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    ABSTRACT: Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system. CSF1, alongside a second ligand, interleukin-34 (IL-34), acts by binding to a cell surface receptor (CSF1R). We previously cloned and expressed pig CSF1 and IL-34. Here we produced a pig CSF1R-Ig+pFUSE Fc fusion protein and used it as an immunogen to produce three monoclonal antibodies (ROS8G11, ROS3A5 and ROS3B10) targeted against porcine CSF1R. Specific binding of each monoclonal antibody was confirmed by ELISA, western blot, flow cytometry and immunocytochemistry. The antibodies did not block CSF1 signalling. The surface expression of CSF1R in pig peripheral blood was restricted to CD14-positive monocytes and was also detected on lung macrophages. These antibodies provided an opportunity to investigate the increase of available CSF1R during pig BMDM differentiation. The new monoclonal antibodies provide useful reagents to support the study of monocyte and macrophage biology in the pig.
    Developmental and comparative immunology. 07/2014;
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    ABSTRACT: The WT1 gene was originally identified through its involvement in the development of Wilms tumours. The gene is characterized by a plethora of different isoforms with, in some cases, clearly different functions in transcriptional control and RNA metabolism. Many different mouse models for Wt1 have already been generated, and these are increasingly providing new information on the molecular roles of Wt1 in normal development and disease. In this review we discuss the different models that have been generated and what they have taught us about the role of Wt1 in the kidney.
    Pediatric Nephrology 11/2013;
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    ABSTRACT: Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is poorly understood. We previously identified eight miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF 20 (cell cycle inhibition), ORF 50 (reactivation) and ORF 73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5 UTRs of ORF 20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8 respectively, and the 3UTR of ORF 50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2 infected bovine T cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF 50 and a smaller but non-significant decrease in ORF 20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation, miRNA inhibition of ORF 20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORFs 50 and 73 play opposing roles in latency, it has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation in conditions that are unfavourable for viral replication, our data would support this hypothesis.
    Journal of General Virology 10/2013;
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    ABSTRACT: Abstract Interest in pluripotent livestock stem cells has been with us for over 2 decades, and although much has been claimed over the years, it is only recently that real progress has been achieved. The recent developments have revolved around the ability to reprogramm somatic cells to become induced pluripotent stem cells (iPSCs). Progress has been achieved in porcine, bovine, equine, and ovine species; but for each species, the extent of progress is different. I review the position of ovine iPSCs and comment on the two main drivers for this work-to produce "better" cells for nuclear transfer and to develop a good in vitro system to study the early development of ruminants.
    Cellular reprogramming. 09/2013;
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    ABSTRACT: SUMMARY The co-occurrence of different pathogen species and their simultaneous infection of hosts are common, and may affect host health outcomes. Co-infecting pathogens may interact synergistically (harming the host more) or antagonistically (harming the host less) compared with single infections. Here we have tested associations of infections and their co-infections with variation in growth rate using a subset of 455 animals of the Infectious Diseases of East Africa Livestock (IDEAL) cohort study surviving to one year. Data on live body weight, infections with helminth parasites and haemoparasites were collected every 5 weeks during the first year of life. Growth of zebu cattle during the first year of life was best described by a linear growth function. A large variation in daily weight gain with a range of 0·03-0·34 kg, and a mean of 0·135 kg (0·124, 0·146; 95% CI) was observed. After controlling for other significant covariates in mixed effects statistical models, the results revealed synergistic interactions (lower growth rates) with Theileria parva and Anaplasma marginale co-infections, and antagonistic interactions (relatively higher growth rates) with T. parva and Theileria mutans co-infections, compared with infections with T. parva only. Additionally, helminth infections can have a strong negative effect on the growth rates but this is burden-dependent, accounting for up to 30% decrease in growth rate in heavily infected animals. These findings present evidence of pathogen-pathogen interactions affecting host growth, and we discuss possible mechanisms that may explain observed directions of interactions as well as possible modifications to disease control strategies when co-infections are present.
    Parasitology 09/2013;
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    ABSTRACT: Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2013 John Wiley & Sons, Ltd.
    Journal of Tissue Engineering and Regenerative Medicine 07/2013;
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    ABSTRACT: Historically, sheep have been selectively bred for desirable traits including wool characteristics. However, recent moves towards extensive farming and reduced farm labour have seen a renewed interest in Easycare breeds. The aim of this study was to quantify the underlying genetic architecture of wool shedding in an Easycare flock. Wool shedding scores were collected from 565 pedigreed commercial Easycare sheep from 2002 to 2010. The wool scoring system was based on a 10-point (0-9) scale, with score 0 for animals retaining full fleece and 9 for those completely shedding. DNA was sampled from 200 animals of which 48 with extreme phenotypes were genotyped using a 50-k SNP chip. Three genetic analyses were performed: heritability analysis, complex segregation analysis to test for a major gene hypothesis and a genome-wide association study to map regions in the genome affecting the trait. Phenotypes were treated as a continuous or binary variable and categories. High estimates of heritability (0.80 when treated as a continuous, 0.65-0.75 as binary and 0.75 as categories) for shedding were obtained from linear mixed model analyses. Complex segregation analysis gave similar estimates (0.80 ± 0.06) to those above with additional evidence for a major gene with dominance effects. Mixed model association analyses identified four significant (P < 0.05) SNPs. Further analyses of these four SNPs in all 200 animals revealed that one of the SNPs displayed dominance effects similar to those obtained from the complex segregation analyses. In summary, we found strong genetic control for wool shedding, demonstrated the possibility of a single putative dominant gene controlling this trait and identified four SNPs that may be in partial linkage disequilibrium with gene(s) controlling shedding.
    Animal Genetics 07/2013;
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    ABSTRACT: Stabilisation of β-catenin, through inhibition of Glycogen synthase kinase 3 (GSK3) activity, in conjunction with inhibition of the enzyme MEK, promotes self-renewal of naïve-type mouse embryonic stem cells (ESC). In developmentally more advanced, primed-type, epiblast stem cells (EpiSC), however, β-catenin activity induces differentiation. We investigated the response of rat ESCs to β-catenin signalling and found that when maintained on feeder-support cells in the presence of a MEK inhibitor alone (1i culture), the derivation efficiency, growth, karyotypic stability, transcriptional profile, and differentiation potential of rat ESC cultures was similar to that of cell lines established using both MEK and GSK3 inhibitors (2i culture). Equivalent mouse ESCs, by comparison, differentiated in identical 1i conditions, consistent with insufficient β-catenin activity. This interspecies difference in reliance on GSK3 inhibition corresponded with higher overall levels of β-catenin activity in rat ESCs. Indeed, rat ESCs displayed widespread expression of the mesendoderm-associated β-catenin targets, Brachyury and Cdx2 in 2i medium, and overt differentiation upon further increases in β-catenin activity. In contrast, mouse ESC were resistant to differentiation at similarly elevated doses of GSK3 inhibitor. Interestingly, without feeder support, moderate levels of GSK3 inhibition were necessary to support effective growth of rat ESC, confirming the conserved role for β-catenin in ESC self-renewal. This work identifies β-catenin signalling as a molecular rheostat in rat ESC, regulating self-renewal in a dose-dependent manner, and highlights the potential importance of controlling flux in this signalling pathway to achieve effective stabilisation of naïve pluripotency.
    Stem Cells 07/2013;
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    ABSTRACT: The genetic architecture underlying nematode resistance and body weight in Blackface lambs was evaluated comparing genome-wide association (GWA) and regional heritability mapping (RHM) approaches. The traits analysed were faecal egg count (FEC) and immunoglobulin A activity against third-stage larvae from Teladorsagia circumcincta, as indicators of nematode resistance, and body weight in a population of 752 Scottish Blackface lambs, genotyped with the 50k single-nucleotide polymorphism (SNP) chip. FEC for both Nematodirus and Strongyles nematodes (excluding Nematodirus), as well as body weight were collected at approximately 16, 20 and 24 weeks of age. In addition, a weighted average animal effect was estimated for both FEC and body weight traits. After quality control, 44 388 SNPs were available for the GWA analysis and 42 841 for the RHM, which utilises only mapped SNPs. The same fixed effects were used in both analyses: sex, year, management group, litter size and age of dam, with day of birth as covariate. Some genomic regions of interest for both nematode resistance and body weight traits were identified, using both GWA and RHM approaches. For both methods, strong evidence for association was found on chromosome 14 for Nematodirus average animal effect, chromosome 6 for Strongyles FEC at 16 weeks and chromosome 6 for body weight at 16 weeks. Across the entire data set, RHM identified more regions reaching the suggestive level than GWA, suggesting that RHM is capable of capturing some of the variation not detected by GWA analyses.Heredity advance online publication, 20 March 2013; doi:10.1038/hdy.2012.90.
    Heredity 03/2013;
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    ABSTRACT: Expression of the cellular prion protein (PrPC) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrPC comprised of 25% more C1 fragment than PrPC from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrPC variants. We propose that the increased α-cleavage of ovine ARR PrPC contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrPC β-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.
    Biochimica et Biophysica Acta 03/2013;
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