Biomedical subjects

Christopher J Secombes

Publications and source records attributed to Christopher J Secombes.

At least 19 recordsLinked to original sources

PubMed2006

Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Amino Acid Sequence↗

PubMed2006

Evolution of the CD4 family: teleost fish possess two divergent forms of CD4 in addition to lymphocyte activation gene-3.

The T cell coreceptor CD4 is a transmembrane glycoprotein belonging to the Ig superfamily and is essential for cell-mediated immunity. Two different genes were identified in rainbow trout that resemble mammalian CD4. One (trout CD4) encodes four extracellular Ig domains reminiscent of mammalian CD4, whereas the other (CD4REL) codes for two Ig domains. Structural motifs within the amino acid sequences suggest that the two Ig domains of CD4REL duplicated to generate the four-domain molecule of CD4 and the related gene, lymphocyte activation gene-3. Here we present evidence that both of these molecules in trout are homologous to mammalian CD4 and that teleosts encode an additional CD4 family member, lymphocyte activation gene-3, which is a marker for activated T cells. The syntenic relationships of similar genes in other teleost and non-fish genomes provide evidence for the likely evolution of CD4-related molecules in vertebrates, with CD4REL likely representing the primordial form in fish. Expression of both CD4 genes is highest in the thymus and spleen, and mRNA expression of these genes is limited to surface IgM- lymphocytes. consistent with a role for T cell functionality. Finally, the intracellular regions of both CD4 and CD4REL possess the canonical CXC motif involved in the interaction of CD4 with p56LCK, implying that similar mechanisms for CD4+ T cell activation are present in all vertebrates. Our results therefore raise new questions about T cell development and functionality in lower vertebrates that cannot be answered by current mammalian models and, thus, is of fundamental importance for understanding the evolution of cell-mediated immunity in gnathosomes.

Amino Acid Sequence↗

PubMed2006

A review of CpGs and their relevance to aquaculture.

CpG oligodeoxynucleotides (ODN) have been described as functioning as natural adjuvants because they promote professional antigen presenting cell (APC) function and co-stimulate lymphocytes. The majority of studies into the immune effects of CpG ODN to date have been carried out on mammals where they are proving very successful at stimulating innate and adaptive immune responses in a variety of species as well as protecting them from bacterial, viral and protozoan pathogens. Fish also possess the ability to raise both innate and adaptive immune responses to invading pathogens and interest in the effect of CpG ODN on the piscine immune system is growing. Various studies have now been carried out to elicit the effects of CpG ODN on diverse fish species showing that 31 different B-class CpG ODN exert various immune responses both in vivo and in vitro in salmonids, cyprinids and pleuronectiformes. These responses include activation of macrophages, proliferation of leucocytes and stimulation of cytokine expression. CpG ODN have also been shown to be protective against bacterial and viral challenge as well as against pathogenic amoebae. As would be expected these effects are all dependent on not only the ODN sequence and length but on the concentration and the species in which it is being used. This review provides the first comprehensive overview of all CpG ODN tested in fish to date and brings together all the work carried out in this field.

Adjuvants, Immunologic↗

PubMed2006

Infectious pancreatic necrosis virus suppresses type I interferon signalling in rainbow trout gonad cell line but not in Atlantic salmon macrophages.

RTG-P1 cells are a rainbow trout fibroblastic cell line permanently transfected with the luciferase gene under the control of the Mx promoter. On exposure to interferon (IFN) or IFN inducing agents, the cells produce luciferase. IPNV did not induce luciferase production up to 24h post-infection but did not suppress constitutive luciferase production. Furthermore, IPNV suppressed luciferase production induced by poly I:C. RT-PCR analysis of IPNV infected cells showed IFN gene transcription from 6h post-infection with increasing expression up to 24h. Housekeeping genes beta-actin and GAPDH were also expressed along with upregulation of IRF1 and slight upregulation of STAT1. When RTG-P1 cells were stimulated with IFN, Mx transcripts, measured by qRT-PCR, peaked at 3-6h and thereafter fell to low levels, but in the presence of IPNV, Mx transcription at this time was significantly suppressed but continued to rise gradually. Luciferase production was lower in infected cells at 12h post-infection but not significantly after 24h. These results indicate that, in non-stimulated RTG-P1 cells, while IPNV induces IFN transcription, activation of Mx expression is suppressed. Furthermore, when stimulated by IFN, the rate of Mx transcription is significantly suppressed by the virus. This would probably give time for the virus to replicate rapidly in the early phases of infection. Contrary to the fibroblastic cell line, IPNV stimulated IFN production by salmon macrophages in vitro at least as strongly as poly I:C, with no suppression of the IFN response to poly I:C, and the virus persisted for up to 9 days without causing CPE.

Animals↗

PubMed2006

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and atlantic salmon (Salmo salar).

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon IV. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Amino Acid Sequence↗

PubMed2005

Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus and member of the genus Isavirus, which contains eight genomic segments coding for ten viral proteins. This study focussed on identifying the function of the largest protein encoded by ISAV genomic segment 7 (7i), which like influenza A segment 7 encodes two proteins, one of which is based on removal of an intron from the primary transcript. Using two independent methods, an Mx1 promoter-driven reporter system and real-time PCR of FACS-sorted transfected cells, we demonstrate that the non-structural ISAV 7i protein is an interferon-signalling antagonist. Other transfection studies indicated a predominantly cytoplasmic localisation of the expressed protein, which is consistent with this role. The demonstration that ISAV segment 7 encodes a putative non-structural IFN system antagonist reveals a difference with influenza A virus, where segment 7, which shares a similar coding strategy, encodes the structural matrix proteins.

Animals↗

PubMed2005

Identification and expression analysis of lymphotoxin-beta like homologues in rainbow trout Oncorhynchus mykiss.

A lymphotoxin-beta (LT-beta) gene has been cloned and sequenced in rainbow trout and provides the first conclusive evidence for the existence of LT-beta in teleost. Two isoforms of LT-beta were isolated. LT-beta1 cDNA was composed of 952 bp (with a 139 bp 5'-UTR and a 201 bp 3'-UTR) and LT-beta2 cDNA was 836 bp (with a 237 bp 5'-UTR and a 197 bp 3'-UTR) both of which translated into a protein of 203 amino acid residues. Both isoforms contained a predicted transmembrane domain of 21 amino acid residues (Leu11-Val31) and the TNF family signature (Val104-Phe120). Homology and phylogenetic analysis of trout LT-beta's with other known TNF family member showed good similarity to TNF-N (teleost) and other LT-beta (mammals and frog). LT-beta1 and TNF-alpha (1 and 2) genes were highly expressed in unstimulated trout head kidney, spleen, gill and intestine, whereas LT-beta2 was weakly expressed only in the gill. The expression of LT-beta1 and -beta2 genes was not found in macrophage (RTS-11) and fibroblast (RTG-2) like cell lines, although the TNF-alpha2 gene was expressed in both cell lines with the TNF-alpha1 gene only expressed in RTS-11 cells. In head kidney cells, expression of LT-beta1 and TNF-alpha (1, 2) genes was increased by stimulation with PHA or LPS. The discovery of trout LT-beta will allow a more complete analysis of fish inflammatory responses.

Adjuvants, Immunologic↗

PubMed2005

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species.

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Amino Acid Sequence↗

PubMed2005

Biological activity of sea bass (Dicentrarchus labrax L.) recombinant interleukin-1beta.

Biological activities of a putative mature sea bass interleukin-1beta peptide, produced as a recombinant protein (rIL-1beta) in Escherichia coli, have been investigated. The rIL-1beta contains a 6-histidine tag at the N-terminus, and protein purification has been achieved through this tag by affinity chromatography. Biological activities have been investigated both at the cellular and gene expression levels. In in vitro assays sea bass rIL-1beta induced the proliferation of murine D10.G4.1 cells and increased yeast phagocytosis by sea bass head kidney leukocytes. The purified cytokine was also tested in a lymphocyte-activation factor assay, where it induced the proliferation of sea bass thymocytes. Finally, in an in vivo assay, rIL-1beta administered intraperitoneally increased expression levels of the IL-1beta gene and activated macrophages to produce a cyclooxygenase 2 homologue (COX-2) gene in the head kidney.

Analysis of Variance↗

PubMed2005

Construction and analysis of a secreting expression vector for fish cells.

A new expression plasmid (pcDNA3-LP) was designed to produce and secrete proteins in fish cells by fusion with the rainbow trout TGF-beta leader peptide. The luciferase reporter gene was used to test the secreting ability of this vector. Secreting (pcDNA3-LP-LUC) and non-secreting (pcDNA3-LUC) constructs were made and compared in transient transfection experiments in salmonid (RTG-2) and cyprinid (EPC) cell lines. The amount of luciferase secreted into the supernatants of RTG-2 or EPC cells transiently transfected with pcDNA3-LP-LUC relative to cells transfected with pcDNA3-LUC was 7- and 85-fold, respectively. Two stable clones of EPC transfected with pcDNA3-LUC and four clones transfected with pcDNA3-LP-LUC were isolated. Approximately 90% of the total luciferase activity produced was secreted by stable EPC clones containing pcDNA3-LP-LUC whereas only 5% of total activity was secreted by clones containing pcDNA3-LUC. The two constructs were injected intra-muscularly into rainbow trout and the luciferase activity present in the serum of fish determined. The luciferase activity in serum from fish injected with pcDNA3-LP-LUC was 2.7-fold higher (P<0.05) than that fish injected with pcDNA3-LUC. This new vector opens up opportunities in fish DNA vaccinology and in the production of fish recombinant proteins.

Amino Acid Sequence↗

PubMed2005

Isolation and characterization of CXC receptor genes in a range of elasmobranchs.

The CXC group of chemokines exert their cellular effects via the CXCR group of G-protein coupled receptors. Six CXCR genes have been identified in humans (CXCR1-6), and homologues to some of these have been isolated from a range of vertebrate species. Here we isolate and characterize CXCR genes from a range of elasmobranch species. One CXCR1/2 gene fragment isolated from Scyliorhinus caniculus (lesser spotted catshark), and two CXCR1/2 copies from each of the elasmobranchs, Cetorhinus maximus (basking shark), Carcharodon carcharias (great white shark), and Raja naevus (cuckoo ray), exhibit high similarity to both CXCR1 and CXCR2. The two copies evident in the cuckoo ray and lamniform sharks provide strong evidence of CXCR1/2 lineage specific duplication in rays and sharks. A CXCR fragment isolated from Lamna ditropis (salmon shark) shows high similarity to a range of CXCR4 genes and strong clustering with CXCR4 gene homologues was apparent during phylogenetic reconstruction.

Amino Acid Sequence↗

PubMed2005

Cloning of the Atlantic salmon (Salmo salar) IL-1 receptor associated protein.

In mammals, the pro-inflammatory cytokine interleukin-1 signals through a receptor complex containing a type I interleukin-1 receptor (IL-1RI) and a receptor associated protein (IL-1RAcP). Previously, we have described a cDNA from Atlantic salmon encoding a molecule with homology to the mammalian IL-RI. This molecule was named IL-1 receptor like protein (IL-1RLP) in the absence of functional data to support its proposed role as the salmon IL-1RI. Here, we describe the cloning and characterisation of a cDNA encoding salmon IL-1RAcP. Like other members of the IL-1R family, the salmon IL-1RAcP encodes three extracellular immunoglobulin-like domains and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain involved in signalling. Specific binding of salmon IL-1RAcP to IL-1RLP was shown by co-immunoprecipitation studies.

Amino Acid Sequence↗

PubMed2005

Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss.

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. It was believed to be restricted to mammals, but here we report the first nonmammalian IL-11 gene, in rainbow trout (Oncorhynchus mykiss). A trout IL-11 cDNA clone was isolated that contains a 5'-untranslated region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3'-UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda library revealed that the trout IL-11 gene has the same five exon/four intron gene organization, as well as the same intron phase, as mammalian IL-11 genes. The 204 amino acid trout IL-11 translation has a predicted signal peptide of 26 amino acids and mature peptide of 178 amino acids, with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83. The mature peptide contains a cysteine residue and a potential N-linked glycosylation site that are not present in mammals. Phylogenetic analysis clearly grouped trout IL-11 with IL-11 molecules from other species and separated from other members of the IL-6 family. The IL-11 gene is highly expressed in intestine and gills in healthy fish and its expression can also be detected in spleen, head kidney, brain, skin and muscle. Bacterial infection of rainbow trout markedly up-regulates IL-11 expression in liver, head kidney and spleen. IL-11 expression is also up-regulated in RTS-11 cells (a trout macrophage cell line), which constitutively expressed the lowest level of IL-11 of the four trout cell lines examined, after stimulation with bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL-1beta. Only a single transcript of 3.2 kb could be detected in lipopolysaccharide or recombinant IL-1beta-stimulated RNA samples by northern blotting. The expression results, showing that IL-11 is widely distributed and modulated by infection and other cytokines, suggest that fish IL-11 is an active player in the cytokine network and the host immune response to infection.

Amino Acid Sequence↗

PubMed2005

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss.

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I x C) also expressed rtCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Animals↗

PubMed2005

Polychlorinated biphenyl 126 affects expression of genes involved in stress-immune interaction in primary cultures of rainbow trout anterior kidney cells.

Stress and immune function are linked in all vertebrates, including teleost fish. Polychlorinated biphenyls (PCBs) are immunotoxic and impair the ability of fish to respond to additional stressors. In this study, we investigated the effects of PCB126 on stress and immune function and the interaction of these systems in fish using primary cultures of rainbow trout anterior kidney cells as a model. Gene expression levels of cytochrome P4501A (CYP1A), interleukin-1beta (IL-1beta), and glucocorticoid receptor (GR) were measured by real-time quantitative polymerase chain reaction. These genes play important roles in detoxification and immune and stress homeostasis, respectively. Incubation with PCB126 led to increased IL-1beta expression between 30 min and 2 h of exposure, with expression back to basal levels after 6 h. Lipopolysaccharide (LPS) incubation evoked normal IL-1beta responses after 2 and 24 h PCB incubation. Gene expression levels of GR and CYP1A increased in a time- and dose-dependent manner, reaching a plateau after 12 h of incubation. Preincubation with cortisol resulted in decreased IL-1beta expression, increased expression of CYP1A and GR, and was accompanied by an abolished PCB responsiveness after more than 4 h of cortisol incubation. We conclude that PCB126 exposure is not "stressful," as increased cortisol levels would result in depressed IL-1beta expression. Incubation with PCB126 evokes a transient stimulation rather than permanent damage of the immune system, as LPS stimulation resulted in increased IL-1beta expression after PCB incubation. Prolonged cortisol preincubation, resembling a chronic stress paradigm, negatively affects the immune responsiveness of the cells as well as their capacity for toxicant metabolization.

Animals↗

PubMed2004

Chemokines.

Chemokines are small proteins that control cellular migration. An extensive family of these molecules has been described in mammals containing nearly 50 members. Within this family are four groups, each defined by the different spacing of two N-terminal cysteines, which form disulphide bonds with two other cysteine residues to create the tertiary structure characteristic of chemokines. Recent evidence shows the chemokine family is not unique to mammals, with several members also identified in birds, amphibians and fish, including a primitive vertebrate, the lamprey. Although there is less evidence to define the roles of chemokines in these lower vertebrates, structural similarities allow some predictions to their function, against which further studies are being made. Additionally, some microorganisms (particularly viruses) appear to have copied genes for chemokines, presumably to confuse the immune system of their host. This review aims to bring together the current information concerning identified chemokines throughout vertebrates and microorganisms.

Amino Acid Sequence↗

PubMed2004

Modelling of fish interleukin-1 and its receptor.

This paper presents original data regarding: the three-dimensional modelling of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) IL-1beta, the modelling of trout IL-1 receptor type I (IL-1RI) and the modelling of trout IL-1beta bound to its receptor. The 3D models of trout and sea bass IL-1beta molecules were predicted by comparison with those already available of human and mouse, and, in both cases, a structure consisting of a beta-trefoil fold was obtained, a motif well conserved during evolution of IL-1beta-related proteins. Moreover, a model for the rainbow trout IL-1 receptor alone and complexed with IL-1beta was predicted and compared to the murine model. Both ligand-receptor complex models were compared with the known crystal structure of the human IL-1beta/IL-1R complex. A cross-interaction of trout IL-1beta with the mouse receptor was also simulated. Such analysis and the predicted interaction energies calculated from the models have helped to explain the different biological efficacies of mammalian and fish IL-1beta molecules in assays based on mammalian target cells. The obtained data are discussed on the basis of evolutionary and applied perspectives.

Amino Acid Sequence↗

PubMed2004

An Mx1 promoter-reporter system to study interferon pathways in rainbow trout.

A rainbow trout interferon (IFN) reporter system has been established by selection of a stable cell line, RTG-P1, transfected with a plasmid expressing the firefly luciferase gene under the control of the promoter for the IFN-induced gene Mx1. After 148 passages, the luciferase expression was still highly induced by polyinosinic:polycytidylic acid (poly I:C) in RTG-P1 cells. Different IFN inducers (dsRNA, viral hemorrhagic septicaemia virus or conditioned medium containing rainbow trout antiviral activity) were able to stimulate the IFN-reporter system in RTG-P1, showing that this cell line can be used to study the activation of the IFN pathway in various contexts. Pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor, significantly blocked poly I:C induced luciferase accumulation in RTG-P1 at intermediate doses (1-10 microM), suggesting that Mx1 induction through the IFN signalling pathway is NF-kappaB-dependent in fish. This inhibition was not observed for doses of 50 microM or higher. The RTG-P1 reporter system constitutes an interesting tool to study the induction and regulation of IFN signalling in teleost fish.

Animals↗