The Turkey Ig-like receptor family: identification, expression and function.

The chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes

EVOLUTION OF ANTIGEN BINDING RECEPTORS

This review addresses issues related to the evolution of the complex multigene families of antigen binding receptors that function in adaptive immunity. Advances in molecular genetic technology now permit the study of immunoglobulin (Ig) and T cell receptor (TCR) genes in many species that are not commonly studied yet represent critical branch points in vertebrate phylogeny. Both Ig and TCR genes have been defined in most of the major lineages of jawed vertebrates, including the cartilaginous fishes, which represent the most phylogenetically divergent jawed vertebrate group relative to the mammals. Ig genes in cartilaginous fish are encoded by multiple individual loci that each contain rearranging segmental elements and constant regions. In some loci, segmental elements are joined in the germline, i.e. they do not undergo genetic rearrangement. Other major differences in Ig gene organization and the mechanisms of somatic diversification have occurred throughout vertebrate evolution. However, relating these changes to adaptive immune function in lower vertebrates is challenging. TCR genes exhibit greater sequence diversity in individual segmental elements than is found in Ig genes but have undergone fewer changes in gene organization, isotype diversity, and mechanisms of diversification. As of yet, homologous forms of antigen binding receptors have not been identified in jawless vertebrates; however, acquisition of large amounts of structural data for the antigen binding receptors that are found in a variety of jawed vertebrates has defined shared characteristics that provide unique insight into the distant origins of the rearranging gene systems and their relationships to both adaptive and innate recognition processes

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass

A comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance

Chickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains. Results: Transcriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection. Conclusions: Taken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken

The duck toll like receptor 7: genomic organization, expression and function.

TLR7 and TLR8 are triggered by antiviral compounds and single-stranded RNA, and are implicated in the immune response to viruses such as influenza. Chickens have a functional TLR7 gene and a genomic disruption in TLR8. PCR evidence suggested that the TLR8 disruption existed in galliform birds, but not anseriform birds. We investigated this due to the different susceptibility of ducks and chickens to influenza. We sequenced a 39 kb duck genomic clone spanning the TLR7/8 locus. Similar to chicken, we detect only fragments of the TLR8 gene in the region downstream of TLR7. Duck TLR7 shares only 85% amino acid identity to chicken TLR7, differing primarily in the ligand-binding LRR domains. TLR7 transcripts were most abundantly expressed in duck spleen, bursa and lung. Duck splenocytes respond to TLR7 agonists with upregulation of mRNA encoding proinflammatory cytokines and IFN alpha, with imiquimod eliciting the greatest response. TLR7 expression in lung, and upregulation of IFN alpha by TLR7 agonists, not typically seen in chickens, could contribute significantly to the antiviral defense of ducks. These differences in TLR7 function, not genomic organization, may contribute to the differential susceptibility of avian species to viral infection

The role of animal surveillance in influenza preparedness: the consequence of inapparent infection in ducks and pigs.

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Purification of duck immunoglobulins: An evaluation of protein A and protein G affinity chromatography

Duck serum proteins binding to protein A Sepharose CL-4B and protein G Sepharose 4 Fast Flow and eluted at pH 2.8 or 11.5 were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, radial/immunodiffusion against defined anti-immunoglobulin (Ig) reagents, and by the reactivity in immunoelectrophoresis of antisera raised in rabbits inoculated with the eluates. The results indicated that IgY (previous nomenclature 7.8S IgG) and IgY (ΔFc) (previously 5.75 IgG) bound to protein A efficiently and to protein G weakly, while IgM bound to protein A and protein G weakly. Some binding of non-Ig proteins also occurred. Attempts to separate the non-Ig proteins from the Igs by elution at different pHs (5.0, 4.0, 3.0 and 2.5) were unsuccessful, but it was found that precipitation of Igs in day-old duck serum with Na 2SO 4, followed by chromatography on protein A Sepharose, yielded relatively pure IgY. The efficient binding of the duck IgYs to protein A resembles high affinity binding of mammalian Igs but cannot be attributed to the Fc, as it is in mammals, since the IgY (ΔFc) does not have an Fc region. Instead, binding probably occurs through unique histidine residues occurring predominantly in the CH I domain.link_to_subscribed_fulltex