Manuel Noya

The anti-inflammatory activity of the polyphenol resveratrol may be partially related to inhibition of tumour necrosis factor-α (TNF-α) pre-mRNA splicing

The present study shows for the first time that the polyphenol resveratrol (RESV) blocks processing of tumour necrosis factor-alpha (TNF-alpha) pre-mRNA in mature mRNA. This study was carried out in turbot (Psetta maxima (L.)), a fish species that we are using to evaluate the effects of RESV on the inflammatory response in vertebrates. Treatment of turbot head kidney leucocytes with polysaccharides from the seaweed Ulva rigida (ulvan) resulted in an increase in TNF-alpha expression. RESV did not inhibit transcription but almost completely inhibited the production of mRNA in ulvan-induced cells and caused a notable increase in the level of unspliced TNF-alpha pre-mRNA. RESV also induced accumulation of IL-1beta pre-mRNA at the expense of mature mRNA, although the effects on IL-1beta were less evident than those on TNF-alpha. However, the housekeeping gene was not affected by RESV. We also evaluated the effects of RESV in vivo under an inflammatory stimulus and found an inhibitory effect on TNF-alpha and IL-1beta pre-mRNA splicing in turbot head kidney at 24 and 48h post-injection. In addition, RESV also reduced migration of cells to the peritoneal cavity under the same inflammatory stimulus. The results show that this fish species may be a useful model for analysing the effects of RESV on TNF-alpha and IL-1beta expression, and suggest that RESV could be used to decrease the levels of pro-inflammatory cytokines in vivo and to reduce inflammatory reactions in certain inflammatory diseases.

Interactions between peritoneal exudate cells (PECs) of gilthead seabream (Sparus aurata) and Pasteurella piscicida. A morphological study

Abstract Peritoneal exudate cells from gilthead seabream (Sparus aurata) weighing 20–30 g and 0.5 g were studied using transmission electron microscopy at several time intervals after intraperitoneal injection with a virulent Pasteurella piscicida strain. Fish of 20–30 g were injected with 0.3 ml of a bacterial suspension containing 1 × 109 cells/ml of P. piscicida. Fish of 0.5 g were injected with 0.1 ml of a suspension of 1 × 106 cells/ml. The microscopic findings showed that peritoneal exudate cells (PECs) of the bigger fish were capable of phagocytosing and killing the bacteria during the first 24 h following injection and, in consequence, of controlling the infection. PECs of seabream of 0.5 g also phagocytosed P. piscicida; however, macrophages appeared to be unable to kill the microorganism. As a result, P. piscicida was able to establish an infection and kill the fish within 5 days. We conclude that the resistance of gilthead seabream to P. piscicida is dependant on the size of the fish and may be related to the efficiency of its phagocytes.

Effect of temperature on the development of pasteurellosis in carrier gilthead seabream (Sparus aurata)

In the present paper we study the effect of water temperature on the development of pasteurellosis in 60-day-old gilthead seabream larvae obtained from asymptomatic carrier broodstock. Fish were exposed to different temperatures and mortalities were recorded over a period of 5 weeks. Mortalities were very low when larvae were kept at 15°C, increased considerably after the temperature was raised to 18°C or 20°C, and decreased again when the temperature was lowered to 15°C. These results suggest that larvae obtained from asymptomatic carrier broodstock are also carriers and can develop pasteurellosis by increasing the water temperature. However, it is possible to control the disease by maintaining larvae at low temperatures.

Marine environment as reservoir of birnaviruses from poikilothermic animals

Abstract In this study 51 aquatic birnaviruses were isolated from environmental samples (molluscs, wild fish and sediments) during routine surveys around fish farms located in Galicia (NW of Spain). These viruses were identified as IPN-like viruses based on their physical and chemical characteristics. Furthermore, these isolates were molecular and serologically compared to the reference strains VR-299, Sp and Ab. Although they caused no mortalities in oysters and showed varying degrees of virulence in trout, all isolates were recovered from the inoculated animals, indicating that oysters can become carriers. The results obtained in this study indicate the role of the molluscs, sediments or fish feed as important agents to consider in the epidemiology of birnaviruses.

Location of superoxide production sites in turbot neutrophils and gilthead seabream acidophilic granulocytes during phagocytosis of glucan particles

Here we show, by spectrophotometry and enzyme cytochemical methods, that turbot neutrophils and gilthead seabream acidophils responded in a similar way when incubated with PMA or with particulate glucans. Cells stimulated with PMA released high amounts of superoxide both intra- and extracellularly. However, O2- was mainly released intracellularly when cells were incubated with particulate glucans. Small glucan particles were quickly phagocytosed and O2- was initially produced in intracellular vesicles and tubular structures that later fused with the phagosome or with the cell membrane. Large glucan filaments that were not phagocytosed also induced cell stimulation and O2- was also produced in intracellular vesicles which then appeared to fuse with the cell membrane. We conclude that, in stimulated turbot neutrophils and gilthead seabream acidophils, superoxide production is carried out initially in intracellular compartments that are very similar to those described in mammalian neutrophils.

Identification of a beta 2 (CD18) molecule in a teleost species, Ictalurus punctatus Rafinesque

Beta 2, in combination with the alpha subunit, is responsible for tight adhesion of leukocytes, especially neutrophils and macrophages, in areas of inflammation. Although identified in mammalian and avian species; the beta 2 or CD18 molecule has yet to be identified in fish. The present investigation has identified a full-length channel catfish, Ictalurus punctatus, cDNA beta 2 molecule composed of 2.8 kb and a deduced amino acid sequence of 772 amino acids. The catfish molecule has an amino acid homology ranging from 54 to 63% with mouse, bovine, rabbit, human and chicken. The channel catfish molecule retains several characteristics of mammalian beta 2 molecules, such as cysteine-rich repeat regions, N-linked glycosylation sites, and several proposed signal sequences. Expression of the beta 2 molecule on the catfish neutrophil cytoplasmic membranes is increased upon phorbol dibutyrate stimulation of the cells. Based on Western blotting and the immunoprecipitation test, the channel catfish beta 2 molecule has a molecular mass of approximately 95 kD, essentially the same as that for mammalian species. However, two additional molecules, perhaps alpha chains, of unexpected molecular mass appear to co-precipitate in the SPIT with the 95 kD CD18 molecule. These results confirm the existence and expression of a beta 2 gene in channel catfish, a species phylogenetically distant from mammalian species.

Response of eosinophilic granule cells of gilthead seabream (Sparus aurata, Teleostei) to bacteria and bacterial products.

Abstract.Eosinophilic granule cells in the gills and peritoneal exudate of gilthead seabream (Sparus aurata L.) are characterized by the presence of prominent eosinophilic granules in their cytoplasm and are here described for the first time. The oval granules of these cells contain an electron-dense inclusion surrounded by a less dense filamentous matrix and are peroxidase- and acid phosphatase-negative. Unlike other granulocytes of gilthead seabream, eosinophilic granule cells do not ingest bacteria in vivo. The intraperitoneal injection of extracellular products of Pasteurella piscicida induces mobilization of eosinophilic granule cells to the blood and other tissues and causes changes in their structure. Shortly after injection, the granules of eosinophilic granule cells become swollen and some fuse with the cell membrane. From 7 h post-injection, many eosinophilic granule cells in the gills degenerate and are then phagocytosed by macrophages, which are especially abundant after 24 h. From 24 h to 72 h, eosinophilic granule cells from the gills contain abundant autolysosomes together with granules of a normal morphology.

MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF CHANNEL CATFISH (ICTALURUS PUNCTATUS) NEUTROPHIL COLLAGENASE

Channel catfish (Ictalurus punctatus) neutrophils, like mammalian neutrophils, contain a variety of enzymes and lytic peptides that participate in pathogen destruction. We have identified and characterized from a channel catfish anterior kidney cDNA library a 1.6 kb cDNA that encodes for channel catfish neutrophil collagenase. The deduced amino acid sequence has a predicted molecular mass of 53 kDa. The putative catfish collagenase has nucleotide and amino acid homology of 51.4% and 45.1%, respectively, with human neutrophil collagenase and 50.4% and 47.1%, respectively, with mouse neutrophil collagenase. Certain regions of the molecule, including the cysteine switch and the putative zinc binding sites, were identical to those in the human and mouse genes. Polyclonal antiserum, prepared to the fusion protein, recognizes proteins from channel catfish neutrophil supernatants with molecular masses of approximately 63, 53 and 28 kDa. Supernatants from phorbol dibutyrate stimulated neutrophils were capable of degrading type I collagen. In addition, the polyclonal antiserum prevented the collagenase activity of the supernatants from stimulated catfish neutrophils; whereas, preimmune serum had no effect on collagenase activity of supernatants. Supernatants from unstimulated cells or the fusion protein did not possess the ability of degrading type I collagen. These results indicate that channel catfish neutrophil collagenases share molecular and functional features with mammalian neutrophil collagenase.

Identification of a channel catfish, Ictalurus punctatus (Rafinesque), leukocyte-specific leucine zipper protein.

Five clones isolated from a channel catfish cDNA library were each reactive with monoclonal antibodies (mAbs) C3-1 and 51A only. The size of the cDNA inserts from C3-1 and 51A positive clones was 2.5 Kb and identical based on sequence analysis. Monoclonal antibodies C3-1 and 51A specifically reacted with the expressed product of the 2.5 Kb cDNA clone. The complete DNA sequence indicated that the 2.5 Kb cDNA encoded an approximately 50 Kd protein molecule consisting of 445 amino acids. Sequence analysis showed that this putative protein was a potential leucine-zipper DNA binding protein. Comparison of the deduced amino acid sequence demonstrated homology (14.6 to 19.5%) throughout the sequence of the catfish protein with a group of cytoplasmic-leucine zipper containing proteins of humans; paraneoplastic cellebellar degeneration related (cdr) antigen 2 and 3 with 39.8 to 56.3% homology in the leucine-zipper motif (amino acids 52 through 175 in the catfish protein). This protein was detected in nuclear extracts. cytoplasmic membrane preparations and cytosolic extracts of neutrophils and lymphocytes when reacted with mAbs C3-1 and 51A in an ELISA. However, the intensity of the reactions was dependent upon the cell type and cellular component. The putative cdr protein was not detected with any appreciable intensity in preparations from other cell types. This finding strongly suggests that this protein is expressed in a leukocyte-specific manner and is unique among the cdr group in that it is being expressed in a site that is not immune privileged.

Replication and morphogenesis of the turbot aquareovirus (TRV) in cell culture

Abstract The ultrastructural aspects of the replication cycle of the turbot aquareovirus (TRV) were studied using CHSE-214 cells. Although this virus in general followed the typical pattern of reovirus replication and morphogenesis, it presented some important differences. Internalisation of virions into the host cell occurred by direct penetration through the plasma membrane (even at temperatures below 4°C), and this step was independent of trypsin treatment. Two size classes of viral particles were detected in the cytoplasm of infected cells: (i) particles of 30 nm diameter, probably corresponding to cores, located inside cytoplasmic viroplasms, and (ii) 45 nm diameter particles, located in vesicles of the endoplasmic reticulum, probably corresponding to single-shelled virions. Double-shelled complete viral particles (75–80 nm diameter) were only detected at the exterior of the cell. Cores formed inside the viroplasms were transformed into 45 nm immature virions by budding from viroplasms to cytoplasmic vesicles. The vesicles carried those subviral particles towards the periphery of the cell. Final maturation occurred by budding of the immature virions through the plasma membrane. During this process, incomplete virions acquired an external protein layer, to form the 75 nm double-shelled complete virions released to the exterior. Thus, release of TRV viral progeny was not necessarily associated with cell lysis. Replication of TRV was cytoplasmic, and some of the ultrastructural changes caused by the viral replication included development of many dense fibrillar bodies, and cell cytoskeleton alterations.