Biomedical subjects

C J Secombes

Publications and source records attributed to C J Secombes.

At least 55 records · Page 3Linked to original sources

PubMed2000

Cloning, sequencing, and analysis of expression of a second IL-1beta gene in rainbow trout (Oncorhynchus mykiss).

The full-length sequence of a second IL-1beta gene (IL-1beta2) in rainbow trout (Oncorhynchus mykiss) has been obtained. As with the first IL-1beta gene, IL-1beta2 is organized into six exons/five introns. There are only small differences in their intron/exon sizes, with the exception of intron 3, which is 334 bp smaller in IL-1beta2. The transcript encoded by the IL-1beta2 gene contains a 5' untranslated region (UTR) of 121 bp, followed by a 762-bp open reading frame and a 518-bp 3'UTR. The 3'UTR contains seven instability attta motifs, typical of inflammatory genes, and a polyadenylation site 11 bp upstream of a 17-bp poly(A) tail. The predicted 254 amino acid sequence of the second IL-1beta gene has 82% similarity to the first gene, 45% similarity to carp IL-1beta, and 40% similarity to human IL-1beta. Comparison of the two trout genes reveals that the IL-1beta2 gene has a deletion of 9 bases in exon 3 and an altered splicing site at the 5' end of exon 4 giving rise to a further 9-bp deletion in the resulting cDNA. As with other nonmammalian IL-1beta genes, no interleukin-converting enzyme (ICE) cut site has been found but the alignment of the amino acid sequence with other species shows a possible cut site between Arg89 and Ala90 that would give arise to a 165-amino acid mature peptide. Expression studies performed by RT-PCR using primers specific for the IL-1beta2 transcript revealed a clear dose-dependent induction of this gene in cultured trout leukocytes by stimulation with lipopolysaccharide.

Amino Acid Sequence↗

PubMed2000

Molecular cloning of the gene for interleukin-1beta from Xenopus laevis and analysis of expression in vivo and in vitro.

The Xenopus cDNA for interleukin-1beta (IL-1B) was cloned and sequenced. The gene contains 1,462 nucleotides that translate in a single reading frame to give a predicted 283-amino acid IL-1beta molecule. The translated molecule contains a single potential glycosylation site, a readily identifiable IL-1 family signature, and has highest homology to chicken IL-1beta by phylogenetic tree analysis and sequence similarity. It lacks a signal peptide in common with other known IL-1B genes, and lacks a clear ICE (caspase) cut site in common with other nonmammalian IL-1B genes sequenced to date. RT-PCR was used to study sites of IL-1B transcript expression, 24 h following injection of lipopolysaccharide (LPS). Expression was detected in the brain, liver, kidney, and spleen, with expression weakest in the brain and strongest in the spleen. No transcript expression was detectable following injection of saline. Northern blot analysis was used to quantify the induction of IL-1B expression in splenocytes following in vivo or in vitro stimulation with LPS. The results are discussed in relation to the potential role of IL-1beta in amphibian immune responses.

Amino Acid Sequence↗

PubMed2000

Immunoprophylaxis in fish by injection of mouse antibody genes.

Antibodies are a crucial part of the body's specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals. The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of infectious diseases. To test this in a fish model, a gene construct encoding a neutralizing single-chain antibody to the fish-pathogenic rhabdovirus VHSV (viral hemorrhagic septicemia virus) was administered to rainbow trout by intramuscular injection of plasmid DNA. Circulating recombinant antibodies could later be detected in the fish, and protective immunity to the viral disease was established.

Animals↗

PubMed2000

The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum.

Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response.

Animals↗

PubMed2000

Invertebrate and fish cytokines.

Cytokine-like molecules are well described in invertebrates, although most recent studies have revealed that there is analogy, rather than homology, between invertebrate and vertebrate cytokine-like activities. Cytokines certainly appeared early in the evolution of vertebrates, dating back some 400 millions years. Here, evidence will be reviewed and updated of the presence of these molecules in jawed fish and in particular, in bony fish, which represent the oldest group displaying true functionality of immune system as known in modern vertebrates. Many studies during the last ten years have confirmed the presence of functional homologues of mammalian cytokines in fish. In this review, particular attention will be focussed on IL-1beta, a very ancient defence cytokine recently sequenced in two species, rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). Original data on the partial peptide sequence of IL-1beta in the mediterranean sea bass Dicentrarchus labrax are also presented.

Amino Acid Sequence↗

PubMed2000

Evolution of adaptive immunity.

Antigen receptors and major histocompatibility molecules, key elements required for adaptive immunity, are first seen in jawed fish. So, how did they evolve and have they changed?

Animals↗

PubMed1999

TGF-beta3 exists in bony fish.

A partial nucleotide sequence of transforming growth factor-beta3 (TGF-beta3) has been isolated from the Siberian sturgeon (Acipenser baeri), rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla), confirming a ubiquitous presence in the ray-finned (Actinopterygian) bony fish. The bony fish TGF-beta3 is highly conserved, with some 83-84% nucleotide identity (coding region) and 90-95% predicted amino acid identity to known homeotherm TGF-beta3's. Far lower homologies are apparent with other known TGF-beta isoforms in fish (e.g. 64-66% and 81-82% amino acid identity to trout TGF-beta 1/5 and carp TGF-beta2 respectively). Phylogenetic tree analysis showed that the fish TGF-beta3's clustered with the known homeotherm TGF-beta3's. The relatively tight clustering of TGF-beta1, TGF-beta2 and TGF-beta3 was in contrast to the TGF-beta5's, which are clearly a more heterogenous group.

Amino Acid Sequence↗

PubMed1999

Fish macrophages express a cyclo-oxygenase-2 homologue after activation.

In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5'-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3'UTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago.

Aeromonas↗

PubMed1999

Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss.

Two rainbow trout chemokine receptors have been sequenced, with homology to CXC-R4 and CC-R7 molecules. The CXC-R4 sequence consisted of 1681 nucleotides, which translated into a mature protein of 357 amino acids, with 80.7% similarity to human CXC-R4. The CC-R7 sequence consisted of 2287 nucleotides, which translated into a 368-amino acid mature protein with 64.5% similarity to human CC-R7. Both sequences contained seven hydrophobic regions, representing the seven transmembrane domains (TM) typical of G-protein-coupled receptors. Extracellular cysteines, transmembrane prolines, and the DRY motif immediately following TM3 were conserved. Phylogenetic tree analysis revealed a tight clustering of trout CXC-R4 with CXC-R3-5 genes. Trout CC-R7 clustered with CC-R6-7 and CXC-R1-2. Reverse transcriptase-polymerase chain reaction analysis demonstrated a wide tissue distribution of CXC-R4 and CC-R7 message in trout, being present in head-kidney leukocytes, blood, gill, brain, spleen, and liver.

Amino Acid Sequence↗

PubMed1999

Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site.

The complete coding sequence of rainbow trout IL-1beta has been obtained. The gene contains a short 5' UTR (97 bp), a 780 bp open reading frame and a 466 bp 3' UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18 bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29 kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1betas and 57% similarity to carp IL-1beta. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala(95)would be the amino terminus. Northern blot analysis showed that whilst no IL-1beta expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4 h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.

3' Untranslated Regions↗

PubMed1999

Binding of soluble immune complexes to fractionated Atlantic salmon (Salmo salar L.) leucocytes.

Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg- enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.

Animals↗

PubMed1999

The rainbow trout Oncorhynchus mykiss interleukin-1 beta gene has a differ organization to mammals and undergoes incomplete splicing.

The rainbow trout interleukin (IL)-1 beta gene consists of six exons/five introns, in contrast to mammals which have seven exons/six introns. The missing exon appears to be at the 5' end of the gene, probably equivalent to exon 1 or 2 of mammals. Fewer and smaller introns make the trout IL-1 beta only half the size of mammalian IL-1 beta genes. Highest homology (> 60% amino acid similarity) is seen between exon 5 of trout and exon 6 of mammals. Reverse transcription-PCR analysis revealed two additional incompletely spliced variants of the trout IL-1 beta gene, containing intron 5 or introns 4 and 5. All three transcripts are detectable in gill, kidney, liver and spleen tissue from bacterially challenged fish but only the fully spliced transcript is detectable in blood. Northern blot analysis revealed that the latter transcript is also induced following stimulation of kidney leucocytes with lipopolysaccharide for 4 h. A second inducible transcript is also detected but is larger (approximately 3 kb) than any of the above, suggesting that it could be from a second gene. Southern blot analysis also suggests at least two copies of the IL-1 beta gene or genes related to the 3' end of the IL-1 beta sequence, are present in trout.

Alternative Splicing↗

PubMed1998

Isolation of rainbow trout neutrophils with an anti-granulocyte monoclonal antibody.

A magnetic cell sorting system has been optimised for the purification of rainbow trout neutrophils using a monoclonal antibody (E3D9) raised against Atlantic salmon neutrophils. The purified neutrophils have good viability (85%) and purity (approximately 92%), and were functional in respiratory burst and migration assays. The isolated neutrophils responded rapidly to PMA stimulation, producing levels of superoxide anion (4.85 nmols superoxide min-1/10(6) cells) approximately twice as high as macrophages from the same species. In the migration assay, there was a four-fold increase in migrating cells using the purified neutrophils compared with unfractionated blood leucocytes, and a relatively high neutrophil migratory activity was seen in the absence of serum.

Animals↗

PubMed1998

Isolation of the first piscine transforming growth factor beta gene: analysis reveals tissue specific expression and a potential regulatory sequence in rainbow trout (Oncorhynchus mykiss).

The nucleotide sequence of a rainbow trout transforming growth factor beta (TGF-beta) peptide is presented, which translates into a 382 amino acid precursor molecule containing a 20 amino acid leader and a mature peptide of 112 amino acids. The mature peptide has nine conserved cysteines and a conserved proline (position 36) and glycine (position 46), all characteristics of TGF-beta superfamily molecules. Within the precursor region are three glycosylation sites, two in common with known TGF-beta s, an integrin binding site (RGD) and the tetrabasic peptide cleavage site (RKKR). The full 3' untranslated region (UTR) consists of 542 nucleotides with a polyadenylation signal 16 nucleotides upstream of the poly(A) tail. The 5' UTR contains an open reading frame with the potential to encode an eleven amino acid peptide, which may have significance for regulation of TGF-beta translation. A wide tissue distribution of TGF-beta message was detected by RT-PCR; in blood leukocytes, kidney macrophages, brain, gill, and spleen tissue but not liver. A phylogenetic tree reveals the trout TGF-beta sequence is most related to xenopus TGF-beta 5, with these sequences and that of chicken TGF-beta 4 grouping with mammalian TGF-beta 1 s. The impact of the trout sequence on current theories of TGF-beta isotype evolution is discussed.

Amino Acid Sequence↗

PubMed1998

Potency testing of a live, genetically attenuated vaccine for salmonids.

Studies have been performed on the use of a live vaccine for immunization of salmonids against the bacterial disease furunculosis. The protection elicited by a kanamycin-resistant aroA mutant of A. salmonicida (Brivax I) and an unmarked aroA deletion mutant (Brivax II) has been examined, and data compared with protection seen using a freeze-dried Brivax II preparation and a commercial, oil-adjuvanted killed vaccine for furunculosis. Whilst high relative percent survival (RPS) values were seen in fish vaccinated with broth-grown Brivax I after a natural exposure to furunculosis (70-100%), much lower RPS values (30-40%) were seen with Brivax II vaccinated fish after an experimental challenge. Nevertheless, the freeze-dried Brivax II formulation performed as well as the broth-grown Brivax II formulation and a commercial vaccine in these studies. In addition, the environmental impact in terms of bacterial shedding into the tank water has been estimated, and shown to approximately 0.03% of the total inoculum used. Lastly, the freeze-dried formulation has been tested for its ability to infect fish and prime for lymphocyte proliferation and antibody production, relative to broth-grown preparations. In all three experiments no significant differences were seen between fish given the broth-grown and freeze-dried formulations. Such data, together with observations that the freeze-dried live preparation had an extended shelf life with the same potency as freshly grown bacteria, show that the potential exists for a commercially viable live vaccine to be produced for use in aquaculture.

Animals↗

PubMed1997

Detection of specific and 'constitutive' antibody secreting cells in the gills, head kidney and peripheral blood leucocytes of dab (Limanda limanda).

The relative immunological importance of the gills of fish was investigated in terms of antibody production by enumerating antibody secreting cells (ASC) in the gills, head kidney and blood of dab (Limanda limanda) using the ELISPOT assay. The contribution of 'constitutive' ASC in the gill appeared more substantial than that of elicited specific ASC. The gills were found to contain a mean (+/- SD) of 4227 +/- 1029 'constitutive' ASC/10(6) cells which was fewer than the head kidney which contained a mean (+/- SD) of 15617 +/- 3723 'constitutive' ASC/10(6) cells but more than peripheral blood leucocytes which contained a mean (+/- SD) of 2650 +/- 212 'constitutive' ASC/10(6) cells. The number of specific anti-human gamma globulin (HGG) ASC following parenteral or oral administration of HGG was also determined. Anti-HGG ASC were detected in all three tissues following parenteral immunization, peaking simultaneously, 4 weeks post-immunization. The strongest response was found in the head kidney. After oral immunization, responses were much weaker: again the head kidney was the most active but the gill response was barely detectable. These data were complemented by measurement of specific antibody in the serum by ELISA. Serum antibody titres following immunization were found to correlate closely with the number of specific ASC in the head kidney following parenteral immunization whereas serum antibody titres after oral administration of antigen most closely followed the number of specific ASC in the blood. In the light of these data it is suggested that the primary immunological role of the leucocytes in the gill may be in the earliest stages of defence against infection.

Animals↗

PubMed1997

Generation and characterisation of monoclonal antibodies against rainbow trout, Oncorhynchus mykiss, leucocytes.

Monoclonal antibodies were raised against head kidney macrophages of the rainbow trout, Oncorhynchus mykiss. Despite the establishment of a significant number of different hybridoma clones, none of these released antibody specific for determinants only found on macrophages. Instead, all the monoclonal antibodies generated reacted with lymphocytes, granulocytes, and monocytes/macrophages, although thrombocytes (the platelet equivalents in fish) and erythrocytes were not recognized by these antibodies. Western blotting of solubilised macrophages revealed that two of the hybridoma lines, designated 21G6 and 21F11, reacted with at least five proteins of 80, 104, 110, 140, and > 170 kDa. Immunocytochemistry was performed on histological sections of trout alimentary canal, gill, liver, spleen, and haemopoietic head kidney using antibodies from several of the hybridoma lines, and all of these showed a similar pattern of reactivity in each tissue. In the alimentary canal, for example, immunoreactive material was found in the eosinophilic granular cells, blood vessel margins, mucus in the lumen, and in the columnar epithelial cells. In the gills, epithelial cells and blood vessels also showed intense immunoreactive products, while in the liver, such reactivity was localised in the sinusoids and adherent macrophages. Both the spleen and head kidney had largely homogenous immunoreactivity.

Animals↗

PubMed1997

In vitro methods for vaccine evaluation.

This paper reviews aspects related to the establishment of in vitro assays for fish vaccine evaluation and presents mainly assays for potency testing where specific immune responses are monitored. Aspects related to the evaluation of currently available bacterial vaccines are focused on, and results are mainly presented where both immune responses and protection are assessed in the same trial. Although there is no agreement on which antigens are protective in such vaccines, results show that there are candidate antigens from the various pathogens that are strongly immunogenic in fish, and a correlation between the immune responses they elicit and protection is striking. For some antigens, a clear relationship between immunizing dose, antibody response and protection is evident, which is an important basis for a potency test. Results presented on the evaluation of live vaccines show that assays to measure specific T-cell responses are required. These assays, like lymphocyte proliferation and detection of macrophage activation factor, are considered to be less specific than antibody assays and are hampered by greater variations between individual responses. However, when reagents become available for the assessment of cytokine release in an ELISA based assay, this will allow easier assessment of cellular immune responses in fish and could form the basis for potency testing of vaccines where protection is correlated with cellular immunity.

Animals↗