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Christopher J Secombes

Publications and source records attributed to Christopher J Secombes.

At least 37 records · Page 2Linked to original sources
PubMed2004

Trout CC chemokines: comparison of their sequences and expression patterns.

Several thousand EST sequences were recently made available in the EMBL sequence database from the rainbow trout Oncorhynchus mykiss. BLAST based searches were utilised to identify sequences resembling mammalian CC chemokines within these ESTs. Fifteen new and unique CC chemokine-like sequences were identified for trout, bringing the total of known CC chemokine sequences in trout to 18 when including those already published. Some of these trout chemokines appeared highly related (in pairs) suggesting recent duplication events or tight evolutionary constraints. Phylogenetically, the trout chemokine sequences grouped with both inducible and constitutive mammalian CC chemokine subtypes, suggesting early divergence of these functional groups. Expression analyses on gill and head kidney show constitutive expression of many of these trout CC chemokines in these lymphoid-rich tissues. However, induction of some of the chemokines structurally related to 'inducible' CC chemokines was observed in a trout macrophage-like cell line (RTS-11) in response to stimulation with recombinant TNFalpha.

Amino Acid Sequence↗
PubMed2004

Expression of immune response genes in rainbow trout skin induced by Gyrodactylus derjavini infections.

By means of semi-quantitative RT-PCR, expression of a number of immune relevant genes was studied in skin of small rainbow trout Oncorhynchus mykiss (Walbaum, 1792) fry during both primary and secondary infections with the ectoparasitic monogenean Gyrodactylus derjavini Mikailov, 1975. The target genes studied included the cyto- and chemokines TNF-alpha1, TNF-alpha2, TGF-beta and IL-8, the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) genes and finally, two cell markers, the beta-chains of TCR and MHC II, from the adaptive arm of the immune system. In general, constitutive expression of all studied genes was apparent. Significant increases in expression of the TNF-alpha1 isoform could be observed at day 8 p.i. in primary infections and although less marked, the alpha2 isoform of TNF showed a similar trend. With the cytokine TGF-beta, 8-10 times increase in the transcription levels was observed in secondary infections compared to uninfected hosts. However, no parasite related changes in expression patterns could be observed for IL-8. Parasite infections elicited strong iNOS expression by 4 days p.i., but significant differences were not detected before day 8 p.i., when transcript levels were increased 5.5-9.6 times compared to uninfected controls. Augmented expression of COX-2 could also be observed in primary, but not secondary, infections at later stages of infections. No clear parasite related changes in transcript levels of the two cell markers TCRbeta and MHC IIbeta could be observed. Although the cellular source(s) was not determined, most of the examined factors appear to take part in a local signalling network of pivotal importance for the initiation, orchestration, effectuation and modulation of immune responses in rainbow trout against the ectoparasite G. derjavini.

Animals↗
PubMed2004

CpG oligodeoxynucleotides stimulate immune cell proliferation but not specific antibody production in rainbow trout (Oncorhynchus mykiss).

Bacterial DNA and CpG ODN have both been shown to have immunostimulatory effects in mammals, activating APCs and inducing a potent Th1 type immune response. They have also been shown to have a strong adjuvant effect and up-regulate MHC class 2 expression in murine cells, augment human and murine NK cell lytic activity, activate human B cells and induce murine B cell proliferation. However, little work has been carried out with regard to their effects on the piscine immune system. Here it is shown that various CpG ODN induce proliferation of peripheral blood leucocytes, spleen and head kidney cells from rainbow trout although, at the range of concentrations tested CpG ODN 2133 lacked the ability to induce specific antibody production to a protein antigen.

Animals↗
PubMed2004

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout.

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3' UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge.

Aeromonas↗
PubMed2004

Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay.

We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.

Animals↗
PubMed2004

Sequencing and expression of the second allele of the interleukin-1beta1 gene in rainbow trout (Oncorhynchus mykiss): identification of a novel SINE in the third intron.

A lambda clone containing a rainbow trout IL-1beta1 gene was isolated by a PCR screening strategy from a genomic library cloned in lambda GEM-11, and an EcoRI fragment from this clone was fully sequenced, and contained 1680 bp 5'-flanking sequence, the whole IL-1beta1 gene open reading frame, and the 3'-flanking region with two potential poly A signals and poly A sites. This clone encoded a protein that shared 99.8% identity to the previously published trout IL-1beta1 cDNA sequence, with only three base substitutions. The main difference was that this clone had an additional complete HpaI SINE insertion in the 3rd intron making intron III 211 bp larger (834 bp via 623 bp). Thus this sequence was designated as allele B (Big intron III) of IL-1beta1 and the previously reported sequence as allele S (Short intron III). Three lines of evidence (allele specific PCR, cloning and sequencing, and direct sequencing of PCR products) revealed that allele B was constitutively expressed and could respond to stimulation with lipopolysaccharide or trout recombinant IL-1beta. Searching of the GenBank database with the HpaI SINE sequence resulted in three additional HpaI loci being identified in rainbow trout. Another SINE retroposition was also identified in the same intron of both alleles of IL-1beta1 by comparison with the trout IL-1beta2 gene. This novel SINE sequence, sharing high homology with the HpaI SINE at the 3'-end region, is present in EST databases of several species including human, mouse and fish. The consensus of this novel SINE shares 57 to 61% identities to tRNA-Leu from different species. Another older retroposition event in the same intron of IL-1beta1 has also been hypothesised, recognised as six adenines, that may function as a RNA polIII terminator. A model for the IL-1beta1 allele formation is proposed. Following the earliest retroposition into one of the two IL-1beta genes that resulted from a genome duplication in salmonids, the proper environment for successive PV SINE retroposition was created. A recent retroposition of the HpaI SINE in IL-1beta1 resulted in the formation of the two alleles of IL-1beta1. Examination of the SINEs insertion and their host gene microenvironments revealed that the SINE retroposition does not appear random, both in the site selection and the direction of insertion. The mechanism governing this outcome is discussed.

Alleles↗
PubMed2003

Complete sequencing and expression of three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway of the complement system in rainbow trout Oncorhynchus mykiss.

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M(r) of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M(r) of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a alpha-chain of 773 aa, a beta-chain of 635 aa and a lambda-chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M(r) of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.

Amino Acid Sequence↗
PubMed2003

Expression in Escherchia coli and purification of sea bass (Dicentrarchus labrax) interleukin 1beta, a possible immunoadjuvant in aquaculture.

Interleukin 1beta (IL-1beta) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses. Our group has recently cloned IL-1beta from sea bass (Dicentrarchus labrax), one of the main Mediterranean aquacultured fish species. The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. As for trout and carp IL-1beta precursor sequence, no candidate cut site for ICE (IL-1beta converting enzyme) enzyme was apparent in the alignments of sea bass IL-1beta with other mammalian IL-1betas. Nevertheless, a possible mature peptide could start at Ala86, giving a protein of 176 amino acids. The nucleotide sequence coding for this polypeptide was cloned into a pQE-30 expression vector. The plasmid was then transformed in Escherichia coli, and the recombinant protein was purified. Finally, we demonstrated that this purified recombinant IL-1beta was able to induce IL-1beta gene expression in a dose-dependent manner on cells purified from sea bass head kidney and could have immunoadjuvant effects in sea bass vaccination experiments.

Animals↗
PubMed2003

Effect of intraperitoneally administered IL-1beta-derived peptides on resistance to viral haemorrhagic septicaemia in rainbow trout Oncorhynchus mykiss.

The present work provides the first information concerning the immunostimulatory activity of trout interleukin (IL)-1beta-derived peptides in vivo. Previous studies have demonstrated the ability of 2 such peptides, referred to as P1 and P3, to up-regulate a range of important immune parameters in vitro. P1 corresponds to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and is analogous to a biologically active mammalian IL-1beta-derived peptide, whilst P3 was synthesised to complex with the IL-1 receptor and corresponds to fragment 197-206 (YRRNTGVDIS) of the trout sequence. Optimal migration of peritoneal leucocytes, peptide induced phagocytosis and intracellular respiratory burst activity occurred following intraperitoneal injection of 3.0 micromol of P3. Furthermore, resistance to viral haemorrhagic septicaemia virus (VHSV) was soon augmented (2 d) post-injection of P3.

Amino Acid Sequence↗
PubMed2003

Peculiar gene organisation and incomplete splicing of sea bass (Dicentrarchus labrax L) interleukin-1beta.

In this paper we describe the gene organisation of sea bass Dicentrarchus labrax interleukin-1beta (IL-1beta) and the presence of incompletely spliced forms of this gene. Interestingly, the sea bass IL-1beta gene comprises five exons and four introns, thus being remarkably different from other known teleost and mammalian genes. The sizes of the introns were typically much shorter than in mammals and this feature, together with the loss of two introns, gave a much smaller gene than in mammals (2.7 vs 6.5-7.0kb). The 5'-end exon-intron organisation seems quite different in the known IL-1beta genes with three exons in human and carp, two in trout and only one in sea bass. The highest percentage of exon identity and similarity is between sea bass exon 4, trout exon 5, carp, mouse, chicken, pig and human exon 6. RT-PCR analysis revealed the presence of two incompletely spliced transcripts. Southern blot analysis suggests the presence of only one copy of IL-1beta gene in sea bass genome.

Amino Acid Sequence↗
PubMed2003

Cloning and expression analysis of rainbow trout Oncorhynchus mykiss interferon regulatory factor 1 and 2 (IRF-1 and IRF-2).

Two rainbow trout (Oncorhynchus mykiss) genes for interferon regulatory factors (IRF) 1 and 2 have been cloned and sequenced. The IRF-1 cDNA contains an open reading frame (ORF) of 996 nucleotides that translates into a 331 amino-acid putative peptide, with a 5' untranslated region (UTR) of 145bp and a 3' UTR of 481bp. The IRF-2 cDNA contains a 1035bp ORF that translates into a 344 amino-acid putative peptide, with a 5' UTR of 146bp and a 3' UTR of 925bp. In vivo, IRF-1 and IRF-2 are constitutively expressed in head kidney, gill and spleen but not liver. Both genes were induced in all the tissues examined. IRF-1 but not IRF-2 expression was significantly increased at the site of injection 1 week after DNA vaccination against viral haemorrhagic septicaemia virus. In vitro, IRF-1 and IRF-2 transcripts are present in unstimulated rainbow trout gonad cells and are up-regulated by poly I/C.

Amino Acid Sequence↗
PubMed2003

The effect of intraperitoneally administered recombinant IL-1beta on immune parameters and resistance to Aeromonas salmonicida in the rainbow trout (Oncorhynchus mykiss).

The present work provides the first information concerning the immunostimulatory activity of trout recombinant interleukin-1 beta (rIL-1beta) in vivo. The predicted rainbow trout mature IL-1beta peptide was produced as a recombinant protein in Escherichia coli. Optimal migration of peritoneal leucocytes and rIL-1beta induced phagocytosis occurred following intraperitoneal injection of 1 microg of the recombinant protein. Moreover, systemic IL-1beta, COX-2 and lysozyme II gene expression was enhanced following >or=1 microg rIL-1beta administration. Finally, resistance to Aeromonas salmonicida, the causative agent of furunculosis, was augmented at early times (2 days) post-injection of 1 microg rIL-1beta.

Adjuvants, Immunologic↗
PubMed2003

Cloning and characterisation of a putative ST2L homologue from Atlantic salmon (Salmo salar).

The ST2L receptor is a member of the interleukin-1 (IL-1) receptor family and has previously been cloned from human, mouse, rat and chicken. This orphan receptor has no known physiological role but has been implicated in T helper cell type 2 effector function. We describe in this report the cloning and characterisation of a cDNA encoding a homologue of ST2L in Atlantic salmon (Salmo salar). The salmon ST2L cDNA is 2364bp in length and has an open reading frame encoding a polypeptide of 582 amino acids. Similar to other members of the IL-1 receptor (IL-1R) family, the predicted protein has a potential signal peptide, extracellular immunoglobulin-like domains, a short transmembrane region and a characteristic cytoplasmic Toll-IL-1R domain. The predicted protein shows 33% identity and 44% similarity to the chicken ST2L homologue. Phylogenetic analyses cluster the putative salmon ST2L with the chicken and the mammalian ST2L homologues, away from the other members of the IL-1R family. Salmon ST2L is constitutively expressed in brain, white and red blood cells, head kidney, liver, gills and muscle, with highest level of expression in spleen. In vivo stimulation of salmon with lipopolysaccaride does not appear to have a significant effect on expression of the ST2L homologue.

Animals↗
PubMed2002

Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss).

The upstream flanking region of the rainbow trout (Oncorhynchus mykiss) IL-1 beta 1 gene has been cloned and characterised functionally using luciferase-based reporter gene constructs, and the transcription start site (TSS) confirmed by RLM-RACE. A TATA box was present 27 bp upstream of the TSS, with an NF-kB site 19 bp upstream of the TATA box. Within 1217 bp of upstream sequence, 3 sites for NF-kB, 10 sites for NF-IL6, 15 sites for AP1, 6 sites for AP4, 2 sites for CHOP/CEBP alpha and 1 site for SP1 and PU.1 were identified. Seven potential sites for the transcription repressor Gfi-1 were also identified. Analysis of eight IL-1 beta 1s promoter luciferase constructs transfected into a trout fibroblast (RTG-2) cell line known to constitutively express IL-1 beta revealed that in the absence of intron 1, very low luciferase activity was detectable. All of the constructs containing intron 1 gave clear luciferase activity, with the highest luciferase activity detected with construct P2-4 containing 617 bp of upstream sequence. As little as 82 bp of upstream sequence gave relatively strong luciferase activity, a region containing both a PU.1 and NF-kB site. That NF-kB is a transcription factor required for expression of the trout IL-1 beta 1 gene was confirmed using inhibitor studies with lipopolysaccharide (LPS)-stimulated macrophages. Both trout recombinant IL-1 beta and LPS were able to increase luciferase activity in the reporter constructs, especially in those containing the most upstream sequence with the lowest constitutive expression. The possibility that an upstream repressor is functioning to inhibit constitutive expression of IL-1 beta in this species is discussed.

5' Flanking Region↗
PubMed2002

Towards selective breeding of Atlantic salmon for sea louse resistance: approaches to identify trait markers.

Several species of sea lice, in particular Lepeophtheirus salmonis (Krøyer), affect the welfare and condition of farmed salmon, with an estimated annual cost to the Scottish industry of 15-30 Pounds million. In Atlantic salmon, some stocks show resistance to L salmonis. Such natural resistance could be utilized for stock improvement using molecular genetic technologies. The development of molecular markers linked to resistance genes, allowing the identification of resistant fish, could increase the efficacy of selective breeding programmes. Various approaches to achieve this goal are described. One way to identify genes conferring resistance is to develop screens for salmon genes that are activated upon louse infection. One such screen--differential display--requires no previous knowledge of gene sequences, involves no preconceptions about which gene families are involved and can therefore identify novel genes. Preliminary results of comparative gene expression in sea-louse-challenged and control fish illustrate the application of differential display.

Animals↗
PubMed2002

Type I-interferon signalling in fish.

Type I interferon (IFN) signalling uses a dual mechanism of action. A Jak-Stat pathway extensively described in mammals involves a cascade of reactions from the interaction of the IFN molecule with its membrane receptor to the stimulation of IFN-induced gene promoters leading in turn to an antiviral state. Regulation of IFN activity is also mediated by two DNA-binding transcription factors called interferon regulatory factor (IRF)-1 and -2, that respectively stimulate and repress the promoter of IFN-induced genes. By gene walking with trout genomic DNA the regulatory sequence of the IRF-1 gene was cloned and sequenced. Sequence analysis showed that this 1 Kb 5' flanking region has a structure which is typical for an IFN-induced gene promoter. It contains a TATA box between -28 to -31, and a NF kappa B site between -41 and -52. No complete interferon stimulatory response element (ISRE) could be found, but ten GAAA motifs, which are characteristic of IFN-induced gene promoters, were found. In rainbow trout gonad (RTG) cells, IRF-1 is expressed constitutively and up-regulated by poly I:C but not by LPS. Transient transfections of RTG cells with a reporter construct based on the luciferase gene show that the IRF1 5' flanking region described above, is sufficient to allow the expression of luciferase and is capable of induction by dsRNA (poly I:C).

Animals↗
PubMed2002

Cloning of a Salmo salar interleukin-1 receptor-like cDNA.

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily, defined by a cytosolic Toll/IL-1R (TIR) signalling domain, participates in host responses to injury and infection. We describe in this study the cloning of a cDNA encoding a Salmo salar interleukin-1 receptor-like protein (SalIL-1RLP). SalIL-1RLP comprises a potential signal peptide, three extracellular immunoglobulin domains, a short transmembrane region and an intracellular region that contains the TIR domain. The predicted amino acid sequence of SalIL-1RLP displays 43-44% similarities and 31% identities to chicken and human IL-1RI sequences. Within the intracellular region, SalIL-1RLP displays highest similarity (59%) and identity (46%) to the chicken IL-1RI sequence. Two different 5' distal UTRs were identified among six salmon IL-1RLP clones. The six clones, however, displayed identical 5' proximal UTRs, coding regions and 3' UTRs. SalIL-1RLP expression is induced in liver, head kidney, spleen and gills upon injection of salmon with bacterial lipopolysaccharide. Sequence comparisons, protein domain structures, expression patterns and phylogenetic analyses indicate that SalIL-1RLP is most closely related to type I interleukin-1 receptors and interleukin-1 receptor related proteins.

Amino Acid Sequence↗