1,721,262 research outputs found
Absolute quantification reveals the stable transmission of a high copy number variant linked to autoinflammatory disease.
No full textBACKGROUND: Dissecting the role copy number variants (CNVs) play in disease pathogenesis is directly reliant on accurate methods for quantification. The Shar-Pei dog breed is predisposed to a complex autoinflammatory disease with numerous clinical manifestations. One such sign, recurrent fever, was previously shown to be significantly associated with a novel, but unstable CNV (CNV_16.1). Droplet digital PCR (ddPCR) offers a new mechanism for CNV detection via absolute quantification with the promise of added precision and reliability. The aim of this study was to evaluate ddPCR in relation to quantitative PCR (qPCR) and to assess the suitability of the favoured method as a genetic test for Shar-Pei Autoinflammatory Disease (SPAID). RESULTS: One hundred and ninety-six individuals were assayed using both PCR methods at two CNV positions (CNV_14.3 and CNV_16.1). The digital method revealed a striking result. The CNVs did not follow a continuum of alleles as previously reported, rather the alleles were stable and pedigree analysis showed they adhered to Mendelian segregation. Subsequent analysis of ddPCR case/control data confirmed that both CNVs remained significantly associated with the subphenotype of fever, but also to the encompassing SPAID complex (p < 0.001). In addition, harbouring CNV_16.1 allele five (CNV_16.1|5) resulted in a four-fold increase in the odds for SPAID (p < 0.001). The inclusion of a genetic marker for CNV_16.1 in a genome-wide association test revealed that this variant explained 9.7 % of genetic variance and 25.8 % of the additive genetic heritability of this autoinflammatory disease. CONCLUSIONS: This data shows the utility of the ddPCR method to resolve cryptic copy number inheritance patterns and so open avenues of genetic testing. In its current form, the ddPCR test presented here could be used in canine breeding to reduce the number of homozygote CNV_16.1|5 individuals and thereby to reduce the prevalence of disease in this breed
Growth factors and hepatic progenitor cells in liver regeneration: translating bench to bedside
No full textUpon severe acute or chronic liver injury, hepatic progenitor cells (HPCs) become activated. HPCs are adult stem cells of the liver and are considered a reserve population acting as second line of defense in liver regeneration. However, in many cases of severe liver disease this repair mechanism falls short and symptoms of liver failure develop. Insight into activation mechanisms of HPCs may provide novel cues for liver regenerative medicine strategies. HPC activation mechanisms have been studied in experimental rodent models and in human liver pathology, but to a far lesser extent in companion animals. In the Netherlands alone, millions of dogs and cats are kept as pet animals which can suffer from spontaneously occurring liver disease similar to human patients. Knowledge of similarities and differences in biology of liver disease may provide new, clinically relevant translational models of liver disease. In part one of the thesis fundamental studies were performed into HPC activation mechanisms. Activated HPCs and their micro-environment (or niche) were molecularly characterized in canine samples of several types of hepatitis and biliary disease. A severe liver disease that occurs specifically in dogs is lobular dissecting hepatitis (LDH). LDH has a rapid clinical course and is associated with prominent HPC activation. Samples of canine LDH were used to study the involvement of the Wnt/β-catenin and Notch signaling pathways in canine HPC activation. To further pinpoint intracellular signals that convey external activation signals into a functional proliferative response, a high throughput siRNA kinome screen was performed in an HPC-like cell line. One kinase was identified that had a inhibitory effect of S phase entry in HPCs. This finding was validated in primary HPCs cultured as liver organoids and in liver organoids expressing one extra allele of the kinase. Exact gene dosage turned out to be essential for balanced S phase entry of HPCs. In part two of the thesis applied studies of growth factors and HPCs in models of liver disease are described. We tested the regenerative potential of Hepatocyte Growth Factor (HGF) treatment in dogs with liver hypoplasia secondary to a congenital portosystemic shunt. It was possible to induce liver growth with HGF, but only for the duration of the treatment. The therapeutic potential of HPCs was investigated in a transplantation study of autologous canine liver organoids. Organoids were cultured from COMMD1-deficient dogs that develop copper toxicosis similar to human Wilson’s disease. HPCs were isolated from biopsies, genetically corrected, massively expanded as organoids in vitro and transplanted either via the portal vein or by intrahepatic injections. Under specific circumstances it was possible to find transplanted cells back in the liver with evidence for in vivo proliferation. In the last chapter of this thesis HPCs cultured as liver organoids were tested for their potential to model fatty liver disease (steatosis), a disease seen in cats and in humans. To this end a feline liver organoid culture was established and characterized. Feline organoids incubated with fatty acids accumulated more fat than human organoids, which is in line with the more severe phenotype of feline hepatic steatosis. In conclusion, this thesis translates fundamental findings in liver regenerative medicine to applications in (pre)clinical models of liver disease to benefit both human and veterinary patients
Mimicking developmental biology to regenerate the intervertebral disc
No full textNearly three-quarters of the human population will be affected by low back pain at some stage in their lives. While this condition is multifactorial, intervertebral disc (IVD) degeneration is one of its major causes, involved in at least 40% of chronic (low) back pain cases. Like humans, also dogs suffer from spontaneous IVD degeneration with similar characteristics. As such, within the concept of One Health, regenerative medicine can benefit both patient populations. In this respect, the dog is considered to be a suitable model for its own species and for human IVD degeneration. As no effective therapies to retard or reverse IVD degeneration have yet been devised, there is huge interest in potential regenerative treatments for both human and veterinary patients. For this reason, new cell- and growth factor-based regenerative strategies for the treatment of human and canine IVD degeneration were investigated in this thesis. In this respect, the value of studying developmental biology was demonstrated, as interesting targets were identified, e.g. caveolin-1, parathyroid hormone related peptide (PTHrP) and indian hedgehog (IHH). Not all proposed agents appeared to exert a positive effect in vitro (mesenchymal stromal cells, Link-N), and promising in vitro results did not always translate to beneficial effects in vivo (bone morphogenetic protein 7; BMP7). However, notochordal cell-based therapies induced regenerative effects across both species susceptible for IVD disease. While identifying the bioactive factors secreted by notochordal cells (e.g. extracellular vesicles) is challenging, it is valuable from a fundamental perspective and will provide innovative insights for the improvement of regenerative treatment strategies. Nonetheless, a more straightforward and promising alternative for bench to (veterinary) bedside translation is the application of notochordal cell-derived matrix (NCM), which exerted regenerative effects on IVD cells and in dogs with IVD degeneration
Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins
No full textCardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human
Canine Vertebral Screw and Rod Fixation System: Design and Mechanical Testing
No full textOBJECTIVES: To develop the canine vertebral screw and rod fixation system (CVSRF) and to compare the biomechanical properties between CVSRF and the screw and polymethylmethacrylate (Screw-PMMA) technique for internal fixation of the vertebral column in dogs. METHODS: The CVSRF consisted of vertebral screws with monoaxial side-loaded head, rods and specific inner screws connecting rod to the screw head. The CVSRF prototype was made from titanium alloy and manufactured by the rapid prototype machine. Vertebrectomy models were simulated by ultra-high-molecular-weight polyethylene blocks and tested with the CVSRF system (n = 8) and the Screw-PMMA technique (n = 8). The models were developed according to the American Society for Testing and Materials (ASTM F-1717-04). The biomechanical parameters were the compressive bending yield load, the compressive bending stiffness, the compressive ultimate load and the load displacement curve. RESULTS: The mean values of the compressive bending yield load, compressive bending stiffness and compressive ultimate load of the CVSRF were significantly higher than those of the Screw-PMMA technique (p < 0.01). The load displacement curve of the CVSRF showed higher rigidity and durability than that of the Screw-PMMA technique. CLINICAL SIGNIFICANCE: This mechanical study indicated that the CVSRF system can be used for canine vertebral stabilization and the biomechanical properties were better than those for the Screw-PMMA device
Erythrocyte copper chaperone for superoxide dismutase and superoxide dismutase as biomarkers for hepatic copper concentrations in Labrador retrievers
Get PDFHereditary hepatic copper accumulation in Labrador retrievers leads to hepatitis with fibrosis and eventually cirrhosis. The development of a non-invasive blood-based biomarker for copper status in dogs could be helpful in identifying dogs at risk and to monitor copper concentrations during treatment. In this study, two cellular copper metabolism proteins, Cu/Zn superoxide dismutase (SOD1) and its chaperone (copper chaperone for SOD1, CCS) were measured in erythrocytes and tested for association with hepatic copper concentrations in 15 Labrador retrievers with normal or increased hepatic copper concentrations. Antibodies against CCS and SOD1 were applicable for use in canine specimens. This was demonstrated by the loss of immune-reactive bands for CCS and SOD1 in siRNA treated canine bile duct epithelial cells. Erythrocyte CCS and CCS/SOD1 ratios were decreased 2.37 (P <0.001) and 3.29 (P <0.001) fold in the high copper group compared to the normal copper group. Erythrocyte CCS and CCS/SOD1 ratio are potential new biomarkers for hepatic copper concentrations in Labrador retrievers and could facilitate early diagnosis and treatment monitoring for copper-associated hepatitis in dogs
A novel VWF variant associated with type 2 von Willebrand disease in German Wirehaired Pointers and German Shorthaired Pointers
Get PDFVon Willebrand disease (VWD), caused by deficiency of the von Willebrand factor (VWF), is the most common bleeding disorder in humans and dogs. The complete cDNA encoding VWF of a German Wirehaired Pointer with type 2 VWD was sequenced, and we found four variants that alter the amino acid sequence. These variants were: c.1657T>G corresponding to p.Trp553Gly; c.1777G>A (p.Glu593Lys); c.4937A>G (p.Asn1646Ser) and c.5544G>A (p.Met1848Ile). A haplotype of the c.1657G, c.1777A and c.4937G alleles co-segregated with the VWF antigen level in a four-generation pedigree with the disease. Healthy dogs of the breed were found that were homozygous for the c.1777A or the c.5544A allele, indicating that these variants do not cause VWD. Dogs that were homozygous for the c.4937G allele and had no signs of a bleeding disorder were observed in the Chinese Crested dog breed. Thus, only the c.1657G variant was found in the homozygous state exclusively in VWD affecteds, and this variant is the strongest candidate to be the cause of VWD type 2 in the German Wirehaired Pointer breed. A screen of German Shorthaired Pointers indicated that the variant also segregates with VWD in this breed
Canine Models for Copper Homeostasis Disorders
No full textCopper is an essential trace nutrient metal involved in a multitude of cellular processes. Hereditary defects in copper metabolism result in disorders with a severe clinical course such as Wilson disease and Menkes disease. In Wilson disease, copper accumulation leads to liver cirrhosis and neurological impairments. A lack in genotype-phenotype correlation in Wilson disease points toward the influence of environmental factors or modifying genes. In a number of Non-Wilsonian forms of copper metabolism, the underlying genetic defects remain elusive. Several pure bred dog populations are affected with copper-associated hepatitis showing similarities to human copper metabolism disorders. Gene-mapping studies in these populations offer the opportunity to discover new genes involved in copper metabolism. Furthermore, due to the relatively large body size and long life-span of dogs they are excellent models for development of new treatment strategies. One example is the recent use of canine organoids for disease modeling and gene therapy of copper storage disease. This review addresses the opportunities offered by canine genetics for discovery of genes involved in copper metabolism disorders. Further, possibilities for the use of dogs in development of new treatment modalities for copper storage disorders, including gene repair in patient-derived hepatic organoids, are highlighted
Transformed canine and murine mesenchymal stem cells as a model for sarcoma with complex genomics
Get PDFSimple SummarySarcomas are rare cancers of mesenchymal origin, the majority of which are characterized by many copy number alterations, amplifications, or deletions. Because of these complex genomics, it is notoriously difficult to identify driver events of malignant transformation. In this study, we show that murine and canine mesenchymal stem cells (MSCs) can be used to model spontaneous malignant transformation towards sarcomas with complex genomics. We show that these MSCs have an abnormal karyotype, many structural variants, and point mutations at whole genome sequencing analysis, and form sarcomas after injection into mice. Our cross-species analysis reveals that p53 loss is an early event in sarcomagenesis, and it was shown that MSCs with a knock-out in Trp53 transform earlier compared to wild-type MSCs. Our study points to the importance of p53 loss in the transformation process towards sarcomas with complex genomics.Sarcomas are rare mesenchymal tumors with a broad histological spectrum, but they can be divided into two groups based on molecular pathology: sarcomas with simple or complex genomics. Tumors with complex genomics can have aneuploidy and copy number gains and losses, which hampers the detection of early, initiating events in tumorigenesis. Often, no benign precursors are known, which is why good models are essential. The mesenchymal stem cell (MSC) is the presumed cell of origin of sarcoma. In this study, MSCs of murine and canine origin are used as a model to identify driver events for sarcomas with complex genomic alterations as they transform spontaneously after long-term culture. All transformed murine but not canine MSCs formed sarcomas after subcutaneous injection in mice. Using whole genome sequencing, spontaneously transformed murine and canine MSCs displayed a complex karyotype with aneuploidy, point mutations, structural variants, inter-chromosomal translocations, and copy number gains and losses. Cross-species analysis revealed that point mutations in Tp53/Trp53 are common in transformed murine and canine MSCs. Murine MSCs with a cre-recombinase induced deletion of exon 2-10 of Trp53 transformed earlier compared to wild-type murine MSCs, confirming the contribution of loss of p53 to spontaneous transformation. Our comparative approach using transformed murine and canine MSCs points to a crucial role for p53 loss in the formation of sarcomas with complex genomics.Molecular tumour pathology - and tumour geneticsMTG
Bioengineering liver transplantation
Get PDFSince the first in-man liver transplantation was performed by Starzl et al [...]
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