No description available.

No description available.

No description available.

No description available.

The Gram-positive bacterium Staphylococcus intermedius is regarded as the major cause of canine pyoderma, a common skin infection of dogs. However, despite its clinical importance, the population genetic structure of S. intermedius is poorly understood. The current study examined the population genetic structure of S. intermedius using a multilocus DNA sequencing approach. A collection of 99 isolates phenotypically identified as S. intermedius and originating from a broad array of animal hosts in several different countries was investigated. Phylogenetic analysis indicated that the isolates belonged to three distinct species including S. intermedius, staphylococcus pseudintermedius, and Staphylococcus delphini, together referred to as the S. intermedius group (SIG). Importantly, it was discovered that all canine isolates investigated belonged to the S. pseudintermedius phylotype and it was concluded that S. pseudintermedius, not S. intermedius, is the common cause of canine pyoderma. Further, it was revealed that S. delphini is more clinically important than was previously thought. The allelic variation of agrD, which encodes the autoinducing peptide (AIP) of the agr quorum sensing system in staphylococci, was determined for all isolates. Four AIP variants were identified, including three which were present in all three species, suggesting that a common quorum sensing capacity has been conserved despite species differentiation in very different niches. Considerable clonal diversity was revealed within the S. pseudintermedius species, including several methicillin-resistant clones which have evolved by recent acquisition of the mecA gene. Using the sequence diversity identified, a simple diagnostic test was developed based on a PCR-RFLP approach to discriminate S. pseudintermedius from S. intermedius and S. delphini. Having established that S. pseudintermedius is the common canine pyoderma pathogen, this study aimed to investugate key host-pathogen interactions involved in colonisation of its canine host. Bioinformatic analysis of the whole genome sequence of a clinical isolate of S. pseudintermedius (strain ED99) revealed 17 genes encoding predicted LPXTG-containing cell wall-anchored (CWA) surface proteins. A diverse collection of S. pseudintermedius isolates and closely related staphylococcal species was screened for the presence of the genes encodng the novel CWA proteins. The majority of genes were widely distributed among the isolates examined, with nine genes being exclusive to S. pseudintermedius and eight being also present in other members of the SIG. In Gram-positive bacteria, a family of CWA proteins called microbial surgace components recognising adhesive matrix molecules (MSCRAMMs)mediates bacterial adherence to extracellular matrix proteins of the host. Three of the 17 predicted novel CWA proteins, designated SpsD, SpsL and SpsO, were selected for further characterisation of their role in host-pathogen interactions and were cloned and expressed on the surface of the surrogate host Lactococcus lactis. Solid phase adherence assays employing host extracellular matrix proteins and canine corneocytes were performed to identify host extracellular matrix proteins and canine corneocytes were performed to identify host receptors for the putative MSCRAMMs. L. lactis expressing SpsD demonstrated binding to fibronectin, fibrinogen and cytokeratin 10, SpsL mediated binding of L. lactis to fibronectin and canine fibrrinogen, and SpsD and SpsO both mediated L. lactis adherence to canine corneocytes. Additionally, a cell culture assay using a commercially available canine epidermal cell line was developed and the adherence of S. pseudintermedius ED99 and the L. lactis constructs to the cell line was tested. S. pseudintermedius ED99, but none of the MSCRAMM-expressing L. lactis strains, adhered to the canine epidermal cells in vitro, suggesting that receptors for S. pseudintermedius adherence which are present in ex vivo corneocytes are not present in undifferentiated canine epidermal cell line preparations. Take together, the present study provides broad new insights into the classification and evolution of the SIG, and the molecular interaction of S. pseudintermedius with its canine host.

Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

Abstract CROWN SIZE COMPARISONS IN PATIENTS WITH PALATALLY DISPLACED CANINES By Joseph L. Eliason, D.D.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Dentistry at Virginia Commonwealth University. Virginia Commonwealth University, 2015 Thesis Director: Steven J. Lindauer, D.M.D., M.Dent.Sc. Professor and Chair, Department of Orthodontics There has been significant debate over the past decades regarding the etiology of palatally displaced canines. Theorized risk factors include agenesis or malformation of the lateral incisors, incisor retroclination, transverse deficiency, or genetic predisposition. The purpose of this study is to compare the linear and volumetric measurements of canines and lateral incisors to determine how tooth size relates to canine impaction. Cone-beam CT images for 40 patients with unilateral palatally displaced canines were utilized to measure the linear dimensions and total crown volume of canines and lateral incisors and to compare those teeth on the impaction side with their isomers on the non-impaction side. Results showed that unilateral palatally impacted maxillary canine crowns were slightly, but statistically significantly wider and larger in volume than their non-impacted isomers. Lateral incisor crowns adjacent to impacted canines were significantly shorter than those adjacent to non-impacted canines.

Osteoarthritis (OA) is a chronic, degenerative condition of articular joints. The prevalence of OA is high in many mammalian populations, though our understanding of the disease is limited, with the initiating factors and the early phenotype of the disease being poorly characterised. Clinically, the early-stage of OA is rarely identified, precluding the identification and treatment of affected individuals. Consequently, in vitro models of OA typically reflect the later stages of the disease, and are rarely validated against the naturally-occurring disease. This project utilised tissue from a naturally-occurring canine disease (medial coronoid process disease) to characterise the transcriptome of early-stage OA, and inform different in vitro models, to try and refine the model conditions. Medial coronoid processes from affected dogs were removed and graded histologically, both manually and through the development of a semi-automated assessment. Early-stage OA was characterised by a decrease in the chondrocyte density, an increase in the thickness of the articular cartilage and a loss of proteoglycan. No histological changes in bone morphology were noted in early-stage OA. A transcriptomic approach was adopted, in which the transcriptome of earlystage canine OA was assessed in the coronoid process samples. The canine data generated were meta-analysed alongside published datasets from in vivo models of early-stage OA. These data were from rodent models of the disease. A panel of genes were identified as being associated with the early stage of the disease across multiple datasets. By immunoassay, synovial fluid was screened for pro-inflammatory cytokines and in affected canine joints, interleukin 8 was found to be increased. Three in vitro models (cytokine stimulation of monolayer cell cultures, cyclic compression of agarose embedded cells and impact loading of osteochondral cores) were refined through modification of their stimuli. An identified panel of differentially expressed genes were used to screen each model under different parameters. Hierarchical clustering analysis was used to cluster the panel of conditions so that those which most closely reflected the naturally occurring disease were selected for more detailed transcriptomic analysis by microarray. Chondrocytes and osteoblasts were stimulated with a range of cytokine conditions, using IL-1β and IL-8 based on use in the literature and immunoassay findings. Monolayers were stimulated for a range of times and conentrations with either a single stimulus or multiple cytokines in the medium. The cells responded differently to the cytokine stimulus, requiring different stimuli to most closely replicate the transcriptomic profile of the natural disease. Microarray profiling revealed that cytokine stimulation enriched genes associated with the extracellular matrix and the extracellular region in both cells types. For the cyclic compression model, cells were embedded in an agarose gel matrix and cyclically compressed for various time periods followed by various incubation periods after compression. Both chondrocytes and osteoblasts responded in a similar manner to the cyclic compression stimulus when a post loading incubation step was included to replicate the transcriptomic profile of the natural disease. Cyclic compression enriched gene clusters associated with response to oxidative stress and the extracellular matrix When osteochondral cores were harvested from joints and impacted to represent a traumatic injury, the model could not replicate the transcriptomic model of the natural disease, although increased sGAG release nitric oxide (NO) production was observed. Degradation of mRNA in both tissues was a feature of this model regardless of the loading condition, which precluded further analysis by microarray, but highlighted the significant limitations that were associated with this model. None of the three models tested could accurately reflect the transcriptomic changes of the early-stage OA phenotype in cartilage or bone. A unified model, combining cytokine stimulation with cyclic compression drove cells towards the diseased phenotype in bone. Inflammatory pathways were activated as well as the proteases MMP3 and MMP13. However, chondrocytes were seemingly unresponsive to the multifactorial model, and this will require further analysis. The chronic nature of OA makes it difficult to match in vitro models to the transcriptomic phenotype identified in naturally occurring OA, particularly with respect to the differential expression of structural genes which were identified in the naturally occurring disease but not the models. This work highlights the limitations of existing models, but proposes a validation process which can be used to direct invitro models towards the naturally occurring phenotype.

No description available.

The measurement of pain has had a growing importance in animals for both privately owned animals and those animals involved in clinical research. Lameness is considered to be 1 aspect of the pain experience. The ability of a veterinarian to assess lameness during a routine orthopedic examination can be difficult given the short amount of time in which the clinician can observe the animal, and the fact that the animal is in a stressful environment. Thus, the input of the owner concerning the animal's well-being over an extended time period may be extremely useful to the clinician in assessing the degree of lameness of the animal. It was the purpose of this study to establish an instrument that was both repeatable and valid in assessing the degree of lameness. The instrument used was a questionnaire containing 39 questions in a visual analog scale format. A force platform was used as the gold-standard for detecting mechanical lameness. Peak vertical, cranial-caudal, and their associated impulses were forces used to determine lameness, along with maximum slope in some cases. A test-retest measure of repeatability was conducted on a subset of 19 dogs that were confirmed to have less than a 10% change in vertical peak force. Nineteen of the 39 questions were found to be repeatable based on a Spearman rank correlation. These 19 questions were then used as predictor variables in several multiple regression models which predicted force plate measurements. The result was 3 different models each containing 7 independent variables that were thought to be valid representations of the forces measured (vertical peak, vertical impulse, and propulsion peak forces). Each reduced model was found to fit the data as well as the full model containing all 19 of the repeatable questions. The composite of 11 questions from the 3 different models was used to calculate a total score. This total score was found to be significantly correlated with force plate measurements. These 11 questions should be useful to a clinician in detecting the degree of lameness in the dog.