3 times higher than in control fish. Within the recipient group, little difference was observed in the studied parameters between non-parasitized and parasitized fish. These data demonstrate that cytotoxic activity may have an important role in the defence of gilthead seabream against the myxosporean E. leei. Immunological implications of E. leei infections are discussed.

Animals↗

The tumor necrosis factor alpha of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner.

Information on the bioactivities of non-mammalian cytokines is scant due to the lack of the recombinant molecules and specific antibodies. We produced the mature predicted peptide of tumor necrosis factor alpha (TNF alpha) from the bony fish gilthead seabream (Sparus aurata L.) (sbTNF alpha), and its biological role was determined in vitro and in vivo. We first demonstrated by analytical size-exclusion chromatography that sbTNF alpha is an oligomeric protein but the dimer appears to predominate over the trimeric form, in contrast to mammalian TNF alpha. Intraperitoneal injection of native sbTNF alpha resulted in (i) priming of the respiratory burst of the peritoneal exudate and head-kidney (HK) leukocytes, the latter being the bone marrow equivalent in fish; (ii) rapid recruitment of phagocytic granulocytes to the injection site, and (iii) induction of granulopoiesis in the HK. Interestingly, sbTNF alpha was able to induce a strong proliferation of HK cells in vitro, whereas human TNF alpha did not. Conversely, sbTNF alpha was not cytotoxic for murine L929 fibroblasts.

Animals↗

Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.).

Immunoglobulin M (IgM) is a major component of the teleost humoral immune system. Despite the significance of IgM levels as an immune parameter, there are relatively few studies on changes induced in its total levels in serum. This study examines the effects of several immunomodulators (vitamin A, chitin, yeast cells or levamisole, which act as immunostimulants, and crowding, hypoxia or anaesthetics, which act as stressors) upon the total serum IgM levels of non-immunized gilthead seabream (Sparus aurata L.). Total serum IgM levels of fish fed with the assayed immunostimulant-supplemented diets were statistically higher than those in fish fed a non-supplemented diet, especially in the case of levamisole. On the other hand, serum IgM levels of fish subjected to different stressors were not affected by crowding, hypoxia or certain anaesthetics. However, benzocaine and a narcotic dose of 2-phenoxyethanol provoked a great reduction, while quinaldine sulphate increased IgM levels to a significant degree. These results show how the seric IgM levels can be differently affected by some immunomodulators and the important role they may play in the regulation of total circulating IgM levels in seabream. The possibility of using total serum IgM for assessing immunostimulation, disease diagnosis and stress symptoms during fish farming is discussed.

Adjuvants, Immunologic↗

Rupatadine 10 mg and ebastine 10 mg in seasonal allergic rhinitis: a comparison study.

BACKGROUND: The aim of this study is to establish the efficacy and safety of rupatadine vs ebastine and placebo in the treatment of seasonal allergic rhinitis (SAR). Rupatadine is a new second generation H(1)-antihistamine with once-daily dosing that may provide better control of symptoms than the currently used H(1)-receptor blockers because of its dual pharmacological profile (anti-PAF and anti-H(1)). METHODS: In a multicentre study, 250 patients with SAR were included in a double-blind, randomized, parallel-group and placebo-controlled study. Patients received either rupatadine 10 mg, ebastine 10 mg or placebo once daily for 2 weeks. The main efficacy outcome was based on the patient's record of severity of nasal symptoms (sneezing, nasal itching, runny nose and nasal obstruction) and nonnasal symptoms (conjunctival itching, tearing and pharyngeal itching). The daily total symptom score (DTSS) was the mean of the DSS recorded for each of the seven symptoms assessed, and the mean DTSS (mDTSS) was the mean of the DTSS values for each study day. RESULTS: Significant differences in mDTSS were detected between rupatadine and placebo (33% lower for rupatadine group; P = 0.005) after 2 weeks of treatment. The TSS for rupatadine were 22% lower than for ebastine, although the differences were not statistically significant. No serious adverse events were reported during the study period. CONCLUSIONS: Rupatadine 10 mg once daily was clearly superior to placebo in alleviating the symptoms of SAR over a 2-week period. In comparison with ebastine, rupatadine shows a trend towards a better profile as regard several secondary efficacy variables.

Adolescent↗

Immunostimulant properties of a cell wall-modified whole Saccharomyces cerevisiae strain administered by diet to seabream (Sparus aurata L.).

The possible differences in the immunostimulant effects between whole wild Saccharomyces cerevisiae and a mutant strain (fks-1) administered in the diet to gilthead seabream (Sparus aurata L.) were studied. Fish were fed diets containing 0 (control) or 10 g whole wild yeast or fks-1 strain per kilogram feed for 2, 4 or 6 weeks. After each sampling, the innate humoral (complement, lysozyme and peroxidases) and cellular (intracellular peroxidases, phagocytosis, respiratory burst activity and cytotoxicity) responses were determined. Among the humoral responses, serum peroxidases and complement activity were significantly decreased after 6 weeks of feeding with the fks-1 strain-supplemented diet, while lysozyme activity increased after 2 and 4 weeks of feeding the fks-1 strain. Of the cellular responses studied, phagocytosis was increased to a significant degree at all the assayed times but only by the fks-1 strain-supplemented diet, while respiratory burst activity (after 4 weeks) and natural cytotoxicity (after 4 and 6 weeks) increased with either yeast strain. The intracellular peroxidase content was not affected by the dietary supplementation of either yeast strain. These results are discussed with a view to assessing the possible use of wild and/or mutant yeast strains for immunostimulant purposes in aquaculture.

Adjuvants, Immunologic↗

Tumouricidal activity of gilthead seabream (Sparus aurata L.) natural cytotoxic cells: the role played in vitro and in vivo by retinol acetate.

The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was evaluated after in vitro incubation with retinol acetate as vitamin A source, and in samples taken from specimens receiving an intraperitoneal injection or a diet supplemented with this vitamin. Isolated leucocytes were incubated with 0 to 10(-10)m all-trans-retinol acetate-supplemented culture medium for 0, 6 or 24h and assayed for their tumouricidal activity which was found to increase for all the assayed concentrations and incubation times. Seabream specimens were intraperitoneally injected with 0 (control), 1.75 or 5.25 micro g retinol acetate 100 g(-1) biomass and sampled 1, 3 or 5 days post-injection. Leucocyte natural cytotoxic activity increased in a dose-dependent manner 1 and 3 days post-injection. When fish were fed a commercial diet supplemented with 0 (control), 50, 150 or 300 mg retinol acetate kg(-1) diet for 1, 2, 4 or 6 weeks, only fish which had been fed the highest supplement for 2 weeks showed any increase in head-kidney leucocyte cytotoxic activity. Serum was isolated and analysed for all-trans-retinol concentration by reverse-phase high-pressure-liquid-chromatography. The normal level was about 0.4 micro g ml(-1) serum, while treatment for 1 to 4 weeks with vitamin A increased this level. In conclusion, retinol acetate increases gilthead seabream head-kidney leucocyte cytotoxic activity both in vitro and in vivo.

Adjuvants, Immunologic↗

The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.).

High dietary doses of the antioxidant vitamins C and E were administered to gilthead seabream (Sparus aurata L.) in an attempt to reduce the stress response in specimens exposed to a multiple stress situation. Fish were fed four different diets for 6 weeks: a commercial feed containing 0.1g vitamin C and 0.1g vitamin E kg(-1) acted as control diet, while experimental diets consisted of the same feed supplemented with 3g vitamin C kg(-1), 1.2g vitamin E kg(-1) or both 3g vitamin C and 1.2g vitamin E kg(-1). After 2, 4 and 6 weeks fish were exposed to stressors typical of aquacultural practices, and serum cortisol levels, complement activity (measured by the alternative pathway), blood glucose level and respiratory burst activity of head-kidney leucocytes were evaluated. The results showed that all stress-induced increases in blood glucose concentration were lower in fish fed the vitamin C and/or E-supplemented diet than in fish fed the control diet after 2 weeks of treatment, although no other differences were found at the rest of the times. Cortisol levels increased in stressed fish and did not suffer depletion as a consequence of administering vitamins C and/or E as a supplement. The natural haemolytic complement activity was not affected by the stressors but enhanced in specimens fed vitamin-supplemented diets at week 6. The respiratory burst activity was depressed by the stressors in fish fed the control diet, although only after 6 weeks of treatment were the differences statistically significant. These results suggest that vitamins C and E are involved in the hypothalamic-sympathetic-chromaffin cell axis and also interfere in tertiary stress responses such as immunodepression, where they protect the leucocyte functions.

Administration, Oral↗

A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes.

In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes. Leucocytes were preincubated for 30min with different concentrations of sugar (alpha-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays. The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry. The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent. More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked. Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed. The results demonstrated that the phagocytosis of yeast was completely blocked by this substance. The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes.

Agglutination↗

A role for acidophilic granulocytes in the testis of the gilthead seabream (Sparus aurata L., Teleostei).

In mammals, a complex interaction between the immune and the reproductive systems has been described, in which testicular immune cells produce cytokines and growth factors which modulate gonad functions, while specific gonad cells influence the immune response in this organ. In this study we describe the presence of acidophilic granulocytes in the testis of the hermaphrodite teleost fish gilthead seabream (Sparus aurata L.) by using a specific monoclonal antibody. During the post-spawning stage of the testis, this cell type appears in the germinal compartment, accumulates interleukin (IL)-1beta and does not seem to be involved in the phagocytosis of degenerating cells. Moreover, in vitro, 11-ketotestosterone and 17beta-oestradiol, the principal fish sexual steroids, regulate the respiratory burst activity of acidophilic granulocytes obtained from the head-kidney (the bone marrow equivalent in fish) and the intracellular accumulation of IL-1beta by these cells. It is likely, therefore, that IL-1beta produced by testicular acidophilic granulocytes regulates important functions of the testis in fish.

Animals↗

Levamisole is a potent enhancer of gilthead seabream natural cytotoxic activity.

Gilthead seabream (Sparus aurata L.) head-kidney (HK) leucocytes were incubated with 10(3) to 10(-4) ng levamisole/ml for 4, 24 or 48 h and then assayed for their natural cytotoxic activity against xenogeneic tumor cells. This activity was slightly increased after 24 h of incubation. In a second experiment, fish specimens were fed 0, 75, 150 or 300 mg levamisole/kg diet for 10 consecutive days. The fish were then fed a commercial non-supplemented diet and sampled 0, 1, 2, 3, 4 and 6 weeks post-administration of levamisole. The cytotoxic activity was found increased along the experiment and remained greatly enhanced at the end. In conclusion, levamisole enhanced seabream natural cytotoxic cell activity both in vitro and in vivo and had a great and lasting action when administered by feeding.

Adjuvants, Immunologic↗

Effects of phenoxyethanol on the innate immune system of gilthead seabream (Sparus aurata L.) exposed to crowding stress.

Phenoxyethanol is routinely used in seabream aquaculture to minimise fish stress response despite the secondary negative effects which have been observed. In this study, two different doses (60 and 200 microl/l) of phenoxyethanol, sedative and narcotic, were tested for their ability to reduce the stress caused in gilthead seabream (Sparus aurata L.) by crowding. Blood glucose and serum cortisol concentrations were measured as stress indicators. In order to study the effects of the treatment on the innate immune system of crowded specimens, two parameters of the innate immune response, serum complement activity and phagocytosis, were assessed. The results show that anaesthesia itself produced a stress response in the fish and affected the immune system, although the effects were greater with the narcotic dose. When the effects of anaesthesia on crowded fish were analysed, the results pointed to a slight reduction in stress as a result of the sedative dose of phenoxyethanol (lower increase in cortisol and lower reduction in phagocytosis). However, additive negative effects were seen in crowded fish when the narcotic dose of phenoxyethanol was used. Since the use of phenoxyethanol is a common practice in aquaculture, the significance of the results should be considered.

Anesthetics↗

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response.

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.

Animals↗

Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.).

Anaesthesia may depress the immune system in mammals, but there is no available information on this topic in fish. In the present work, four anaesthetics that are used in aquaculture, MS222 (0 19 mM), benzocaine (0.21 mM), 2-phenoxyethanol (16 mM) and quinaldine sulphate (0.083 mM), were tested in order to observe their effects on the gilthead seabream (Sparus aurata L.) innate immune system. The results showed that the four anaesthetics produced increased blood glucose levels after an hour. In addition, benzocaine and 2-phenoxyethanol depressed complement activity and phagocytosis, while MS222 and quinaldine sulphate did not. Some anaesthesia is a common practice in aquaculture, the data obtained should be taken into account to avoid possible immunodepression in farmed fish.

Aminobenzoates↗

Natural cytotoxic cells of gilthead seabream: maximum percentage of lysis.

The maximum percentage of lysis of head-kidney non-specific cytotoxic cells (NCC) against mammalian tumour cells (L1210 and K562) in the marine teleost gilthead seabream (Sparus aurata L.) was studied. The present data indicate the short period of time necessary for gilthead seabream NCC to form conjugates and deliver a lethal hit. The maximum percentage of lysis observed demonstrates that seabream NCC activity against L1210 tumour cells is faster than against K562 tumour cells. This kinetic parameter suggests that fish NCC show a less efficient cytotoxic activity than their mammalian counterparts. The possibility of applying theoretical treatments to systems consisting of lower vertebrate non-specific cytotoxic cells and tumour targets, similar to those applied to mammals, is considered, and the phylogenetic implications of our findings are discussed.

Animals↗