Search

Background: Chemicals of Emerging Concern (CECs) include a very wide group of chemicals that are suspected to be responsible for adverse effects on health, but for which very limited information is available. Chromato-graphic techniques coupled with high-resolution mass spectrometry (HRMS) can be used for non-targeted screening and detection of CECs, by using comprehensive annotation databases. Establishing a database focused on the annotation of CECs in human samples will provide new insight into the distribution and extent of exposures to a wide range of CECs in humans. Objectives: This study describes an approach for the aggregation and curation of an annotation database (CECscreen) for the identification of CECs in human biological samples. Methods: The approach consists of three main parts. First, CECs compound lists from various sources were aggregated and duplications and inorganic compounds were removed. Subsequently, the list was curated by standardization of structures to create & ldquo;MS-ready & rdquo; and & ldquo;QSAR-ready & rdquo; SMILES, as well as calculation of exact masses (monoisotopic and adducts) and molecular formulas. The second step included the simulation of Phase I metabolites. The third and final step included the calculation of QSAR predictions related to physicochemical properties, environmental fate, toxicity and Absorption, Distribution, Metabolism, Excretion (ADME) processes and the retrieval of information from the US EPA CompTox Chemicals Dashboard. Results: All CECscreen database and property files are publicly available (DOI: https://doi.org/10.5281/zenodo. 3956586). In total, 145,284 entries were aggregated from various CECs data sources. After elimination of du-plicates and curation, the pipeline produced 70,397 unique & ldquo;MS-ready & rdquo; structures and 66,071 unique QSAR-ready structures, corresponding with 69,526 CAS numbers. Simulation of Phase I metabolites resulted in 306,279 unique metabolites. QSAR predictions could be performed for 64,684 of the QSAR-ready structures, whereas information was retrieved from the CompTox Chemicals Dashboard for 59,739 CAS numbers out of 69,526 inquiries. CECscreen is incorporated in the in silico fragmentation approach MetFrag. Discussion: The CECscreen database can be used to prioritize annotation of CECs measured in non-targeted HRMS, facilitating the large-scale detection of CECs in human samples for exposome research. Large-scale detection of CECs can be further improved by integrating the present database with resources that contain CECs (metabolites) and meta-data measurements, further expansion towards in silico and experimental (e.g., MassBank) generation of MS/MS spectra, and development of bioinformatics approaches capable of using cor-relation patterns in the measured chemical features.

Organoids are three-dimensional culture systems in which adult stem cells and their progeny grow and represent the native physiology of the cells in vivo. Organoids have been successfully derived from several organ systems in both animal models and human patients. Organoids have been used for fundamental research, disease modeling, drug testing, and transplantation. In this review, we summarize the applications of liver-derived organoids and discuss their potential. It is likely that organoids will provide an invaluable tool to unravel disease mechanisms, design novel (personalized) treatment strategies, and generate autologous stem cells for gene editing and transplantation purposes. Significance Organoids derived from the liver have hepatocellular differentiation potential and can be an unlimited source for hepatocytes for application in in vitro toxicology testing and for transplantation purposes as an alternative to orthotopic liver transplantation. The in vitro representation of the native physiology and epigenetic background of the adult liver stem cells makes the organoid technology an excellent tool to study and model liver diseases, for drug screening, and for the design of personalized treatments. This review summarizes the applications of liver organoids and discusses their potential in the study and modeling of liver diseases, and in the development and testing of novel drugs.

During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.

Background Intervertebral disc (IVD) disease is a common spinal disorder in dogs and degeneration and inflammation are significant components of the pathological cascade. Only limited studies have studied the cytokine and chemokine profiles in IVD degeneration in dogs, and mainly focused on gene expression. A better understanding is needed in order to develop biological therapies that address both pain and degeneration in IVD disease. Therefore, in this study, we determined the levels of prostaglandin E2 (PGE2), cytokines, chemokines, and matrix components in IVDs from chondrodystrophic (CD) and non-chondrodystrophic (NCD) dogs with and without clinical signs of IVD disease, and correlated these to degeneration grade (according to Pfirrmann), or herniation type (according to Hansen). In addition, we investigated cyclooxygenase 2 (COX-2) expression and signs of inflammation in histological IVD samples of CD and NCD dogs. Results PGE2 levels were significantly higher in the nucleus pulposus (NP) of degenerated IVDs compared with non-degenerated IVDs, and in herniated IVDs from NCD dogs compared with non-herniated IVDs of NCD dogs. COX-2 expression in the NP and annulus fibrosus (AF), and proliferation of fibroblasts and numbers of macrophages in the AF significantly increased with increased degeneration grade. GAG content did not significantly change with degeneration grade or herniation type. Cytokines interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, immune protein (IP)-10, tumor necrosis factor (TNF)-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) were not detectable in the samples. Chemokine (C-C) motif ligand (CCL)2 levels in the NP from extruded samples were significantly higher compared with the AF of these samples and the NP from protrusion samples. Conclusions PGE2 levels and CCL2 levels in degenerated and herniated IVDs were significantly higher compared with non-degenerated and non-herniated IVDs. COX-2 expression in the NP and AF and reactive changes in the AF increased with advancing degeneration stages. Although macrophages invaded the AF as degeneration progressed, the production of inflammatory mediators seemed most pronounced in degenerated NP tissue. Future studies are needed to investigate if inhibition of PGE2 levels in degenerated IVDs provides effective analgesia and exerts a protective role in the process of IVD degeneration and the development of IVD disease. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0635-6) contains supplementary material, which is available to authorized users.

Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their native environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect on extracellular matrix production. Methods: Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 μg/mL Link N. Furthermore, BM and NCCM samples were injected with 106 BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, glycosaminoglycan [GAG], hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining. Results: Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC-injected groups was not higher than in the other groups after 4 weeks of culture. Discussion: This study shows that, indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of an NCCM-based treatment for intervertebral disc regeneration.

Liver-derived multipotent stromal cells (L-MSCs) may prove preferable for treatment strategies of liver diseases, in comparison to the widely studied bone marrow-derived MSCs (BM-MSCs). Canines are a large animal model, in which the pathologies of liver diseases are similar to man. This study further promotes the implementation of canine models in MSC-based treatments of liver diseases. L-MSCs were characterized and compared to BM-MSCs from the same individual. Both cell types demonstrated a spindle-shaped fibroblast-like morphology, possessed the same growth potential, and demonstrated similar immunomodulation gene expression of CD274, PTGS-1, and PTGS-2. Marked differences in cell surface markers, CD105 and CD146, distinguished these two cell populations, and L-MSCs retained a liver-specific imprinting, observed by expression of CK18 and CK19. Finally, both populations differentiated toward the osteogenic and adipogenic lineage; however, L-MSCs failed to differentiate into the chondrogenic lineage. In conclusion, characterization of canine L-MSCs and BM-MSCs demonstrated that the two cell type populations are highly comparable. Although it is still unclear which cell source is preferred for clinical application in liver treatment strategies, this study provides a foundation for future controlled studies with MSC therapy in various liver diseases in dogs before their application in man.

Background Preceding intervertebral disc (IVD) degeneration, the cell phenotype in the nucleus pulposus (NP) shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). Microarray analysis showed a correlation between caveolin-1 expression and the phenotypic transition of NCs to CLCs. With a clinical directive in mind, the aim of this study was to determine the role of caveolin-1 in IVD degeneration. As a scaffolding protein, caveolin-1 influences several signaling pathways, and transforming growth factor (TGF)-β receptors have been demonstrated to colocalize with caveolin-1. Therefore, the hypothesis of this study was that caveolin-1 facilitates repair by enhancing TGF-β signaling in the IVD. Methods Protein expression (caveolin-1, apoptosis, progenitor cell markers, extracellular matrix, and phosphorylated Smad2 [pSmad2]) was determined in IVDs of wild-type (WT) and caveolin-1-null mice and canine IVDs of different degeneration grades (immunofluorescence, immunohistochemistry, and TUNEL assay). Canine/human CLC microaggregates were treated with chondrogenic medium alone or in combination with caveolin-1 scaffolding domain (CSD) peptide and/or caveolin-1 silencing RNA. After 28 days, gene and protein expression profiles were determined. Results The NP of WT mice was rich in viable NCs, whereas the NP of caveolin-1-null mice contained more collagen-rich extracellular matrix and fewer cells, together with increased progenitor cell marker expression, pSmad2 TGF-β signaling, and high apoptotic activity. During canine IVD degeneration, caveolin-1 expression and apoptotic activity increased. In vitro caveolin-1 silencing decreased the CLC microaggregate glycosaminoglycan (GAG) content, which could be rescued by CSD treatment. Furthermore, CSD increased TGF-β/pSmad2 signaling at gene and protein expression levels and enhanced the anabolic effects of TGF-β1, reflected in increased extracellular matrix deposition by the CLCs. Conclusions Caveolin-1 plays a role in preservation of the NC phenotype. Additionally, it may be related to CLC apoptosis, given its increased expression in degenerated IVDs. Nevertheless, CSD enhanced CLC GAG deposition in vitro, and hence the increased caveolin-1 expression during IVD degeneration may also facilitate an ultimate attempt at repair. Further studies are needed to investigate how caveolin-1 modifies other signaling pathways and facilitates IVD repair. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0960-y) contains supplementary material, which is available to authorized users.

The first aim of this study was to determine whether vitamin D supplementation influenced the effects of high vitamin A intake on new bone formation in adult cats. The second aim was to determine whether high vitamin A intake in cats caused liver pathology and, if so, whether the current upper limit for the dietary intake of vitamin A for healthy adult cats would be safe. Twenty-four healthy adult cats were divided into four groups that received a control diet supplemented with peanut oil (control), or peanut oil containing a 100-fold increase in vitamin A (HA), or a 100-fold increase in vitamin A and a fivefold increase in vitamin D (HAMD), or a 100-fold increase in vitamin A and a 65-fold increase in vitamin D (HAHD) over a period of 18 months. Cats did not show abnormal locomotion or clinical signs of liver failure after 18 months of supplementation but did show subtle skeletal changes and liver pathology, suggesting that the current National Research Council (2006) safe upper limit for vitamin A for cats is too high. The addition of vitamin D did not seem to influence bone pathology. While moderately elevated dietary vitamin D levels (HAMD) seemed to protect cats against the liver pathology caused by the consumption of large amounts of vitamin A, higher dietary levels of vitamin D (HAHD) did not seem to be protective.

Hip as well as elbow dysplasia (HD, ED) are developmental disorders leading to malformation of their respective joints. For a long time both disorders have been scored and targeted for improvement using selective breeding in several Dutch dog populations. In this paper all scores for both HD and ED, given to pure bred dogs in the Netherlands from 2002 to 2010, were analyzed. Heritabilities and correlations between HD and ED were calculated for the 4 most frequently scored breeds. Heritabilities ranged from 0.0 to 0.37 for HD related traits (FCI-score, osteoarthritis, congruity, shape and laxity (Norberg angle); FCI: Federation Cynologique Internationale) and from 0.0 to 0.39 for ED related traits (IEWG score, osteoarthritis, sclerosis and indentation; IEWG: International Elbow Working Group). HD related traits showed high genetic and residual correlations among each other but were only to a minor extent correlated with ED related traits, which also showed high correlations among each other. Genetic correlations were higher than residual correlations. Phenotypic and genetic trends since 2001 for the four most scored breeds were slightly positive but decreasing overtime, indicating that selection over the past decade has not been effective. (C) 2014 Published by Elsevier B.V.

BACKGROUND: Degenerative lumbosacral stenosis is a common problem in large breed dogs. For severe degenerative lumbosacral stenosis, conservative treatment is often not effective and surgical intervention remains as the last treatment option. The objective of this retrospective study was to assess the middle to long term outcome of treatment of severe degenerative lumbosacral stenosis with pedicle screw-rod fixation with or without evidence of radiological discospondylitis. RESULTS: Twelve client-owned dogs with severe degenerative lumbosacral stenosis underwent pedicle screw-rod fixation of the lumbosacral junction. During long term follow-up, dogs were monitored by clinical evaluation, diagnostic imaging, force plate analysis, and by using questionnaires to owners. Clinical evaluation, force plate data, and responses to questionnaires completed by the owners showed resolution (n = 8) or improvement (n = 4) of clinical signs after pedicle screw-rod fixation in 12 dogs. There were no implant failures, however, no interbody vertebral bone fusion of the lumbosacral junction was observed in the follow-up period. Four dogs developed mild recurrent low back pain that could easily be controlled by pain medication and an altered exercise regime. CONCLUSIONS: Pedicle screw-rod fixation offers a surgical treatment option for large breed dogs with severe degenerative lumbosacral stenosis with or without evidence of radiological discospondylitis in which no other treatment is available. Pedicle screw-rod fixation alone does not result in interbody vertebral bone fusion between L7 and S1.

Introduction Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs. Methods Canine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture. Results NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. Conclusions No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.

Currently, there are no established treatments to prevent, stop, or even retard the degeneration of articular cartilage in osteoarthritis (OA). Biological repair of the degenerating articular cartilage would be preferable to surgery. There is no benign site where autologous chondrocytes can be harvested and used as a cell source for cartilage repair, leaving mesenchymal stem cells (MSCs) as an attractive option. However, MSCs from OA patients have been shown to constitutively express collagen type X (COL-X), a marker of late-stage chondrocyte hypertrophy. We recently found that naproxen (Npx), but not other nonsteroidal anti-inflammatory drugs, can induce collagen type X alpha 1 (COL10A1) gene expression in bone marrow-derived MSCs from healthy and OA donors. In this study, we determined the effect of Npx on COL10A1 expression and investigated the intracellular signaling pathways that mediate such effect in normal human MSCs during chondrogenesis. MSCs were cultured in standard chondrogenic differentiation media supplemented with or without Npx. Our results show that Npx can regulate chondrogenic differentiation by affecting the gene expression of both Indian hedgehog and parathyroid hormone/parathyroid hormone-related protein signaling pathways in a time-dependent manner, suggesting a complex interaction of different signaling pathways during the process.

Dogs with a congenital portosystemic shunt (CPSS) often have enlarged and hyper-filtrating kidneys. Although expression of different growth factors has been well-described in the livers of dogs affected with a CPSS, their expression in the kidneys has yet to be determined. Bone morphogenetic protein 7 (BMP-7), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β have been implicated in renal development (BMP-7, HGF) or the onset of renal fibrosis (TGF-β). Moreover, BMP-7 and HGF have protective properties in renal fibrosis. In this study, the expression and activity of BMP-7 were investigated in renal biopsies obtained from 13 dogs affected with a CPSS and compared to similar samples from age-matched healthy control dogs. Both quantitative reverse-transcriptase PCR and Western blotting showed up-regulated BMP-7 signalling in kidneys of CPPS-affected dogs. These research findings may help to explain the renal pathology/dysfunction in dogs affected with a CPSS.

Introduction Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain. Methods Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue. Results In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7. Conclusions An intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.

The prevalence of patellar luxation (PL) and genetic factors potentially involved in the disorder were investigated in Dutch Kooiker dogs. A cohort of 842 Kooiker dogs, the offspring of 195 sires and 318 dams, was screened for PL from 1994 to 2011. The cohort was included in a pedigree of 1737 Kooiker dogs comprising nine generations. PL was present in 24% of screened dogs, with unilateral and bilateral luxation being observed equally frequently. Medial PL was more common (61%) than lateral PL (32%) or bidirectional PL (7%). The frequency of PL was similar in male and female dogs, with a female:male relative risk of 1.15 (95% confidence interval, CI, 0.90-1.48). The heritability of PL in the screened population was 0.27 +/- 0.07. Since the start of the screening programme, the prevalence of PL decreased from 28% to 19%. A genome-wide association study of PL with 48 cases and 42 controls suggested the possible involvement of a region on chromosome 3 (P-raw = 1.32 x10(-5), P-genome = 0.142), but the involvement of this region could not be confirmed in a validation group. Breeding programmes for complex diseases, such as PL, would benefit from combining pedigrees, phenotypes and genotypes, i.e. from genomic selection, as is currently the method of choice for breeding of production animals. (C) 2014 Elsevier Ltd. All rights reserved.

Diseases of the coxofemoral (hip) joint are infrequently diagnosed in horses. Most cases are presented as an unilateral condition and usually are of traumatic origin. This case report describes a Friesian foal with a clinically obvious thoracolumbar kyphosis, combined with a weight-shifting stance and a shortened stride of both hindlimbs. General clinical and lameness examinations, computed tomographic examination of the pelvis, and macroscopic and histopathological examinations of the coxofemoral joints were performed. This revealed a final, phenotypical diagnosis of a primary osteochondral dysplasia of both coxofemoral joints with secondary osteoarthritis. Similar to the occurrence of this condition in other species and considering the small genetic basis of the Friesian horse breed, a genetic predisposing factor is suspected to play a key role in the developing mechanism of dysplastic coxofemoral joint disease in horses as illustrated with this case. Computed tomography scanning appears to be a useful imaging technique in the detection of coxofemoral joint disease in small horses and foals.

Disparities in longitudinal growth within a species can be partly explained by endocrinological differences. We hypothesized that regulatory networks acting locally in the growth plate may also be important. We tested this hypothesis by evaluating the IGF/IGFBP expression, the vitamin D pathway, and the PTHrP-Indian hedgehog (IHH) feedback loop in rib growth plates from 10- and 21-wk-old small- (Miniature Poodles, MP) and large-breed dogs (Great Danes, GD) using immunohistochemistry and quantitative (q)PCR. The rib growth plates of GD were 1.7 times thicker compared with those of MP, with larger proliferative (in absolute terms) and larger hypertrophic (in absolute and relative terms) zones. IGF/IGFBP gene expression profiling of the growth plates revealed decreased gene expression of igfbp2, -4, and -6 and an unaltered expression of igf-I and igf-II and their respective receptors in GD vs. MP. Immunohistochemistry and qPCR findings showed that the vitamin D pathway was more active in GD than in MP. Staining for 1α- and 24-hydroxylase was more abundant and intense in GD and the gene expressions of 1α-hydroxylase and the vitamin D receptor-driven 24-hydroxylase were six- and eightfold higher in GD vs. MP, respectively. Consistent with the immunohistochemistry findings, the expression of mRNA for components of the parathyroid hormone-related peptide (PTHrP)-IHH loop was different in GD compared with MP, with there being a relative threefold downregulation of Pthrp and a tenfold upregulation of Ihh in GD vs MP . These differences suggest that the effects of IHH in the regulation of chondrocyte proliferation and hypertrophy, both independently of PTHrP, can become more dominant during rapid growth rates. In conclusion, our data suggest that, in addition to modest endocrine differences, more pronounced changes in the expression of locally acting regulatory networks, such as the IGF system, vitamin D pathway, and PTHrP-IHH feedback loop are important contributors to within-species disparities in growth rates.

Cushing's disease (CD) or pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, with an estimated prevalence of 1 or 2 in 1000 dogs per year. It is caused by an adrenocorticotropic hormone secreting adenoma in the pars distalis or pars intermedia of the pituitary gland. The pituitary gland is a small endocrine gland located in the pituitary fossa. In the postnatal individual, the hypothalamus-pituitary axis plays a central role in maintaining homeostatic functions, like control of metabolism, reproduction, and growth. Stem cells are suggested to play a role in the homeostatic adaptations of the adult pituitary gland, such as the rapid specific cell-type expansion in response to pregnancy or lactation. Several cell populations have been suggested as pituitary stem cells, such as Side Population cells and cells expressing Sox2 or Nestin. These cell populations are discussed in this review. Also, stem and progenitor cells are thought to play a role in pituitary tumorigenesis, such as the development of pituitary adenomas in dogs. There are limited reports on the role of stem cells in pituitary adenomas, especially in dogs. Further studies are needed to identify and characterize this cell population and to develop specific cell targeting therapeutic strategies as a new way of treating canine CD.

Introduction: Medial coronoid disease (MCD) is a common heritable disease in young large-breed dogs and is characterized by fissures and/or fragmentation of the medial coronoid process (MCP), cartilage, and/or subchondral bone. MCD development has also been attributed to disturbed endochondral ossification. Vitamin D influences skeletal development and endochondral ossification by stimulating the terminal differentiation of chondrocytes and the mineralization of cartilage and the newly formed osteoid. The aim of this study was to investigate whether vitamin D supplementation of the standard puppy diet given to puppies after weaning can prevent the development of medial coronoid disease (MCD) by stimulating endochondral ossification, including terminal differentiation of chondrocytes, and mineralization of the cartilaginous template of the developing medial cornoid process (MCP). Animals, material and methods: A litter of Labrador puppies was on purpose bred by mating a dam and a sire with MCD; these dogs are known to produce offspring with MCD. The puppies received a diet supplemented with 50,000 IU vitamin D per kg as fed. Development of MCD was monitored by computed tomography (CT) and plain radiographs every two weeks, and post-mortem by microCT, necropsy, histology, and immunohistochemistry and compared with data from a previous study. Results and discussion: Vitamin D supplementation did result in increased plasma levels of 25- vitamin D and 1.25-vitamin D, but did not prevent development of MCD in growing Labradors. Instead, vitamin D supplementation resulted in increased collagen X staining ofthe MCP and irregular costal growth plates, demonstrating disturbed endochondral ossification. Conclusion: Vitamin D supplementation does not prevent MCD development. Other causes for MCD rather than relative vitamin D deficiency causing disturbed endochondral ossification are more likely.

New therapeutic concepts developed in rodent models should ideally be evaluated in large animal models prior to human clinical application. COMMD1-deficiency in dogs leads to hepatic copper accumulation and chronic hepatitis representing a Wilson's disease like phenotype. Detailed understanding of the pathogenesis and time course of this animal model is required to test its feasibility as a large animal model for chronic hepatitis. In addition to mouse models, true longitudinal studies are possible due to the size of these dogs permitting detailed analysis of the sequence of events from initial insult to final cirrhosis. Therefore, liver biopsies were taken each half year from five new born COMMD1-deficient dogs over a period of 42 months. Biopsies were used for H&E, reticulin, and rubeanic acid (copper) staining. Immunohistochemistry was performed on hepatic stellate cell (HSC) activation marker (alpha-smooth muscle actin, α-SMA), proliferation (Ki67), apoptosis (caspase-3), and bile duct and liver progenitor cell (LPC) markers keratin (K) 19 and 7. Quantitative RT-PCR and Western Blots were performed on gene products involved in the regenerative and fibrotic pathways. Maximum copper accumulation was reached at 12 months of age, which coincided with the first signs of hepatitis. HSCs were activated (α-SMA) from 18 months onwards, with increasing reticulin deposition and hepatocytic proliferation in later stages. Hepatitis and caspase-3 activity (first noticed at 18 months) increased over time. Both HGF and TGF-β1 gene expression peaked at 24 months, and thereafter decreased gradually. Both STAT3 and c-MET showed an increased time-dependent activation. Smad2/3 phosphorylation, indicative for fibrogenesis, was present at all time-points. COMMD1-deficient dogs develop chronic liver disease and cirrhosis comparable to human chronic hepatitis, although at much higher pace. Therefore they represent a genetically-defined large animal model to test clinical applicability of new therapeutics developed in rodent models.

Background The liver has a large regenerative capacity. Hepatocytes can replicate and regenerate a diseased liver. However, as is the case in severe liver diseases, this replication may become insufficient or exhausted and hepatic progenitor cells (HPCs) can be activated in an attempt to restore liver function. Due to their bi-potent differentiation capacity, these HPCs have great potential for regenerative approaches yet over-activation does pose potential health risks. Therefore the mechanisms leading to activation must be elucidated prior to safe implementation in the veterinary clinic. Wnt/β-catenin and Notch signalling have been implicated in the activation of HPCs in mouse models and in humans. Here we assessed the involvement in canine HPC activation. Gene-expression profiles were derived from laser microdissected HPC niches from lobular dissecting hepatitis (LDH) and normal liver tissue, with a focus on Wnt/β-catenin and Notch signalling. Immunohistochemical and immunofluorescent studies were combined to assess the role of the pathways in HPCs during LDH. Results Gene-expression confirmed higher expression of Wnt/β-catenin and Notch pathway components and target genes in activated HPC niches in diseased liver compared to quiescent HPC niches from normal liver. Immunofluorescence confirmed the activation of these pathways in the HPCs during disease. Immunohistochemistry showed proliferating HPCs during LDH, and double immunofluorescence showed downregulation of Wnt/β-catenin and Notch in differentiating HPCs. Vimentin, a mesenchymal marker, was expressed on a subset of undifferentiated HPCs. Conclusions Together these studies clearly revealed that both Wnt/β-catenin and Notch signalling pathways are enhanced in undifferentiated, proliferating and potentially migrating HPCs during severe progressive canine liver disease (LDH).

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Hepatic progenitor cells (HPCs) are an adult stem cell compartment in the liver that contributes to liver regeneration when replication of mature hepatocytes is insufficient. In this study, laser microdissection was used to isolate HPC niches from the livers of healthy dogs and dogs with lobular dissecting hepatitis (LDH), in which HPCs are massively activated. Gene expression of HPC, hepatocyte and biliary markers was determined by quantitative reverse transcriptase PCR. Expression and localisation of selected markers were further studied at the protein level by immunohistochemistry and immunofluorescent double staining in samples of normal liver and liver from dogs with LDH, acute and chronic hepatitis, and extrahepatic cholestasis. Activated HPC niches had higher gene expression of the hepatic progenitor markers OPN, FN14, CD29, CD44, CD133, LIF, LIFR and BMI1 compared to HPCs from normal liver. There was lower expression of albumin, but activated HPC niches were positive for the biliary markers SOX9, HNF1β and keratin 19 by immunohistochemistry and immunofluorescence. Laminin, activated stellate cells and macrophages are abundant extracellular matrix and cellular components of the canine HPC niche. This study demonstrates that the molecular and cellular characteristics of canine HPCs are similar to rodent and human HPCs, and that canine HPCs are distinctively activated in different types of liver disease.

Pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, caused by an adrenocorticotrophic hormone secreting pituitary tumour of the anterior or intermediate lobe. The prognosis of intermediate lobe adenomas is worse than that of anterior lobe adenomas, indicating the possible usefulness of melanotropic markers as prognosticators. Another possible origin of pituitary adenomas is found in cancer stem cells. The aim of the present study was to investigate the expression of melanotroph specific transcription factor paired box protein 7 (Pax7) and stem cell marker and reprogramming factor sex determining region Y-box 2 (Sox2) and to relate their expression to clinical parameters. The mean ± SD of labelling index (LI) for Pax7 was 8.6% ± 21.7% in the adenomas; 1/6 controls had positive staining (LI, 15.2%). For Sox2, the LI in the adenomas was 16.9% ± 15.2% and 19.5% ± 11.6% in the controls. Pax7 expression was significantly higher in enlarged pituitaries, compared to non-enlarged pituitaries (P = 0.05), but Pax7 or Sox2 immunopositivity did not correlate to other clinical parameters such as histological diagnosis, survival time or disease-free interval. Gene expression of Pax7 target genes, such as proconvertase 2 (PC2), pro-opiomelanocortin (POMC), and dopamine D2 receptor (DRD2), was significantly lower in the adenoma samples compared to normal tissue, indicating that Pax7 signalling was not activated in adenomas. It was suggested that Pax7 and Sox2 remain interesting targets for molecular investigations into their role in pituitary tumorigenesis, but were unsuitable as clinical prognosticators in dogs.

Following an endochondral approach to bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for endochondral bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to bone formation with bone marrow cavities. In addition, rat-specific bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for endochondral bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf bone regenerative substitutes.

BACKGROUND: Transsphenoidal hypophysectomy is an effective treatment for dogs with pituitary-dependent hypercortisolism (PDH). However, long-term recurrence of hypercortisolism is a well-recognized problem, indicating the need for reliable prognostic indicators. OBJECTIVES: The aim of this study was to evaluate the prognostic value of perioperative plasma ACTH and cortisol concentrations for identifying recurrence of hypercortisolism after transsphenoidal hypophysectomy. ANIMALS: A total of 112 dogs with PDH that underwent transsphenoidal hypophysectomy met the inclusion criteria of the study. METHODS: Hormone concentrations were measured preoperatively and 1-5 hours after surgery. Both absolute hormone concentrations and postoperative concentrations normalized to preoperative concentrations were included in analyses. The prognostic value of hormone concentrations was studied with Cox's proportional hazard analysis. RESULTS: Median follow-up and disease-free period were 1096 days and 896 days, respectively. Twenty-eight percent of patients had recurrence, with a median disease-free period of 588 days. Both absolute and normalized postoperative cortisol concentrations were significantly higher in dogs with recurrence than in dogs without recurrence. High ACTH 5 hours after surgery, high cortisol 1 and 4 hours after surgery, high normalized ACTH 3 hours after surgery, high normalized cortisol 4 hours after surgery and the random slope of cortisol were associated with a shorter disease-free period. CONCLUSIONS AND CLINICAL IMPORTANCE: Individual perioperative hormone curves provide valuable information about the risk of recurrence after hypophysectomy. However, because no single cutoff point could be identified, combination with other variables, such as the pituitary height/brain area (P/B) ratio, is still needed to obtain a good estimate of the risk for recurrence of hypercortisolism after hypophysectomy.

Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a genome-wide association study in Dutch Labrador Retrievers, comparing data of nearly 18,000 single nucleotide polymorphisms (SNPs) in 48 cases and 30 controls using two different statistical methods. An individual SNP analysis based on comparison of allele frequencies with a χ(2) statistic was used, as well as a simultaneous SNP analysis based on Bayesian variable selection. Significant association with canine hip dysplasia was observed on chromosome 8, as well as suggestive association on chromosomes 1, 5, 15, 20, 25 and 32. Next-generation DNA sequencing of the exons of genes of seven regions identified multiple associated alleles on chromosome 1, 5, 8, 20, 25 and 32 (p

Pain due to spontaneous intervertebral disc (IVD) disease is common in dogs. In chondrodystrophic (CD) dogs, IVD disease typically develops in the cervical or thoracolumbar spine at about 3-7 years of age, whereas in non-chondrodystrophic (NCD) dogs, it usually develops in the caudal cervical or lumbosacral spine at about 6-8 years of age. IVD degeneration is characterized by changes in the biochemical composition and mechanical integrity of the IVD. In the degenerated IVD, the content of glycosaminoglycan (GAG, a proteoglycan side chain) decreases and that of denatured collagen increases. Dehydration leads to tearing of the annulus fibrosus (AF) and/or disc herniation, which is clinically characterized by pain and/or neurological signs. Current treatments (physiotherapy, anti-inflammatory/analgesic medication, surgery) for IVD disease may resolve neurological deficits and reduce pain (although in many cases insufficient), but do not lead to repair of the degenerated disc. For this reason, there is interest in new regenerative therapies that can repair the degenerated disc matrix, resulting in restoration of the biomechanical function of the IVD. CD dogs are considered a suitable animal model for human IVD degeneration because of their spontaneous IVD degeneration, and therefore studies investigating cell-, growth factor-, and/or gene therapy-based regenerative therapies with this model provide information relevant to both human and canine patients. The aim of this article is to review potential regenerative treatment strategies for canine IVD degeneration, with specific emphasis on cell-based strategies. © 2014 Bach et al.; licensee BioMed Central Ltd.