The relationship between haemoglobin A1c measured in a novel immunoturbidity assay, other markers of glycaemic control and outcomes of diabetes mellitus in dogs
The relationship between the clinical control of canine diabetes mellitus and markers of diabetic control, such as average blood glucose, blood glucose curves and fructosamines, is not well established. In humans a form of glycated haemoglobin called haemoglobin A1c (HbA1c) has been shown to be a highly specific and reliable biomarker for the long term control of diabetes mellitus. Increasing values of HbA1c in humans correspond to increased risk of diabetic complications. HbA1c has two advantages over fructosamines; it is less affected by other clinical conditions and provides a longer term estimate of average glycaemic control. Previous studies in dogs have generally measured glycated haemoglobin and these assays are no longer available.
Recently we have validated a new immunoturbidity assay that measures HbA1c in dogs. The aim of this study is to compare HbA1c, fructosamines and mean blood glucose measurements as markers of outcome (cataracts, hypoglycaemic episodes, ketoacidosis, pancreatitis, urinary tract infections) in recently stabilised diabetic dogs. The ultimate goal is to identify appropriate intervention points for use in practice but without identifying an effective measure of outcome first such intervention points are necessarily only estimates.
Hypertrophic cardiomyopathy: Exploration of signalling pathways to unravel disease mechanisms that drive fibrosis and ventricular hypertrophy
Background: Hypertrophic cardiomyopathy (HCM) has a high prevalence of over 15% in cats. Interstitial fibrosis is both a hallmark of HCM and importantly a key driving force for left ventricular hypertrophy (LVH). Upregulation of crucial pro-fibrotic signalling pathways including the transforming growth factor beta (TGFβ)/SMAD pathway and the lumican and lysyl oxidase (LOX)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) pathway have been identified in left ventricular (LV) tissue from rodent models of HCM and human HCM patients.
Hypothesis: Cardiac fibrosis in feline HCM is regulated by signalling through the TGFβ/SMAD and Lumican - LOX/NFĸB pathways.
Pilot Data: Our pilot data (shown later) indicates that lumican and LOX (important molecular drivers of cardiac fibrosis) are increased in atrial tissue from cats with HCM
Aims and methods: We aim to extend these results by examining the expression of a larger number of pro-fibrotic mediators (based on published data from rodent models and human patients) including lumican, LOX, TGFβ and NFĸB at the transcriptional and translational level. To do this we will use Multiplex PCR, immunohistochemistry (IHC) and Western Blot (WB) in LV samples from 15 HCM affected and 15 normal cats. Exploring the activation of these pathways in the LV (as opposed to atria) is crucial since the influence of HCM causing mutations on pro-fibrotic pathways is most apparent within the high pressure LV. This proposal will benefit from our ongoing exciting collaboration with Dr David Hodson Senior Fellow at the Institute of Metabolism and Systems Research University of Birmingham exploring intracellular signalling pathways that drive pathological LV remodelling in feline HCM.
Clinical importance: Identifying which pro-fibrotic signalling pathways are active in feline HCM will form the foundation for future studies to explore their interactions. Manipulation of these pathways with novel agents such as selective NFĸB and SMAD inhibitors represents a powerful therapeutic approach for the management of the fibrosis and LVH associated with HCM.
Resistance of canine Staphylococcus pseudintermedius to selected topical and systemic antimicrobials in the UK.
Staphylcococcus pseudintermedius is the main mucosal coagulase positive staphylococcal commensal of dogs, but also a major opportunistic pathogen and potentially zoonotic for immune-suppressed people. Recently, meticillin resistant S. pseudintermedius (MRSP) clones have emerged and disseminated globally. These clones are multidrug resistant (resistant to three or more antimicrobial classes) and often pan-resistant (Perreten et al 2010). Treatment options are often limited to topical antimicrobials for localised and/or superficial skin infections, but for deep, severe and/or widespread skin infections or systemic infections therapeutic options are lacking. Extra-label use of unauthorised systemic antimicrobials is permitted under the cascade but pharmacokinetic (PK) and pharmacodynamics (PD) data for these drugs is limited or unavailable.
This study would aim to investigate and characterise resistance to topical and systemic antimicrobials including fusidic acid, mupirocin and chlorhexidine, minocycline and florfenicol amongst UK MRSP and methicillin susceptible (MSSP) isolates. This study will provide important local and national resistance patterns for this canine pathogen that can be used to guide treatment. Providing the appropriate treatment of canine MRSP infections is an animal welfare issue. Furthermore the results will provide data that can be used in future studies for the determination of antimicrobial resistance breakpoints (cut offs) in dogs for the tested antimicrobials.
Erythropoietin in Canine Transmissible Venereal Tumour Disease
Canine Transmissible Venereal Tumour (CTVT) is unusual in that the cancer causing agent is a dog cell line that in essence uses the affected dogs as a culture vessel. The cancer is spread by direct contact (usually via sexual transmission) and is found in many parts of the world where there are large numbers of free ranging dogs. Dr Murchison has preliminary data demonstrating that the tumour causes higher levels of erythropoietin (a hormone that encourages the production of red blood cells) in affected dogs. This is probably a way in which the tumour manipulates its host to increase its own blood supply. This project will focus on confirming that the increased erthryopoetin is being produced by the tumour cells. If proved this potentially opens up new areas of cancer therapeutics in dogs.
Is primary tumour growth associated with a shift in tau protein status in the canine brain?
Brain tumours are a common reason for dogs to be referred to a specialist neurology service. This typically happens at a late disease stage – a stage which is difficult to treat and significantly compromises patient welfare. Brain tumours are also challenging to diagnose and classify to a level that can inform clinicians of the best treatment option. Often, the definitive diagnosis relies on post-mortem analysis. Furthermore, we currently have a poor understanding of how and why tumours develop in the canine brain, although there has been increasing evidence to support the ‘cancer stem cell hypothesis’ in which a rare group of tumour cells with stem cell features (including the capacity to self renew) is responsible for tumour growth, resistance, and recurrence. We urgently need to deepen our understanding of brain tumour development in order to identify new markers that will help us to diagnose these lesions faster and treat them in a more targeted manner. Any novel diagnostic markers must first be validated against the pathological diagnosis of the tumour using a brain tissue sample. We have recently developed a brain bank for companion animals that can support research of this nature. Brains are collected and archived from animals that have been euthanased on welfare grounds for a terminal condition with full consent of their owner. Diagnostic evaluation of the tissue is performed by qualified veterinary pathologists.
One protein that requires further study in the canine brain is tau. Tau protein is highly abundant in nerve cells and undergoes modifications during brain development, under certain physiological conditions that promote brain protection, and in the disease state. Certain changes in tau have been linked to both neurodegenerative disorders (e.g. Alzheimer’s disease) and also tumours. It might seem counterintuitive for this protein to play a role in seemingly opposite disease processes, however there is evidence to suggest that tau interacts with overlapping biological pathways involved in both the failed regeneration of degenerating nerve cells and the aberrant growth of cells observed in cancer. This study asks whether tumours that develop within the canine brain are associated with changes in tau, and whether these changes in tau are specific for tumour development or reflect more general changes that might be expected in for any lesion that affects brain tissue. In order to answer this question, we will characterize and compare tau expression in post-mortem brain tissue from three groups of dogs: (1) with brain tumours, (2) with other brain lesions and (3) with normal brains. If specific changes in tau can be mapped to specific disease processes, tau profiling, either via tissue biopsy or by analysis of the fluid that bathes the brain, may become a useful tool with which to diagnose these conditions sooner in live patients. Furthermore, it may pave the way for tau-targeted treatments to help manage certain aspects of tumour growth. Ultimately, rapid diagnosis and a better understanding of the disease process will advance our capacity to treat brain tumours and improve the welfare of pet dogs.
3D Quantification and Characterisation of spinal cord dorsal horn neuronal population (lamine I to V) in Cavalier King Charles Spaniels with Syringomyelia
Syringomyelia is a painful condition characterised by fluid filled cavitation off the spinal cord. This disease has high prevalence in toy breed dogs and is a serious welfare concern. A classic sign of the severe syringolmyelia is tendency to scratch towards one shoulder, often when the dog is walked or on a lead. Commonly referred to as phantom scratching, affected dogs are often unable to walk normally. The sign is easy to recognise however the underlying mechanism is not understood. In previous PetSavers funded research we showed phantom scratching is associated with a large dorsolateral syrinx that extends to the superficial dorsal horn in the cervical spinal cord (C3-C6 spinal segments corresponding to C2-C5 vertebrae) WE propose that phantom scratching was due to damage to superficial dorsal horn neuron and consequently inhibitory interneurons which influence on the lumbosacral scratching central pattern generator (I.e. the neural network that produces a rhythmical scratching action of the hind limb). An extension of that work to be undertaken by the same student researcher will examine the quantitative histopathology of the dorsal horn neurons damage by syringolmyelia in Cavalier King Charles Spaniels (CKCS) with and without phantom scratching.
Developing novel biocompatible and antimicrobial coatings for orthopaedic implants in dogs
Many millions of orthopaedic surgeries including arthroplasties take place worldwide each year in dogs. Bacterial infections leading to implant failure occur in around 5% of cases, often resulting in implant removal due to infections each year. Infections, often caused by Staphylococcus aureus bacteria, may occur during surgery or much later (up to one year) via the blood. Bacteria are protected from antibiotics and the body’s immune defence system by the implant material. This project will examine whether dog bone marrow stem cells, collected from the bone removed during routine surgery, can grow in the presence of a novel titanium dioxide “smart” implant coating, developed by the Engineering Department at Bristol, which can prevent bacterial colonisation.
Immunohistochemical characterisation of feline idiopathic lymphocytic-plasmacytic anterior uveitis
The project looks at a disease in cats called “idiopathic anterior uveitis” (IAU), which is when the cats own immune system attacks part of the eye causing it to become inflamed and painful. Without treatment it can lead to blindness, and often patients will have to have an eye removed because of the pain. What triggers IAU isn’t understood, and the purpose of this project was to look at some of the types of immune cells present in the eyes of cats suffering from this disease to get a better idea of its cause and hopefully inform better treatments. I got some samples of eyes of cats that had them removed because of IAU, and used a staining technique called immunohistochemistry to highlight the cells I was interested in and count them. We compared the number of cells between eyes that had different levels of inflammation. We found that regulatory T cells, a type of immune cell that acts to stop other kinds of immune cells from damaging the body, were more common in more inflamed eyes. This means that higher numbers of these cells does not protect against the IAU like we thought may be the case, and that the disease probably isn’t caused by a lack of these cells causing the body to attack itself. We also showed that a marker for a kind of inflammatory cell, IL17A, was present in eyes with IAU, but not in eyes with inflammation caused by a virus. This means that this type of cell might be particularly important in causing IAU, because it isn’t present in a disease that looks similar but has a different cause, and so treatments that target this cell type might be particularly effective in managing the disease.
Effect of hyperthyroidism on serum SDMA concentrations and the utility of SDMA as a marker of CKD in hyperthyroidism
Serum symmetrical dimethylarginine (SDMA) concentrations are reported to be a better test of kidney function in cats when compared with more traditional tests of kidney function, such as serum creatinine concentrations. We know that hyperthyroidism can influence the serum creatinine concentrations, which in turn makes the diagnosis of chronic kidney disease (CKD) in hyperthyroid cats more difficult. However, to date no study has reported the effect of hyperthyroidism itself on serum SDMA concentrations. This is important because hyperthyroidism and CKD are common co-morbidities in older cats. In human patients, thyroid dysfunction (both hyper- and hypothyroidism) can influence arginine metabolism (including SDMA). If hyperthyroidism affects serum SDMA concentrations, independent of the presence of concurrent CKD, this could lead to misdiagnosis of CKD in some hyperthyroid cats.
In addition, currently there is no reliable test of renal function in hyperthyroid cats available, because of the effects of hyperthyroidism on traditional markers like serum creatinine concentrations. Therefore, evaluation of serum SDMA concentrations as a marker of CKD in hyperthyroid cats is also warranted.
Evaluation of the microenvironment and immune function in Histiocytic Sarcoma, a tumour of dendritic cells
To evaluate the relationship between tumour, microenvironment and immune system in canine histiocytic sarcoma, using flow cytometry, immunohistochemistry and mRNA expression analysis.
1. To explore the relationship between tumour (of dendritic cell origin) and regulatory T cells and the role of the latter in promoting immune-tolerance within the tumour milieu.
2. To explore the expression of tolerogenic immune mediators in histocytic sarcomas.
3. To establish and validate a panel of antibodies for flow cytometry to profile T cells in peripheral blood and samples from tumour and draining lymph node of affected dogs.
4. To examine T cell profiles (including T-regs) in the peripheral blood of dogs bearing histiocytic sarcoma by flow cytometry, to document changes in these profiles during the course of treatment and at relapse, and determine whether these correlate with prognosis.
The immune system has an important role in both the development and progression of cancer. This can be a dual role, on the one hand the immune system can eradicate emerging malignant cells, but on the other it can promote the growth, invasion and metastasis of malignant cells. In recent years the tumour microenvironment and the presence of infiltrating immune cells (T cells and macrophages) has become of increasing importance to our understanding of the relationship between cancer and the immune system. Of particular interest is the recognition of regulatory T cells within tumours that appear to down regulate the immune system and are associated with a poorer outcome in many human tumours. These cells may offer a target for future cancer management strategies so their role in cancer progression is important to understand.
The flat-coated retriever (FCR) is one of the dog breeds at risk of developing histiocytic sarcoma (HS) and although the precise cellular origin of this tumour is unknown, the immunophenotype is suggestive of a myeloid dendritic antigen presenting cell (APC) lineage. We have previously shown that HS in FCR contain a prominent infiltrate of T cells 1, and recently that these were regulatory T cells phenotypically. It is interesting that this prominent T cell infiltrate should be present in a tumour comprising cells that modulate the immune response, dendritic cells. It is known that dendritic cells play a pivotal role in determining immune-tolerance versus immunity. Thus a key step in better understanding the relationship between the tumour, its microenvironment and the immune system is essential to understanding how the tumour influences immune function. The finding that a significant proportion of tumour infiltrating T cells expressed FOXP3, suggesting them to be regulatory T cells, raises interesting questions of cause and effect which we aim to address in the proposed study
Neutering and Acquired Urinary Incontinence in the Bitch
An increased risk of acquired urinary incontinence due to sphincter mechanism incompetence in bitches has been attributed to timing of ovariohysterectomy (Holt 1987, Holt & Thrusfield 1993). Proposed mechanisms of action of acquired urinary incontinence include reduced levels of endogenous oestrogen, which may reduce tone in the urethral sphincter, increased gonadotropin levels, decreased gonadotropin or cyclooxygenase-2 receptor expression, decreased amounts of smooth muscle in the urethra and bladder, changes to collagen structure and shortening of the urethra (Gregory et al. 1992, Byron et al. 2007, Noël et al. 2010). However existing evidence is equivocal with limited response rates, losses to follow-up and small sample sizes being frequently seen. As such a recent systematic review, though identifying some evidence of an association between neutering per se and timing of neutering and development of incontinence, highlighted that the existing evidence was of moderate strength at best and further work was required to more fully address this important issue (Beauvais et al. 2012).
In a recent survey of UK veterinarians, urinary incontinence was the second most commonly stated disadvantage of neutering bitches (Diesel et al 2010). Work by O’Neill and colleagues (2014) estimated that approximately 1.7% of practice-attending dogs were diagnosed with urinary incontinence. While many affected bitches respond to oral therapy, this treatment generally has to be continued for life (Shiel et al. 2008). Furthermore, a recent study suggested that urinary incontinence in bitches was a cause of disharmony in 10-20% of affected households, with individual owners reporting feelings of anger and frustration (de Bleser et al, 2011). Hence, although the direct welfare impact may be considered minor for the affected animal, the potential impact on a large group of dogs, the owner-animal bond, as well as the perceived importance of the condition in the neutering decision-making process, in the context of evidence that needs strengthening, suggest further evaluation of the condition is merited. The aim of this study is to revisit this important topic, build on existing work and more completely evaluate the role of neutering and timing of neutering on the onset of the condition using a large existing primary practice database (VetCompass).
Variation in biological behaviour of canine cutaneous mast cell tumour: a study of prognostic variants between tumour grades in Labrador and Golden retrievers
Mast cell tumour (MCT) is a common tumour of the canine skin, and the second most frequently diagnosed canine malignancy (Dobson et al. 2002). Its prevalence in the UK dog population, across all breeds, is around 0.27% (Shoop et al. 2015), which is equivalent to 27,000 cases in the population at any given time. Several breeds, including the Labrador retriever and Golden retriever have an increased risk of developing cutaneous MCTs (Warland and Dobson 2013; Shoop et al. 2015). Despite advances in treatment, canine cutaneous MCTs are associated with substantial morbidity and mortality. They have variable behaviour, and histopathology is currently used to predict prognosis. The commonly employed Patnaik system categorises MCTs into grade I (well differentiated), II (intermediately differentiated) or III (poorly differentiated) tumours (Patnaik et al. 1984). Grade III tumours show aggressive behaviour with more than an 80% metastatic rate and they frequently cause death. Grade II tumours are the more frequently reported, and cause death in 17–56% of cases. Less than 10% of grade I MCT metastasise, and in general they carry a good prognosis. A more recently proposed Kiupel two tier system categorises cutaneous MCT as high or low-grade (Kiupel et al. 2011). However, inherent variability in histological grading of MCTs can make providing an accurate prognosis to clients problematic.
Several recent studies have demonstrated a significant inherited susceptibility to cancers in dogs (Shearin et al. 2012; Karlsson et al. 2013; Tonomura et al. 2015). The presumed genetic risk factors for MCT are largely unknown, although somatic mutations in the proto-oncogene c-kit have been detected in some cases (Letard et al. 2008). Germline mutations in genes encoding for metabolism of hyaluronic acid have also recently been reported in affected Golden retrievers (Arendt et al. 2015), and a candidate genome region for MCT risk has been identified on chromosome 36 in Labrador retrievers (Hayward et al. 2016). The identification of multiple genome regions associated with MCT risk indicates it has a complex genetic basis.
To explore this complex genetic basis we have come together as a multi-disciplinary team of researchers with expertise in clinical oncology (Dr Nicholas Bexfield – University of Nottingham), clinical genetics (Dr Sarah Blott - University of Nottingham) and epigenetics (Dr Victoria James - University of Nottingham), leadership in bioinformatics (Professor Richard Emes - University of Nottingham Advanced Data Analysis Centre http://www.nottingham.ac.uk/adac/index.aspx), and extensive experience of histopathology (Dr Tim Scase – Bridge Pathology Ltd). The investigators have a strong proven track record performing high quality basic and clinical research. The team is focused on understanding how genetic variants confer MCT risk, and the interaction of genetic and environmental factors to modulate both germline risk and tumour behaviour. This project would form part of this overarching research area, building on data already generated by the group. The Scholar would therefore benefit from being part of an active multi-disciplinary team, undertaking a research project arising from a currently unmet clinical need.