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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.

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.

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.

The effects of high doses of cortisol, biologically the most active corticosteroid in the circulating blood of teleost fish, on gilthead seabream (Sparus aurata L.) leucocytes were determined. Leucocytes isolated from the head-kidney (the principal haemopoietic organ of this fish species) were incubated with five different concentrations (ranging from 0 to 10(-1) mM) of cortisol for 30, 120, 240, 360, and 480 min and their effects on leucocyte viability and some of the main innate cellular immune responses were evaluated. The viability and the cytotoxic activity of leucocytes against tumor cells were not significantly affected by in vitro incubation with cortisol, at any of the assayed concentration or incubation times. With cortisol concentrations of 10(-9), 10(-7) or 10(-5) mM, the respiratory burst activity of head-kidney leucocytes were significantly depressed after 30 min of incubation. On the other hand, with cortisol concentrations of 10(-1) mM, the phagocytosis of yeast cells and the total peroxidase content of leucocytes were significantly depressed at incubation times longer than 240 min. To corroborate that the observed effects were due to the cortisol treatment, assays were developed using both cortisol and cycloheximide, which blocked the inhibitory effects of cortisol. The present results demonstrate that cortisol plays an important role in the down-regulation of phagocytic but not of cytotoxic activity in seabream leucocytes.

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1beta (IL-1beta) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1beta. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1beta intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1beta following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1beta were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed., (Copyright 2004 Springer-Verlag)

Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.

The immunostimulant potential of the whole fungus Mucor circinelloides administered in the diet to gilthead seabream (Sparus aurata L.) was studied. Three different lyophilised strains were used: the wild-type (R7B) and two mutant strains in the carotene-production pathway which are enriched in lycopene (MU224) or beta-carotene (T31). Fish were fed diets containing 0 (control) or 10 g of one of the whole M. circinelloides strains per kg feed. After 2, 4 or 6 weeks of treatment the growth rate as well as humoral (lysozyme activity) and cellular (phagocytosis and cytotoxicity) immune responses were determined. The specific growth rate increased slightly with all the M. circinelloides-supplemented diets. Serum lysozyme activity increased slightly (P>0.05) in fish fed the mutant strain-supplemented diets. Of the cellular responses, phagocytosis significantly increased after 6 weeks, in fish fed the wild-type strain-supplemented diets while cytotoxicity increased after 4 weeks in fish fed the beta-carotene-enriched strain-supplemented diet. The immune responses were increased to some degree by diets containing whole M. circinelloides strains. These results are discussed in the light of the on-going search for new microorganisms, wild or mutant-type, for use as immunostimulants in fish farming.

The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1beta. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification.

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.

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.

Several common situations in gilthead seabream (Sparus aurata L.) farming, such as air exposure, crowding and the use of anaesthetics, have been demonstrated to be stressful. In the present study, these conditions were simulated in the laboratory, after which head-kidney natural cytotoxic cell (NCC) activity was evaluated. For this, several specimens were air exposed for 2 min, returned to the aquarium and sampled from 0 to 4 days after exposure. NCC activity was significantly lower on the day following air-exposure compared with the control (rested fish) but not at any other time studied. Other fish were crowded (100 kg biomass m(-3), 2 h), returned to an aquarium with the same density as the control group (9 kg m(-3)) and sampled from 0 to 4 days after treatment. Head-kidney NCC activity was statistically increased compared with the control (resting) fish, 1 day after crowding. Anaesthesis for 1 h with 60 or 200 microl 2-phenoxyethanol l(-1)had no significant effect on NCC activity, while the use of 50 mg MS222 l(-1)for 1 h reduced such activity (by about 40%) compared with the control. In other experiments, fish were consecutively treated with crowding and anaesthetics. When treated with the lowest 2-phenoxyethanol concentration after crowding, the NCC activity inhibition was abolished compared with the activity in fish treated either with crowding or anaesthetic alone, while the use of the highest concentration increased such inhibition. The use of MS222 after crowding did not produce any differential effect compared with the fish treated with only one of the factors. In conclusion, NCC activity is affected differently according to the stress factor applied (hypoxia, crowding and/or anaesthetics). Differences in the effects provoked by these stressors on other seabream innate immune parameters are discussed.

The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied. Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM. Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones. Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated. Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test. Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected. As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner. Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected. To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.

A flow cytometric method was adapted to evaluate phagocytosis by gilthead seabream leucocytes after incubation with yeast cells (Saccharomyces cerevisiae). Head-kidney leucocytes were incubated in vitro for different times in different proportions with heat-killed fluorescein isothiocyanate (FITC)-labeled yeast cells to study the kinetics of phagocytosis. Attached and internalized yeast cells were differentiated by quenching FITC-labeled S. cerevisiae with trypan blue dye. Only internalized cells kept their FITC fluorescence after quenching. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity, as demonstrated by transmission electron microscopy (TEM). From the ultrastructural features of the phagocytic process, it was observed that cytoplasmic granule membranes fused with the phagocyte membrane at the point where the yeast cell was attached to the phagocyte surface. This observation led us to adapt a colorimetric method to study peroxidase (myeloperoxidase and eosinophil peroxidase) release, since both are considered to be markers of the degranulation that occurs in seabream head-kidney leucocytes in response to yeast cells. Head-kidney leucocytes were incubated with calcium ionophore (CaI), phorbol myristate acetate (PMA), or yeast cells for different periods of time (0-30 min) to study the kinetics of peroxidase release. The results obtained indicate that CaI and yeast cells, but not PMA, stimulate the degranulation (by about 44.51% and 21.04%, respectively, at 30 min) of seabream head-kidney leucocytes., (Copyright 2003 Wiley-Liss, Inc.)

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.

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.

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.

The possible expression of FasL in gilthead seabream leucocytes was studied by flow cytometry, immunoblotting and immunocytochemistry, using an anti-mouse FasL monoclonal antibody. The results pointed to a cytosolic FasL-like, but not a membrane-like form, in resting leucocytes from head-kidney, thymus, spleen, blood and peritoneal exudate. Immunoblotting revealed a 19kDa band in resting leucocytes, while activated leucocytes showed the same band and another of 39kDa. The FasL-like molecule is identified in lymphocytes, monocyte-macrophages and acidophilic granulocytes. Phylogenetical and functional implications are suggested.

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., (Copyright 2002 Elsevier Science B.V.)

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.

The effects of high doses of dietary or intraperitoneally (i.p.) injected retinol acetate on the gilthead seabream (Sparus aurata L.) innate immune system were studied. Gilthead seabream specimens were fed a commercial non-supplemented diet containing 1.75 mg of vitamin A kg(-1) (as control) or the same diet supplemented with 50, 150 or 300 mg of retinol acetate kg(-1) (as vitamin A source). After 1, 2, 4 or 6 weeks, serum samples and head-kidney leucocytes were obtained from each fish. Serum lysozyme activity and myeloperoxidase (MPO) content were unaffected by the vitamin A diet content. The phagocytic and respiratory burst activities of head-kidney leucocytes were established, as well as their myeloperoxidase content. While phagocytosis was not enhanced by dietary vitamin A intake and was even slightly decreased after 2 weeks, respiratory burst activity was enhanced in specimens fed supplements of 150 and 300 mg retinol acetate kg(-1) diet for 1 or 2 weeks. Leucocyte MPO content was also enhanced when seabream were fed the highest vitamin A dose for 2 or 4 weeks and after being fed the 150 or 50 mg supplemented diets for 4 or 6 weeks, respectively. Three different groups of seabream were i.p. injected with 1 ml of phosphate buffer containing an amount of retinol acetate equivalent to the daily dietary supplements from the first experiment (0-control-, 0.05 or 0.30 mg 100 g(-1) biomass). Both injection doses of retinol acetate were toxic for the gilthead seabream which showed hypervitaminic effects. These data show that retinol acetate plays an important role in the gilthead seabream nonspecific cellular immune system due to its antioxidant properties. They also point to the importance of the way in which it is administered, by dietary uptake or intraperitoneal injection.

Isolated gilthead seabream head-kidney leucocytes were incubated in a culture medium supplemented with vitamin C (from 0-control-to 2 mg ml(-1)) or with a combination of different amounts of vitamin C (0.002 to 2 mg ml(-1)) and vitamin E (0.01 to 10 microg ml(-1)) for 6, 24 or 48 h. Afterwards, the cellular ascorbic acid content and the natural cytotoxic activity of leucocytes were evaluated. Leucocyte ascorbic acid levels were enhanced after incubation for 6-24 h with 0-2 mg of vitamin C ml(-1) and above. The natural cytotoxic activity of leucocytes after incubation with vitamin C was also increased for all the assayed concentrations and incubation times except in the case of the highest vitamin C concentration (2 mg ml(-1)) and the longest incubation time (48 h). No statistically significant differences in leucocyte cytotoxic activity were observed when vitamin E was added to the vitamin C, compared with the results of vitamin C alone. For the in vivo study, fish were fed diets supplemented with vitamin C (2.9 g kg(-1) diet) without or with vitamin E (1.2 g of vitamin E kg(-1) diet) for 2, 4 or 6 weeks. Serum lysozyme activity was enhanced to a statistically significant degree when fish were fed with the vitamin C + E supplemented diet for 2 weeks and with the vitamin C and vitamin C + E supplemented diets for 4 weeks. Both groups of fish showed a statistically significant increase in the natural cytotoxic activity of head-kidney leucocytes after 6 weeks of treatment although no differences were observed between treatments incorporating vitamin C alone or vitamin C combined with vitamin E.

Introduction and Objectives: Analysis and comparison of the clinical performance and hemodynamics in vivo of 21 mm Carpentier-Edwards supra-annular (CESA) and Perimount (CEPM) aortic bioprostheses., Methods: A follow-up study was made of 40 patients implanted a 21 mm CESA (n = 21) or CEPM (n = 19) prosthesis between October 1992 and September 1997. All eligible survivors (14 CESA, 12 CEPM) were assessed echocardiographically., Results: There were no significant differences between models in the effective orifice area (1.6 cm2 for CESA, 1.44 cm2 for CEPM), peak flow rate (rest: 2.5 m/s for CESA, 2.3 m/s for CEPM; post-dobutamine: 3.4 m/s for CESA, 3.3 m/s for CEPM), mean flow rate (rest: 1.7 m/s for CESA, 1.6 m/s for CEPM; post-dobutamine: 2.5 m/s for CESA, 2.2 m/s for CEPM), peak gradient (rest: 28.3 mmHg for CESA, 21.6 mmHg for CEPM; post-dobutamine: 48.4 mmHg for CESA, 41.6 mmHg for CEPM), and mean gradient (rest: 15.8 mmHg for CESA, 12.0 mmHg for CEPM; post-dobutamine: 28.5 mmHg for CESA, 22.5 mmHg for CEPM)., Conclusion: In our experience, these two prosthetic models have similar hemodynamic characteristics in small aortic annuli.

Temporary epicardial pacing wires are routinely placed in patients undergoing cardiac surgery. Eighteen suitable patients undergoing elective surgery were prospectively studied. Their sensing and stimulating characteristics were studied at various locations. Subepicardial pacing leads were applied to the lateral wall of the LV, apex of the LV, anterior wall of the RV, diaphragmatic wall of the RV, and diaphragmatic wall of the LV. Impedance, R wave amplitude, slew rate, and stimulation thresholds were measured on postoperative days 1 and 5. Impedance remained unchanged in time with no significant differences between locations. R waves and slew rates were significantly lower in the anterior RV wall. Stimulation thresholds displayed no differences on day 1, but they increased significantly in all locations on day 5. These thresholds were significantly lower in the lateral and diaphragmatic LV walls on day 5, and the rate of voltage increase was also lower in these two locations. Five patients presented phrenic nerve stimulation when stimulating the lateral LV wall. The authors advocate the diaphragmatic wall of the LV as the best location for placing temporary leads. The anterior wall of the RV is not recommended for pacing purposes.

Background and Aim of the Study: Clinical experience with the Omnicarbon mechanical valve implanted in the aortic position is reported in this retrospective survey., Methods: A total of 110 patients (mean age 56 years) undergoing isolated aortic valve replacement with Omnicarbon mechanical prostheses at the authors' institution between November 1991 and October 2000 was included. Follow was available in 94.5% of patients (mean follow up 4 years per patient; total follow up was 415 patient-years (pt-yr))., Results: Hospital mortality was 3.6%. There were no instances of valve thrombosis or structural failure. The incidence of valve-related complications (linearized rates in events per 100 pt-yr/actuarial freedom at 9 years, in %) was: valve-related mortality 2.4+/-0.7 / 80.3+/-8.0; non-structural failure 0.7+/-0.4 / 93.8+/-4.2; thromboembolism 0.2+/-0.2 / 99.0+/-1.0; anticoagulant-related hemorrhage 0.9+/-0.5 / 93.7+/-3.3; prosthetic valve endocarditis 0.2+/-0.2 / 99.0+/-1.0; and reoperation 0.7+/-0.4 / 93.8+/-4.2., Conclusion: In the authors' experience the Omnicarbon mechanical prosthesis provides a good clinical performance for up to nine years in the aortic position.

Thrombocytes and platelets, beyond their primary function in hemostasis, seem to play an active role in inflammation. As regards their phagocytic ability, the results to date are confusing, incomplete, and somewhat contradictory. Whereas the interaction of avian thrombocytes or mammalian platelets with bacteria both in vitro and in vivo has received wide attention, almost no information exists on the topic in "lower" vertebrates. The aim of this work is to review the available information on the phagocytic properties of thrombocytes and platelets. Particular attention is payed to the ontogeny of these cells, the soluble factors involved in the inflammatory process derived from them, and their interaction with particulate material, mainly with bacteria., (Copyright 2002 Wiley-Liss, Inc.)

The tumor necrosis factor alpha ( TNF alpha) gene from the marine fish, gilthead seabream (Sparus aurata L.), has been isolated by RT-PCR using degenerate primers designed against vertebrate TNF alpha conserved motifs and subsequent rapid amplification of cDNA ends (RACE). The TNF alpha cDNA consists of a 142 bp 5' untranslated region (5'UTR), a single open reading frame of 762 bp, which could code for a 253 amino acid protein, and a 476-bp 3'UTR. The protein sequence deduced from seabream TNF alpha gene shows a high degree of homology with the Japanese flounder TNF alpha (65.6% identity and 78.9% similarity) and, more important, it is more homologous to mammalian TNF alphas (41.1-48.6% similarity) than to TNF betas (36.0-43.5% similarity). The prediction of a transmembrane domain between residues 37 and 54 of seabream TNF alpha and the presence of a conserved Thr-Leu sequence, which is associated with cleavage of the mouse TNF alpha molecule, suggest that seabream TNF alpha exists in two forms, a membrane-bound and a soluble form. RT-PCR shows that the seabream TNF alpha messenger was widely and constitutively accumulated. Lastly, stimuli known to up-regulate seabream IL-1 beta, lipopolysaccharide and lymphocyte-derived macrophage-activating factor, failed to up-regulate TNF alpha in cultured macrophages. The putative role of three AU-rich endotoxin-responsive motifs (AREs) of seabream TNF alpha mRNA, found within two phylogenetically conserved protein binding regions, is discussed.

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.

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream (Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.

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.

A complex stressful event, which commonly occurs in modern aquacultural practices, was broken down into factors that were analysed both individually and jointly to assess their effect on two stress indicators (blood glucose and serum cortisol levels) and two activities of the innate immune response (serum complement and head-kidney leukocyte respiratory burst). For this, gilthead seabream (Sparus aurata L.) specimens were exposed to the following stressors: physical disturbance, crowding, anaesthesia with 2-phenoxyethanol and air exposure. At 0, 1, 2, 3 and 4 days post-stress, blood and serum samples were collected to measure glucose concentration and cortisol and complement levels, respectively. Head-kidney leukocytes were isolated and assayed to evaluate respiratory burst activity. The results show that physical disturbance, crowding and anaesthesia produced an occasional increase in glucose and cortisol concentrations. Crowding and anaesthesia induced a depression in complement activity, while hypoxia by air exposure caused a reduction in the respiratory burst. When all factors were jointly applied both humoral and cellular defences were compromised and cortisol values remained high throughout the experimental period. Any long-term effects of this abnormal serum cortisol levels on the immune system remain unknown.

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.

The genomic sequencing of hundreds of organisms including homo sapiens, and the exponential growth in gene expression and proteomic data for many species has revolutionized research in biology. However, the computational analysis of these burgeoning datasets has been hampered by the sparse successes in combinations of data sources, representations, and algorithms. Here we propose the application of symbolic toolsets from the formal methods community to problems of biological interest, particularly signaling pathways, and more specifically mammalian mitogenic and stress responsive pathways. The results of formal symbolic analysis with extremely efficient representations of biological networks provide insights with potential biological impact. In particular, novel hypotheses may be generated which could lead to wet lab validation of new signaling possibilities. We demonstrate the graphic representation of the results of formal analysis of pathways, including navigational abilities, and describe the logical underpinnings of the approach. In summary, we propose and provide an initial description of an algebra and logic of signaling pathways and biologically plausible abstractions that provide the foundation for the application of high-powered tools such as model checkers to problems of biological interest.

The gilthead seabream IL-1beta gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5' untranslated region (UTR), a single open reading frame of 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3'UTR with a polyadenylation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has the highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1beta sequences. The IL-1beta message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1beta was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation., (Copyright 2001 Academic Press.)

Cell adhesion molecules play a key role in the inflammatory response. Selectins, integrins and immunoglobulin gene superfamily adhesion receptors mediate the different steps of leukocyte migration from the blood-stream towards inflammatory foci. In addition to their adhesive function, these receptors modulate major cellular processes such as cell activation, growth, differentiation and death. To characterise the fish molecules involved in cell adhesion, a panel of mAbs was raised by immunising mice with macrophages from the marine fish gilthead seabream (Sparus aurata L.). One of these mAbs, which we named anti-Aggregatin, was found to induce a rapid heterotypic aggregation of seabream leukocytes. Anti-Aggregatin defined a 140-kDa cell surface receptor which was highly expressed by macrophages and was up-regulated after co-stimulation with LPS and MAF. Functionally, the cell adhesion which occurred upon exposure to anti-Aggregatin required Ca(2+), an intact cytoskeleton and an active cell metabolism. More importantly, Aggregatin engagement resulted in strong inhibition of the phagocyte respiratory burst, although the cells showed neither loss of viability nor DNA fragmentation. The results are discussed in relation to the potential role of cell adhesion molecules in fish immune responses.

The effect of the oral administration of high dosages of vitamins C and E on the innate immune system of the seabream was investigated. Gilthead seabream (Sparus aurata L.) were fed four different diets for 45 days: a commercial diet as control, a 3g/kg vitamin C-supplemented diet, a 1.2g/kg vitamin E-supplemented diet or a diet containing both vitamin supplements. After 15, 30 and 45 days, serum ascorbic acid and alpha-tocopherol levels, growth, complement titers and head-kidney leucocyte phagocytic and respiratory burst activities were evaluated. The results showed that serum vitamin levels reflected dietary input. Fish fed the vitamin C-supplemented diet showed an enhanced respiratory burst activity, while fish fed the vitamin E-supplemented diet exhibited increased complement and phagocytic activities. All of these positive effects were found in fish fed the joint diet, as well as a synergistically enhanced respiratory burst activity at day 30. The results demonstrate that in vivo vitamins C and E exert a synergistic effect enhancing the respiratory burst activity of seabream phagocytes.

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0 mg (control), 25, 50 or 100 mg kg(-1) chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.

The natural cytotoxic activity of head-kidney leucocytes from gilthead seabream (Sparus aurata L.), after in vitro and in vivo vitamin E treatment, against tumor cells was studied by flow cytometry. Leucocytes were incubated in culture medium with different vitamin E supplementations (0.01-10 microg ml(-1)) for 6, 24 or 48 h and the results demonstrate that all the assayed vitamin E supplementations significantly enhanced the natural cytotoxic activity of leucocytes. To determine the effect of a high dietary level of vitamin E on this activity, fish were fed with 0 (control), 600, 1200 or 1800 mg of vitamin E supplementation kg(-1) diet for 2, 4 or 6 weeks. After 2 and 4 weeks of treatment, the natural cytotoxic activity was significantly enhanced at the highest (1.8 g kg(-1) diet) and lowest (600 mg kg(-1) diet) vitamin E supplement dosage, respectively. No effect of the vitamin E supplemented diet on seabream leucocyte natural cytotoxic cell activity was observed after 6 weeks of treatment.

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D--glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.

To determine the effects of three high levels of dietary intake of alpha-tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100 mg alpha-tocopherol kg-1) as control, or vitamin E supplemented diets (600, 1200 or 1800 mg alpha-tocopherol acetate kg-1) for 15, 30 or 45 days. Growth, serum alpha-tocopherol levels, natural haemolytic complement activity and head-kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between alpha-tocopherol acetate intake and serum alpha-tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600 mg vitamin E kg-1 showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200 mg vitamin E kg-1 presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head-kidney leucocytes were enhanced by the dietary intake of 1200 mg vitamin E kg-1 after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained unaffected. The highest vitamin E dietary dose used, 1800 mg kg-1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200 mg kg-1) stimulates the seabream's non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so effective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.

Studies of fish blood cells made to date presented numerous problems derived from both the nomenclature and the techniques used. A combination of quantitative and morphological methods is needed if the classification of fish blood cells is to advance from it present provisional state. The aim of the present paper was first to isolate sea bass blood cell populations by flow cytometry and second to characterize then microscopically. Blood cell populations from sea bass (Dicentrarchus labrax L.) were isolated according to their FSC (size) and SSC (granularity) properties by flow cytometry. The isolated populations were then processed for light and transmission and scanning electron microscopic characterization. Sea bass blood leukocytes isolated by flow cytometry consisted of two main cell subpopulations. Subsequent microscopic study of these cells revealed that the first subpopulation was composed of small cells (3-5 microm) of low granularity and consisted of thrombocytes and lymphocytes whereas, the second subpopulation was formed of 6-9 microm sized cells of high granularity consisting of granulocytes and monocyte/macrophages. The combined use of flow cytometry and electron microscopy makes it possible to characterize the different cell types present in sea bass peripheral blood with a high degree of certainty. Although sea bass basically follows the common vertebrate hematological pattern, significant modifications such as the presence of circulating immature erythrocytes, plasma cells and monocyte/macrophages and different forms of thrombocytes can be established with respect to this pattern., (Copyright 2000 Wiley-Liss, Inc.)

This paper describes an easy and sensitive flow cytometric assay for assessing the non-specific cytotoxic activity of gilthead seabream (Sparus aurata L.) head-kidney leucocytes against tumor target cells. Concomitantly, the cytotoxic process and the cell types involved were microscopically studied. The assay was based on the consecutive use of two fluorochromes. The targets were preincubated with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and then mixed with effectors. At the end of incubation time propidium iodide (PI) was added. While live effectors were non-fluorescent, live and dead targets retained the DiO (green) fluorescence and non-viable targets and effectors showed PI (red) fluorescence staining. The kinetics of the cytotoxic activity was studied from 10 to 240 min. Lymphocytes, monocyte-macrophages and granulocytes showed non-specific cytotoxic activity, as demonstrated by light and electron microscopy. In conclusion, the technique presented validates the effectiveness of a dual-color flow cytometric assay for assessing the activity of non-specific cytotoxic cells in fish.

The presence of infective endocarditis with appearance of vegetations over the tricuspid valve in patients with permanent pacemaker is an uncommon complication. We report here three patients with permanent cardiac pacemakers who developed a picture of infective endocarditis over the electrode with appearance of vegetations over the native valve. The microorganism recovered from blood cultures in the three patients was Staphylococcus. The presence of retained pacemaker leads and repetitive surgical procedures over the pacemaker pouch are two risk factors reported in literature also present in our patients. Infective endocarditis is an uncommon difficult-to-diagnose complication, with a poor prognosis and which requires the removal of the whole infected material, with implantation of an epidermal pacemaker. Some diagnostic and therapeutic issues are discussed.

The phagocytic cell function is an important factor of resistance to infection in fish. Although it is known that several nutritional factors affect phagocyte function and that vitamins C and E modulate the vertebrate immune system, there are no data on the direct effect of these vitamins or on their possible synergistic effects on the fish phagocyte functions. We report the in vitro effect of vitamin C and/or E on the functions and ultrastructure of gilthead seabream (Sparus aurata L.) head-kidney phagocytic leukocytes. Head-kidney leukocytes were incubated with different concentrations of vitamins C and/or E for 48 h and then their migration, respiratory burst, phagocytosis and ultrastructure were studied. Leukocyte migration and phagocytosis increased when vitamin C or E were added, while the respiratory burst was synergistically enhanced when both vitamins were used together. Noticeable ultrastructural changes not previously reported were induced by these vitamins in gilthead seabream phagocytes (acidophilic granulocytes and macrophages). These results indicated that vitamins C and E added in vitro modulate the main functions of gilthead seabream phagocytes and induced ultrastructural changes characteristic of cell activation on such cells.

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.

Background: Patch angioplasty is an alternative surgical technique in cases of left main coronary artery stenosis. We report our experience with this technique, with particular mention of the use of spiral computed tomography for the follow-up of our patients., Methods: In this retrospective study we analyzed the results obtained in all 7 patients (3 women and 4 men) who were operated on with this technique in our institution between July 1992 and August 1994. Five consenting patients also underwent graft patency assessment with spiral computed tomographic imaging., Results: The operation was uneventful in all patients and there were no hospital deaths. Two patients required reoperation (1 of them dying at reoperation), 1 because of restenosis of the graft and 1 because of the presence of a new stenosis in the proximal anterior descending coronary artery. The remaining 5 patients were asymptomatic after 51 +/- 2 months. Spiral computed tomographic images were artifacted and of poor quality., Conclusions: Patch angioplasty of the left main coronary artery can be a valuable therapeutic method in selected cases. Conventional spiral computed tomography is not an optimal noninvasive method for the assessment of graft patency.

Background and Aims of the Study: Between 1st January 1988 and 30th June 1996, a total of 165 patients (115 males, 50 females; mean age 66.8 years) underwent isolated aortic valve replacement with Carpentier-Edwards supra-annular porcine bioprostheses in our unit. Our clinical experience with this device is reported., Methods: In this retrospective study, mortality and complications observed in these patients were analyzed. Complications appearing with a non-biased time-course were expressed by means of linearized rates. Actuarial analysis was performed using the Kaplan-Meier method., Results: Cumulative follow up was 551.2 patient-years, with a mean follow up of 3.5 years per patient. The hospital mortality rate was 6.7%. Linearized rates (events per 100 patient-years) and actuarial probabilities of freedom from the different complications at 8.5 years follow up, respectively, were: late mortality, 4.5 and 58.6%; valve-related mortality, 1.1 and 82.6%; thromboembolism, 1.3 and 92.7%; prosthetic endocarditis, 0.5 and 97.4%; non-structural dysfunction, 0.5 and 94.7%; structural deterioration, 0.5 and 91.6%; reoperation, 0.5 and 94.2%. Actuarial freedom from any valve-related mortality or morbidity at 8.5 years was estimated at 66.3%., Conclusions: The Carpentier-Edwards supra-annular bioprosthesis, when implanted in the aortic position, presents a low rate of valve-related complications at this length of follow up. However, the limited life expectancy of our patients undergoing aortic valve replacement with bioprostheses (usually elderly subjects) may overshadow the potential good performance of this device.