Publications by authors named "René J M Stet"

27 Publications

  • Page 1 of 1

Varying disease-mediated selection at different life-history stages of Atlantic salmon in fresh water.

Evol Appl 2011 Nov 4;4(6):749-62. Epub 2011 Jul 4.

Scottish Fish Immunology Research Centre, University of Aberdeen Zoology Building, Tillydrone Avenue, Aberdeen, UK.

Laboratory studies on associations between disease resistance and susceptibility and major histocompatibility (MH) genes in Atlantic salmon Salmo salar have shown the importance of immunogenetics in understanding the capacity of populations to fight specific diseases. However, the occurrence and virulence of pathogens may vary spatially and temporally in the wild, making it more complicated to predict the overall effect that MH genes exert on fitness of natural populations and over several life-history stages. Here we show that MH variability is a significant determinant of salmon survival in fresh water, by comparing observed and expected genotype frequencies at MH and control microsatellite loci at parr and migrant stages in the wild. We found that additive allelic effects at immunogenetic loci were more likely to determine survival than dominance deviation, and that selection on certain MH alleles varied with life stage, possibly owing to varying pathogen prevalence and/or virulence over time. Our results highlight the importance of preserving genetic diversity (particularly at MH loci) in wild populations, so that they have the best chance of adapting to new and increased disease challenges as a result of projected climate warming and increasing aquaculture.
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http://dx.doi.org/10.1111/j.1752-4571.2011.00197.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352546PMC
November 2011

Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family.

BMC Genomics 2010 Dec 9;11:697. Epub 2010 Dec 9.

Scottish Fish Immunology Research Centre, University of Aberdeen, UK.

Background: Several novel immunoglobulin-like transcripts (NILTs) which have previously been identified in the salmonid species rainbow trout (Oncorhynchus mykiss) contain either one or two extracellular Ig domains of the V-type. NILTs also possess either an immunoreceptor tyrosine-based activating motif (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region resulting in different signalling abilities. Here we report for the first time the genomic organisation and structure of the multigene family of NILTs in Atlantic salmon (Salmo salar) using a BAC sequencing approach.

Results: We have identified six novel Atlantic salmon NILT genes (Ssa-NILT1-6), two pseudogenes (Ssa-NILTp1 and Ssa-NILTp2) and seven genes encoding putative transposable elements in one BAC covering more than 200 kbp. Ssa-NILT1, 2, 4, 5 and 6 contain one Ig domain, all having a CX₃C motif, whereas Ssa-NILT3 contains two Ig domains, having a CX₆C motif in Ig1 and a CX₇C motif in Ig2. Atlantic salmon NILTs possess several ITIMs in the cytoplasmic region and the ITIM-bearing exons are in phase 0. A comparison of identity between the amino acid sequences of the CX₃C Ig domains from NILTs varies from 77% to 96%. Ssa-NILT1, 2, 3 and 4 were all confirmed to be expressed either by their presence in EST databases (Ssa-NILT1) or RT-PCR (Ssa-NILT2, 3, and 4) using cDNA as template. A survey of the repertoire of putative NILT genes in a single individual revealed three novel genes (Ssa-NILT7-9) represented by the Ig domain, which together with Ig domains from Ssa-NILT1-6 could be divided into different groups based on specific motifs.

Conclusions: This report reveals a tightly clustered, multigene NILT family in Atlantic salmon. By screening a highly redundant Atlantic salmon BAC library we have identified and characterised the genomic organisation of six genes encoding NILT receptors. The genes show similar characteristics to NILTs previously identified in rainbow trout, having highly conserved cysteines in the Ig domain and several inhibitory signalling motifs in the cytoplasmic region. In a single individual three unique NILT Ig domain sequences were discovered at the genomic DNA level, which were divided into two different groups based on a four residue motif after the third cysteine. Our results from the BAC screening and analysis on the repertoire of NILT genes in a single individual indicates that many genes of this expanding Ig containing NILT family are still to be discovered in fish.
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http://dx.doi.org/10.1186/1471-2164-11-697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022915PMC
December 2010

Rainbow trout (Oncorhynchus mykiss) possess multiple novel immunoglobulin-like transcripts containing either an ITAM or ITIMs.

Dev Comp Immunol 2009 Apr 12;33(4):525-32. Epub 2008 Nov 12.

Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK.

The recognition of pathogens by the innate immune system relies on a wide range of inhibitory and activating receptors. Some of these non-rearranging receptors belong to the immunoglobulin superfamily (IgSF) and in teleost fish the novel immune-type receptor (NITR) and the novel immunoglobulin-like transcript (NILT) have been reported. Here we describe the identification and characterisation of three new NILTs from rainbow trout (Oncorhynchus mykiss), with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain. The cytoplasmic regions contain either immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or an immunoreceptor tyrosine-based activating motif (ITAM) for downstream signalling. Alignment of the various NILT Ig domains revealed a high similarity, especially between Ig domains from NILTs found in this study. Furthermore, a phylogenetic tree showed that NILTs are more closely related to the triggering receptor expressed on myeloid (TREM) cells and NKp44 than to NITRs. The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues. Expression of NILTs was also shown to be an early event in development, with both eyed eggs and embryos expressing all four genes. The results obtained in this study and future experiments will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in rainbow trout.
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http://dx.doi.org/10.1016/j.dci.2008.10.002DOI Listing
April 2009

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles.

Proc Biol Sci 2009 Feb;276(1656):559-66

School of Biological Sciences, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, UK.

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.
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http://dx.doi.org/10.1098/rspb.2008.1257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592554PMC
February 2009

Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes.

Fish Shellfish Immunol 2009 Mar 6;26(3):352-8. Epub 2008 Sep 6.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Gołysz, Zaborze, ul. Kalinowa 2, 43-520 Chybie, Poland.

In cyprinids, two paralogous groups of major histocompatibility (MH) class II B genes, DAB1 and DAB3, have been reported but have not been studied in detail. In our study on MH association with immune responsiveness in common carp (Cyprinus carpio L.) we have taken a long-term approach using divergent selection for antibody production. We report the co-segregation of Cyca-DAB1-like and Cyca-DAB3-like genes with antibody response, in backcrosses to high- and low-responsive parental carp lines. We show that the presence of Cyca-DAB1-like, but not Cyca-DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp. Background genes other than Cyca-DAB genes also influenced the level of antibody response. Our data support the hypothesis of a genetic control by Cyca-DAB genes on the antibody response measured. We could not detect an association of the Cyca-DAB genes with disease resistance to the parasite Trypanoplasma borreli. Sequence information, constitutive transcription levels and our co-segregation data indicate that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. Previously defined differences in allelic diversity between Cyca-DAB1-like genes, especially, identify Cyca-DAB1 as the most interesting DAB gene for further study in common carp.
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http://dx.doi.org/10.1016/j.fsi.2008.08.011DOI Listing
March 2009

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.).

Fish Shellfish Immunol 2008 Jun 5;24(6):734-44. Epub 2007 Dec 5.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Golysz, Zaborze, ul. Kalinowa 2, Chybie, Poland.

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.
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http://dx.doi.org/10.1016/j.fsi.2007.11.015DOI Listing
June 2008

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity.

Fish Shellfish Immunol 2008 Apr 11;24(4):459-66. Epub 2008 Jan 11.

Department of Food Science and Nutritional Engineering, China Agricultural University, 100083, Beijing, PR China.

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of beta2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasabeta2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.
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http://dx.doi.org/10.1016/j.fsi.2008.01.003DOI Listing
April 2008

Peptide-binding motif prediction by using phage display library for SasaUBA*0301, a resistance haplotype of MHC class I molecule from Atlantic Salmon (Salmo salar).

Mol Immunol 2008 Mar;45(6):1658-64

Department of Food Science and Nutritional Engineering, China Agricultural University, 100083 Beijing, PR China.

The structure of the peptide-binding specificity of major histocompatibility complex (MHC) class I has been analyzed extensively in human and mouse. For fish, there are no crystallographic models of MHC molecules, neither are there data on the peptide-binding specificity. In this study, we describe for the first time the identification of a fish class I peptide-MHC ligand binding motif. Phage display technology using both 7 mer and 12 mer libraries enabled us to identify peptide ligands with unique specificity that interacts with the recombinant Salmon MHC class I molecule. The recombinant proteins, beta 2m/SasaUBA*0301, were produced in Escherichia coli, in which the carboxyl terminus of beta 2-microglobulin is joined together with a flexible (GGGGS)3 linker to the amino terminus of the heavy chain. One hundred and seven individual phages bound to beta 2m/SasaUBA*0301 were isolated after four rounds of panning from the 7 mer random-peptide library. The peptide encoding sequences were determined and peptide alignment led to the prediction of position-specific anchor residue. A prominent proline at position 2 was observed and we predict that it might be one of the anchors at the N-terminus. Meanwhile, phage display peptide library encoding random 12 mer peptides was also screened against beta 2m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon class I molecule might have a very similar binding motif at the C-terminus compared with a known mouse class I molecule H2-Kb which has L, or I, V, M at p8. Previous work showed that Atlantic Salmon carrying the allele SasaUBA*0301 are resistant to infectious Salmon aneamia virus and there is a significant association between MHC polymorphism and the disease resistance. Therefore, our study might contribute to designing a peptide vaccine against this viral disease.
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http://dx.doi.org/10.1016/j.molimm.2007.10.014DOI Listing
March 2008

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II beta chain from sea bass (Dicentrarchus labrax L.).

Fish Shellfish Immunol 2007 Oct 24;23(4):853-66. Epub 2007 Mar 24.

Dipartimento di Scienze Ambientali, University of Tuscia, Largo dell'Università snc, I-01100, Viterbo, Italy.

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.
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http://dx.doi.org/10.1016/j.fsi.2007.03.013DOI Listing
October 2007

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon.

Fish Shellfish Immunol 2007 Sep 19;23(3):504-13. Epub 2006 Sep 19.

Cell Biology and Immunology Group, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands.

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.
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http://dx.doi.org/10.1016/j.fsi.2006.08.018DOI Listing
September 2007

Natural selection acts on Atlantic salmon major histocompatibility (MH) variability in the wild.

Proc Biol Sci 2007 Mar;274(1611):861-9

Aquaculture and Catchment Management Services, Marine Institute, Newport, Co Mayo, Ireland.

Pathogen-driven balancing selection is thought to maintain polymorphism in major histocompatibility (MH) genes. However, there have been few empirical demonstrations of selection acting on MH loci in natural populations. To determine whether natural selection on MH genes has fitness consequences for wild Atlantic salmon in natural conditions, we compared observed genotype frequencies of Atlantic salmon (Salmo salar) surviving in a river six months after their introduction as eggs with frequencies expected from parental crosses. We found significant differences between expected and observed genotype frequencies at the MH class II alpha locus, but not at a MH class I-linked microsatellite or at seven non-MH-linked microsatellite loci. We therefore conclude that selection at the MH class II alpha locus was a result of disease-mediated natural selection, rather than any demographic event. We also show that survival was associated with additive allelic effects at the MH class II alpha locus. Our results have implications for both the conservation of wild salmon stocks and the management of disease in hatchery fish. We conclude that natural or hatchery populations have the best chance of dealing with episodic and variable disease challenges if MH genetic variation is preserved both within and among populations.
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http://dx.doi.org/10.1098/rspb.2006.0053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2093966PMC
March 2007

Head kidney-derived macrophages of common carp (Cyprinus carpio L.) show plasticity and functional polarization upon differential stimulation.

J Immunol 2006 Jul;177(1):61-9

Cell Biology and Immunology, Wageningen Institute of Animal Sciences, Wageningen University, 6700 AH Wageningen, The Netherlands.

Cells from the myeloid lineage are pluripotent. To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we phenotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synthase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish.
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http://dx.doi.org/10.4049/jimmunol.177.1.61DOI Listing
July 2006

Biocomplexity in a highly migratory pelagic marine fish, Atlantic herring.

Proc Biol Sci 2006 Jun;273(1593):1459-64

Dalhousie University, Department of Biology, Halifax, NS B3H 4J1, Canada.

The existence of biologically differentiated populations has been credited with a major role in conferring sustainability and in buffering overall productivity of anadromous fish population complexes where evidence for spatial structure is uncontroversial. Here, we describe evidence of correlated genetic and life history (spawning season linked to spawning location) differentiation in an abundant and highly migratory pelagic fish, Atlantic herring, Clupea harengus, in the North Sea (NS) and adjacent areas. The existence of genetically and phenotypically diverse stocks in this region despite intense seasonal mixing strongly implicates natal homing in this species. Based on information from genetic markers and otolith morphology, we estimate the proportional contribution by NS, Skagerrak (SKG) and Kattegat and western Baltic (WBS) fish to mixed aggregations targeted by the NS fishery. We use these estimates to identify spatial and temporal differences in life history (migratory behaviour) and habitat use among genetically differentiated migratory populations that mix seasonally. Our study suggests the existence of more complex patterns of intraspecific diversity than was previously recognized. Sustainability may be compromised if such complex patterns are reduced through generalized management (e.g. area closures) that overlooks population differences in spatial use throughout the life cycle.
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http://dx.doi.org/10.1098/rspb.2005.3463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1560315PMC
June 2006

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005.

Immunogenetics 2006 Jan 3;57(12):953-8. Epub 2006 Jan 3.

Immunology Division, Institute for Animal Health, Compton, RG20 7NN, UK.

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Göttingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.
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http://dx.doi.org/10.1007/s00251-005-0071-4DOI Listing
January 2006

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44.

Immunogenetics 2005 Apr 9;57(1-2):77-89. Epub 2005 Feb 9.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands.

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.
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http://dx.doi.org/10.1007/s00251-005-0771-9DOI Listing
April 2005

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species.

Immunogenetics 2005 Mar 8;56(12):894-908. Epub 2005 Feb 8.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands.

The 16 African 'large' barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.
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http://dx.doi.org/10.1007/s00251-005-0767-5DOI Listing
March 2005

The molecular evolution of the interleukin-1 family of cytokines; IL-18 in teleost fish.

Dev Comp Immunol 2004 May;28(5):395-413

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands.

The cytokine network is an important homeostatic system with potent activities in immune surveillance, growth, developmental and repair processes. Although interleukin-1beta (IL-1beta) is considered a pivotal pro-inflammatory cytokine, merely focussing on its inflammatory role would be too narrow an approach. Elucidation of the human, the mouse and the Fugu rubripes (pufferfish) genome now enables a more comprehensive overview of this cytokine family and its receptors in several vertebrate classes. Phylogenetic analyses of the IL-1 family members, comprising over 80 sequences of various fish, amphibian, avian and mammalian species, reveal that for only a few mammalian IL-1 family members unambiguous orthologues have been found in fish, indicating a recent origin of some of the mammalian IL-1 family members. Interestingly, the Fugu genome did reveal teleost orthologues for IL-18 and its putative receptor complex. All teleost IL-1beta sequences cluster separately from IL-1beta sequences of other species. In contrast, a number of IL-1 receptor family members have well conserved fish orthologues. This supports the concept of an ancestral role of this family, possibly in the brain.
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http://dx.doi.org/10.1016/j.dci.2003.09.005DOI Listing
May 2004

Major histocompatibility lineages and immune gene function in teleost fishes: the road not taken.

Crit Rev Immunol 2003 ;23(5-6):441-71

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands.

It has become increasingly clear over the course of the past decade that the immune system genes of teleosts and tetrapods are plainly derived from common ancestral genes. The last 5 years, however, have also made it abundantly clear that in the teleost genome some of these genes are organized in a manner very different from that seen in mammals. These differences are probably the result of differences in life history traits, such as fecundancy, within each group of species when faced with an evolutionary fork in the road shortly after their divergence from each other. One group, the tetrapods, including mammals, chose a highly organized linked major histocompatibility complex, while in teleosts the major histocompatibility genes remained unlinked. In this review we will discuss the structural and functional implications of this different organization, particularly for major histocompatibility genes, but drawing on the current knowledge of some other genes for further support for the hypothesis that each group took a different road, one more traveled and one less taken.
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http://dx.doi.org/10.1615/critrevimmunol.v23.i56.50DOI Listing
October 2004

Analysis of genomic and expressed major histocompatibility class Ia and class II genes in a hexaploid Lake Tana African 'large' barb individual (Barbus intermedius).

Immunogenetics 2004 Feb 15;55(11):770-81. Epub 2004 Jan 15.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands.

Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC class I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some class II B loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed class II MHC genes in a hexaploid African 'large' barb. This prompted us to study the number of MHC genes present in the genome of an African 'large' barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic class Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of class I and II B genes. Our study revealed three beta2-microglobulin, five class Ia, four class II A, and four class II B genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The class Ia and class II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.
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http://dx.doi.org/10.1007/s00251-003-0635-0DOI Listing
February 2004

Differential expression and haplotypic variation of two interleukin-1beta genes in the common carp (Cyprinus carpio L.).

Cytokine 2003 Apr;22(1-2):21-32

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O. Box 338, 6700 NH Wageningen, The Netherlands.

Interleukin-1beta (IL-1beta) is a central component in innate immunity and the inflammatory response of mammals. Only recently, the first non-mammalian IL-1beta sequences were published. In this study, we describe a second IL-1beta sequence (IL-1beta2) in carp with 74% amino acid identity to the carp IL-1beta1 sequence. The existence of two IL-1beta copies in the carp genome probably originates from the tetraploid nature of the species. In contrast to the first carp IL-1beta sequence, IL-1beta2 is represented by multiple genes with 95-99% identity. Detection of several IL-1beta2 sequences within individual homozygous fish suggests the presence of multiple copies of the IL-1beta2 gene in the carp genome, possibly as a result of subsequent gene duplication of IL-1beta2. In vivo, constitutive mRNA expression of both IL-1beta genes was found in healthy carp. IL-1beta2 mRNA expression could be up-regulated in head kidney cells similar to carp IL-1beta1, in vivo by infection with Trypanoplasma borreli and in vitro by stimulation with lipopolysaccharide (LPS). Cortisol, the major glucocorticoid in fish, is an endocrine-derived fator mediating IL-1beta expression. Although constitutive IL-1beta expression was inhibited by a physiological dose of cortisol, cortisol synergistically enhanced LPS-induced IL-1beta expression in carp. Involvement of the transcription factor nuclear factor (NF)-kappaB in expression of IL-1beta1 and IL-1beta2 was demonstrated. Ratio of IL-1beta expression was determined and this showed IL-1beta1 mRNA expression to be at least tenfold higher compared with IL-1beta2. The possibilities of IL-1beta2 being a functional gene or approaching pseudogene status are discussed.
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http://dx.doi.org/10.1016/s1043-4666(03)00102-9DOI Listing
April 2003

MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar); facing pathogens with single expressed major histocompatibility class I and class II loci.

Immunogenetics 2003 Jul 17;55(4):210-9. Epub 2003 Jun 17.

Department of Morphology, Genetics and Aquatic Biology, Section of Genetics, Norwegian School of Veterinary Science, Ullevaalsveien 72, P.O. Box 8146 Dep., 0033 Oslo, Norway.

Few studies have yet addressed the functional aspects of MHC molecules in fish. To lay the foundation for this, we evaluated the association between disease resistance and MHC class I and class II polymorphism in Atlantic salmon. Standardized disease challenge trials were performed on a semi-wild Atlantic salmon population with subsequent MHC typing and statistical analysis. The pathogens employed were infectious salmon anaemia virus (ISAV) causing infectious salmon anaemia and the Aeromonas salmonicida bacteria causing furunculosis. The material consisted of 1,182 Atlantic salmon from 33 families challenged with A. salmonicida and 1,031 Atlantic salmon from 25 families challenged with ISAV. We found highly significant associations between resistance towards infectious diseases caused by both pathogens and MH class I and class II polymorphism in Atlantic salmon. The observed associations were detected due to independently segregating MH class I and class II single loci, and inclusion of a large number of fish allowing an extensive statistical analysis.
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http://dx.doi.org/10.1007/s00251-003-0567-8DOI Listing
July 2003

Molecular evolution of CXC chemokines: extant CXC chemokines originate from the CNS.

Trends Immunol 2003 Jun;24(6):307-13

Cell Biology and Immunology Group, Wageningen University, PO Box 338, 6700 AH, The Netherlands.

The mammalian CXC chemokine system comprises 16 ligands and six receptors, and its actions stretch well beyond the immune system. Recent elucidation of the pufferfish genome, a representative of an evolutionary ancient vertebrate class, has enabled analysis of the mammalian CXC chemokine system in a phylogenetic context. Comparison of the phylogenies of vertebrate CXC chemokines revealed that fish and mammals have found different solutions to similar problems, grafted on the same basic structural motif. Phylogenetic analyses showed that the large, highly redundant CXC chemokine family is a very recent phenomenon that is exclusive to higher vertebrates. Moreover, its ancestral role is found within the central nervous system and not within the immune system.
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http://dx.doi.org/10.1016/s1471-4906(03)00120-0DOI Listing
June 2003

Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance?

Dev Comp Immunol 2003 Jan;27(1):29-41

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands.

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3' UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.
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http://dx.doi.org/10.1016/s0145-305x(02)00064-2DOI Listing
January 2003

The major histocompatibility class I locus in Atlantic salmon (Salmo salar L.): polymorphism, linkage analysis and protein modelling.

Immunogenetics 2002 Nov 2;54(8):570-81. Epub 2002 Oct 2.

Department of Morphology, Genetics and Aquatic Biology, Section of Genetics, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., 0033 Oslo 1, Norway.

A cDNA library screening using the conserved exon 4 of Atlantic salmon Mhc class I as probe provided the basis for a study on Mhc class I polymorphism in a breeding population. Twelve different alleles were identified in the 82 dams and sires studied. No individual expressed more than two alleles, which corresponded to the diploid segregation patterns of the polymorphic marker residing within the 3'-untranslated tail. Close linkage between the Sasa-UBA and Sasa-TAP2B loci strengthens the claim that Sasa-UBA is the major Mhc class I locus in Atlantic salmon. We found no evidence for a second expressed classical or non-classical Mhc class I locus in Atlantic salmon. A phylogenetic analysis of salmonid Mhc class I sequences showed domains conserved between rainbow trout, brown trout and Atlantic salmon. Evidence for shuffling of the alpha(1) domain was identified and lineages of the remaining alpha(2) through the cytoplasmic tail gene segment can be defined. The coding sequence of one allele was found associated with two different markers, suggesting recombination within the 3'-tail dinucleotide repeat itself. Protein modelling of several Sasa-UBA alleles shows distinct differences in their peptide binding domains and enables a further understanding of the functionality of the high polymorphism.
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http://dx.doi.org/10.1007/s00251-002-0499-8DOI Listing
November 2002

Unique haplotypes of co-segregating major histocompatibility class II A and class II B alleles in Atlantic salmon (Salmo salar) give rise to diverse class II genotypes.

Immunogenetics 2002 Aug 4;54(5):320-31. Epub 2002 Jul 4.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands.

Sequence-based typing of a breeding population (G1) consisting of 84 Atlantic salmon individuals revealed the presence of 7 Sasa-DAA and 7 Sasa-DAB expressed alleles. Subsequent typing of 1,182 individuals belonging to 33 families showed that Sasa-DAA and Sasa-DAB segregate as haplotypes. In total seven unique haplotypes were established, with frequencies in the population studied ranging from 0.01 to 0.49. Each haplotype is characterized by a unique minisatellite marker size embedded in the 3' untranslated region of the Sasa-DAA gene. These data corroborate the fact that Atlantic salmon express a single class II locus, consisting of tightly linked class II A and class B genes. The seven haplotypes give rise to 15 genotypes with frequencies varying between 0.01 and 0.23; 21 class II homozygous individuals were present in the G1 population. We also studied the frequency distribution in another breeding population (G4, n=374) using the minisatellite marker. Only one new marker size was present, suggesting the presence of one new class II haplotype. The marker frequency distribution in the G4 population differed markedly from the G1 population. The genomic organizations of two Sasa-DAA and Sasa-DAB alleles were determined, and supported the notion that these alleles belong to the same locus. In contrast to other studies of salmonid class II sequences, phylogenetic analyses of brown trout and Atlantic class II A and class II B sequences provided support for trans-species polymorphism.
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http://dx.doi.org/10.1007/s00251-002-0477-1DOI Listing
August 2002

A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains.

J Immunol 2002 Aug;169(4):1936-47

Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands.

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.
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http://dx.doi.org/10.4049/jimmunol.169.4.1936DOI Listing
August 2002

Structures of two major histocompatibility complex class I genes of the rainbow trout (Oncorhynchus mykiss).

Immunogenetics 2002 Jun 5;54(3):193-9. Epub 2002 Apr 5.

Department of Structural Biology, Sherman Fairchild Building, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.

Here we describe two rainbow trout major histocompatibility complex (MHC) class I genes characterized from lambda phage genomic clones prepared from a single fish. Clone GC71 contains all exons except a leader peptide-encoding exon. An open reading frame is maintained, and thus the gene MhcOnmy-U71 could be expressed in this individual. The class I gene found on clone GC41 lacks exons encoding the leader peptide and cytoplasmic domain. This gene, MhcOnmy-U41p, is a pseudogene due to a deletion in the alpha(2) domain-encoding exon causing premature termination. Both the Onmy-U71 and Onmy-U41p genes are distinguished by long introns between the exons encoding the alpha(1) and alpha(2) domains. Clone GC41 also contains the 3' exons of the LMP7/ PSMB8 gene encoding the gamma-interferon-induced proteosome subunit of rainbow trout.
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http://dx.doi.org/10.1007/s00251-002-0450-zDOI Listing
June 2002