Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 219.32K | Year: 2017
A latticework of bony trabeculae stiffens the internal spaces of many bones. Trabeculae transmit forces between joint surfaces and bone shafts and maintain the integrity of vertebrae. Bone loss is a normal physiological response to reduced mechanical load, occurring during bed rest, in sedentary lifestyles, and during the weightlessness of spaceflight. Bone loss is also a normal, undesirable, part of ageing which occurs particularly rapidly in women after menopause. Reduced bone mass is strongly associated with increased fracture risk. Peak bone mass is obtained in humans in early adulthood. Strategies to increase peak bone mass and to slow its physiological loss are of great interest to ensure healthy ageing. In addition to bone mass (bone volume fraction, BV/TV), bone architecture is thought to play a crucial role in trabecular bones force transmission and fracture resistance. Bone architecture is defined by standard measurements such as trabecular thickness (Tb.Th), spacing (Tb.Sp), interconnectedness (Conn.D), anisotropy (DA) and rod-plate geometry (structure model index, SMI). The bulk behaviour of trabecular bone in physiological loading is almost entirely determined by BV/TV: this is intuitive because the more bone matrix there is per unit volume, the greater the overall volume can resist applied load. Once a higher failure load has been reached, however, the behaviour of trabecular elements dominates collapse of the overall structure. Each trabecula might fail by buckling, shear or crumpling. Bone biologists believe that plate-like trabeculae resist higher loads than rod-like trabeculae and that plate-like trabeculae convert to rod-like trabeculae during bone loss, creating a double-whammy: reduced bulk resistance to load due to decreased BV/TV and reduced resistance to trabecular element failure due to rod-like geometry. Whether a plate-to-rod transition actually occurs in bone loss is now in serious doubt, because the paradigm is based on flawed SMI measurements. I recently showed that variation in SMI during bone loss is dominated by increasing amounts of concave portions of the bone surface, which the SMI theory assumes are not there or have a negligible contribution. There is a high correlation between SMI and BV/TV even when the underlying architecture has not changed. SMI is a fundamentally broken measurement that does not do what bone bioscientists use it for. Over 850 papers have cited SMI and a common interpretation when seeing BV/TV and SMI results together is that bone loss is associated with a plate-to-rod transition, however, this is merely an artefact of SMIs design that obscures any true relationship. Statements linking bone loss and plate-rod transition have been repeated often enough to have become an accepted dogma in the bone field. This project aims to overturn the dogma there is a plate to rod transition in bone loss using 3D X-ray microtomography (XMT) image data sets archived from previous experiments. To measure rods and plates independent of BV/TV, the PDRA will validate a new method, ellipsoid factor (EF), that defines rods and plates by the shape of the biggest ellipsoid that fits within each bony region. Rods can fit long, javelin-shaped ellipsoids, while plates can fit flat, discus-shaped ellipsoids. The PDRA will use image data collected on RVCs XMT instrument, images from my project partner Phil Salmon at Bruker microCT, and from other collaborators worldwide, and aim to increase our current pool of ~150 images from 3 studies to ~1000 images from ~20 studies. Having identified rods and plates in bone samples, the PDRA will correlate rods and plates with mechanical behaviour using finite elements analysis in the physiological load range for bulk behaviour and overload range for trabecular element failure behaviour. The primary benefit of the work is improved understanding of true geometric and mechanical changes in bone loss, along with the new EF method and free software.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 359.77K | Year: 2016
Female reproductive lifespan is established during fetal life through the formation of a cohort of primordial follicles in the developing ovary. Primordial follicles consist of an egg (oocyte) surrounded by a layer of pre-granulosa cells, which support, maintain and control the growth of the oocyte. Women are born with a finite number of follicles which decline in number throughout life, and no new follicles are formed after birth. The menopause occurs when the follicular reserve is exhausted, which normally happens around the age of 50. However, in 1% of women the menopause occurs before the age of 40, a condition known as premature ovarian insufficiency (POI). In addition to being a devastating diagnosis for women yet to complete their families, POI is associated with significant post-menopausal health risks, such as osteoporosis and cardiovascular disease. Furthermore, as societal trends move increasingly towards starting families later in life, and the supply and quality of oocytes in the ovary declines with age, the number of women experiencing sub-fertility is likely to rise. Despite the critical importance of the follicle formation process in establishing reproductive lifespan in humans, and other mammals (such as livestock species), we know very little about how the stock of follicles is formed in the fetal ovary. Improving our knowledge of this fundamental process is essential if we are to investigate how genetic, environmental or lifestyle factors can disrupt follicle formation and curtail reproductive lifespan. The cell to cell signalling molecule Activin A (ActA) is a possible key regulator of follicle formation. Elevated ActA signalling in fetal/neonatal mouse ovaries results in the formation of greater numbers of primordial follicles. The mechanism by which this occurs is unclear, but is likely to involve changes to pre-granulosa cell number or behavior, as ActA only signals to this cell type in the developing ovary. Pre-granulosa cells also express a protein called FoxL2, which activates genes important for ovarian development. In humans, mutations in the FOXL2 gene cause POI, infertility and granulosa cell tumours (a rare type of ovarian cancer). In mice, pre-granulosa cells that lack FoxL2 cannot function correctly, and so fail to form follicles (or form follicles that cannot mature). In the pituitary gland, ActA and FoxL2 work together to activate genes that are required for cells to respond to, and produce, reproductive hormones, and a similar FoxL2/ActA interaction activates genes in the granulosa cells of the adult ovary. Whether ActA and FoxL2 work together to regulate pre-granulosa cell function and follicle formation in the fetal ovary is not known, however. The aim of this project is to address this question, using the neonatal mouse ovary and human pre-granulosa cells as models. We will establish whether the number of FoxL2-expressing pre-granulosa cells formed in the ovary limits the number of follicles that can be formed, and determine whether activin increases follicle number by altering the size of the pre-granulosa cell pool. To gain insight into the biochemical processes they regulate in pre-granulosa cells, we will identify the genes that ActA and FoxL2 jointly-control, and investigate whether loss of either factor prevents these genes from being switched on or off correctly. Finally, we will investigate why primordial follicles fail to form correctly in FoxL2-deficient mouse ovaries, determine whether aberrant signalling by activin (or other signals) is the cause of this defect, and establish whether this can be corrected by restoring normal levels of signalling between cells. These studies will shed new light on how the number of follicles (and thus female reproductive lifespan) is established, provide insight into how mutations in FOXL2 in humans lead to infertility, and inform future studies to develop of new strategies to manage fertility.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 901.73K | Year: 2016
Bovine tuberculosis (BTB) is a persistent problem in certain areas of the UK. The current control strategy is to test herds for the presence of BTB at timely intervals and slaughter test-positive animals. However, the observation that BTB is spreading in both geographical area and prevalence suggests that the existing control strategy is not working effectively. The cost of the control programme is £100 million annually, therefore not only is the policy ineffective, it is also a significant economic burden. In addition, BTB can cause tuberculosis in humans, and while this is currently rare, the escalating prevalence of BTB has the potential to develop into a zoonotic (the passage of disease from animals to humans) risk. BTB is caused by a bacterium called Mycobacterium bovis. This is very closely related to Mycobacterium tuberculosis, a bacterium that commonly causes tuberculosis. Tuberculosis in cattle and humans show great similarities and, like the human disease, vaccination is an alternative strategy to control BTB. The vaccine against human tuberculosis is a strain of Mycobacterium bovis called Mycobacterium bovis BCG, however this provides limited protection against the disease in both cattle and humans. Mycobacterium bovis is transmitted by aerosol, once within the animal it enters into cells called macrophages. These cells, normally dedicated to the killing and removal of bacterial pathogens, are unable to kill the bacteria. Instead, Mycobacterium bovis adapts and survives within the lungs in structures called granulomas. However, we do not know much about the genes that allow the bacteria to survive and cause disease in the host. We will knock-out the function of every single gene in the genome of Mycobacterium bovis (approximately 4000) and determine those genes that are required for survival in bovine macrophages and in the whole cow. We will achieve this by using a technique called Transposon Directed Insertion Seqeuncing (TraDIS). This techniquehas been applied successfully to the study of other bacterial pathogens of medical and veterinary importance, but this will be the first time it has been used to study the genetic determinants of disease in Mycobacterium bovis. We will make libraries of mutants where the function of every gene in the genome has been knocked-out. We will then put the libraries into screens. Mutants that are recovered after being through a screen are compared to the original pre-screened mutant pool by DNA sequencing. Mutants that are unable to survive the screen are identified and as a result we will identify all the genes necessary for survival in the host. Those mutants that are unable to survive in the host represent potential vaccine candidates. We will be able to assess the function of essential genes using a combination of computational analysis, literature searching and comparisons to screens performed on different types of bacteriological media (in vitro). The functions that are essential for survival in the host also reflects the conditions within the host. At the end of this project we will have filled in key gaps in the knowledge of BTB in the area of host pathogen interactions and have identified several potential vaccine candidates to be considered for future development.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 1.77M | Year: 2015
Brucellosis is the worlds most widespread zoonosis, imposing a substantial burden on the livelihoods of poor people as a result of human disease and reduced livestock productivity. However, brucellosis is rarely a priority for health systems and for this reason the WHO classifies brucellosis as a neglected endemic zoonosis. The main routes by which people can be infected are consumption of contaminated dairy products and direct contact with infected animals. Therefore, the control of human brucellosis depends on its control in animals, mainly ruminants, while its detrimental effects include both animal and human disease. Although a range of tools (diagnostics, vaccines) are available and have been used, sometimes successfully, to control brucellosis in different parts of the world the disease remains present at high levels in others. It is likely that failure to control the disease is due to a combination of factors, some of them technical (e.g. quality and appropriateness of different vaccines for different settings), others socioeconomic (livestock keepers perceptions towards the disease and incentives for its control) or institutional (priority given by veterinary services to the disease, lack of interaction between animal health and public health services). In this project we target one of the livestock systems in which Brucellosis is likely to pose the heaviest burden on vulnerable populations in Sub-Saharan Africa: dairy farms supplying milk to rapidly growing urban populations in West and Central Africa, and we will do it by integrating a range of disciplines so that we consider the different biological, social and institutional dimensions of the disease that are relevant for its control. We will focus on the first phase of control of the disease: the reduction of its prevalence by means of vaccination, since total eradication requires conditions that are not currently met in the target area. The so-called peri-urban dairy farms are an important source of wealth in poor settings and a source of protein in increasingly urbanised African populations. Furthermore, there is evidence that this region has the highest prevalence of ruminant brucellosis in Sub-Saharan Africa. We will carry out a series of field studies to measure the burden of brucellosis in livestock productivity and human health in the main areas for peri-urban dairy production in the 15 target countries, to identify routes by which people become infected, to quantify the economic consequences of the disease for farmers and to understand farmers perceptions and attitudes toward the disease. In parallel we will carry out a series of training activities and we will work with local institutions to identify barriers for effective and sustainable control and to overcome those. The results will be used to design and carry out 2-year control programs based on vaccination in a small number of settings. The effectiveness of these control programs will be carefully monitored and the results widely shared within and outside the region. Central to this project is the Interstate School of Veterinary Science and Medicine of Dakar (EISMV) a regional educational hub that involves 15 West and Central African countries. EISMV academics will work in partnership with UK-based researchers from two leading veterinary and medical educational institutions: Royal Veterinary College and London School of Hygiene and Tropical Medicine and with the international reference laboratory for brucellosis at Animal Health Veterinary Laboratories Agency. The Global Alliance for Livestock Veterinary Medicines (GALVmed), an organization which works globally to facilitate access of poor livestock keepers to affordable livestock vaccines, medicines and diagnostics will also be an essential part of the consortium contributing to the translation of our research findings into tangible benefits for vulnerable populations and livestock keepers in poor countries.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 400.10K | Year: 2016
Yeast species such as Saccharomyces cerevisiae are useful tools for the high yield production of recombinant proteins and have a Generally Regarded As Safe (GRAS) status. They are capable of performing several complex post-translational modifications that are not achieved in many other expression systems, and are easily grown to very high densities producing large quantities of stable particles. Recently, the idea of using S. cerevisiae as a delivery vehicle for cancer, viral, and bacterial vaccines has been explored, inducing robust humoral and cellular immune responses. In addition to using yeast to produce a vaccine antigen of interest, the yeast cell itself has been shown to have adjuvant-like properties and has the potential to activate both inflammatory and phagocytic receptors expressed on antigen-presenting cells. Our preliminary data demonstrates that freeze-drying recombinant yeast cultures expressing viral protein at their surface renders the recombinant yeast completely non-viable (unpublished). However, the freeze-drying process does not alter conformation of these proteins, as surface expression is equal in live and freeze-dried yeast as quantified by flow cytometry and Western blotting. This has interesting implications in vaccine design as a non-viable S. cerevisiae is not categorised as a genetically modified organism (GMO) and such a killed vaccine would not be subject to GMO regulations. Additionally there would be no need for refrigeration of the freeze-dried yeast, reducing transport and storage costs. Vaccines based upon S. cerevisiae are likely to be particularly valuable against diseases of farmed poultry, where safety, scalability, stability, delivery and cost are crucial. In one example modern poultry production relies on effective control of Eimeria, but current approaches using drugs and live vaccines require improvement. In recent years a panel of coccidial antigens have been identified as vaccine candidates, each individually capable of inducing up to a 65% reduction in oocyst output. Now, the focus is shifting from antigen discovery to antigen formulation and delivery with yeast being a leading option, especially given the importance of T-cell mediated responses in anticoccidial immunity.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 417.93K | Year: 2016
Notwithstanding current world population infertility in humans is on the rise. 1 in 10 couples is infertile and in about a third of the cases, the defect arises from the male. Testicular defects are among the principal causes of reproductive deficit in males. Perhaps due to environmental effects male infertility in animals is also on the rise. This has most likely contributed to the reduction in numbers of many animal species in the wild. Understanding mechanisms that regulate testicular development and function is therefore essential for development of therapeutic strategies to alleviate male infertility disorders. The two major functions of the testis are to produce testosterone and sperm throughout the reproductive life of the species. Generally this begins at puberty when testicular size increases. Increased testicular size is an indicator of active generation of sperm and testicular function. There is, however, a need for age-related waning of testis function so that mutations accumulated with age are not propagated in the population. For seasonal breeders an additional requirement is the cyclical increase and decrease of the testis with the beginning and end of each breeding season. Sertoli cells (SC) in the testis allow for the duplications and development of the cells that give rise to sperm and generally the total number of germ cells produced depends on the number of SC in a testis. This number is fixed very early in life and normally does not change with age. What is not clear is how the number of SC is determined and whether SC numbers in a testis can be increased later on in life. It is also unclear how testis size reduction is controlled in an age or season dependent manner. We have found that a natural cell product, follistatin-like 3 (FSTL3) might be crucial in regulating testicular development. Our findings in genetically modified mice lacking FSTL3 in all cells show increased testis size, and a lack of testis size reduction with age. Also, within the testis there is an increase in SC numbers and related increase in cells that give rise to sperm. We, therefore, hypothesise that testicular FSTL3 action is essential for limiting testicular size and age-related decline in testicular function. There are only two other mouse models with increased testis size, but neither of these have a block in age-dependent reduction of testicular size. Our FSTL3 deletion mouse model, therefore, allows us to ask several crucial questions regarding testis development and function. Here we will first determine whether SC multiplication can be increased by reducing FSTL3 in mice and in tissue culture cells. We will then investigate whether SC numbers, can be increased by deleting FSTL3 only in the SC. This will demonstrate whether SC numbers are controlled by local FSTL3 expressed in the SC or whether there is a systemic requirement for FSTL3 production. Furthermore, we will address whether removing FSTL3 after puberty increases SC numbers. This will demonstrate whether SCs can multiply beyond the first few days after birth, therefore providing the possibility of a therapeutic strategy of inducing SC growth and fertility in infertile males with limitingly small testes. Finally, we will identify which genes and cellular signalling pathways FSTL3 may influence to limit testicular size and help achieve reduction of testis size with age. Our research will, therefore, identify how FSTL3 regulates testis development and function and helps limit reproduction with age. We will be identify the molecular mechanisms of these actions, whether FSTL3 dependent SC number regulation is achieved through its expression solely in SC and whether SC can be induced to grow, by removing FSTL3 at any time after birth. The contributions from this work to our understanding of the processes underlying testis development may support development of preventative and therapeutic approaches to dealing more effectively with male infertility.
Agency: GTR | Branch: BBSRC | Program: | Phase: Fellowship | Award Amount: 304.82K | Year: 2017
Epilepsy is a complex brain disease, in which individuals are predisposed to spontaneous seizures, and is common in both humans and dogs. Epilepsy is estimated to affect 0.6% of dogs, with prevalence markedly higher in some breeds e.g. 17-33% in the Belgian Shepherd. Although seizures may be the signature symptom of epilepsy, epilepsy may have a variety of manifestations, not limited to seizure activity. Co-morbid psychiatric and neurodevelopmental disorders are commonly seen in people with epilepsy, and have been reported to have a greater impact upon the quality of life (QoL) of the individual patient. To date, the behavioural comorbidities of epilepsy in the dog have been little studied, with the main focus in veterinary medicine being upon seizure control (reducing seizure frequency/severity). This approach may leave many dogs vulnerable to the negative effects of undiagnosed comorbid disorders including anxiety, depression, attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). This research programme will focus on two recognised neurodevelopmental comorbidities of epilepsy, ADHD and ASD. ADHD is a neurodevelopmental disorder that affects one third of human epilepsy patients, with hallmarks of ADHD including easy distraction, impulsivity, hyperactivity and slow learning. ADHD-like behaviour is seen in rat models of epilepsy and has recently been recognised as a comorbidity of canine epilepsy. ASD is characterised by social deficits and communication difficulties and stereotyped/repetitive behaviours. ASD symptoms occur in 15-35% of children with epilepsy, and ASD-like behaviours are seen in rodent models, but has not yet been considered as a comorbidity of canine epilepsy. Children with these disorders frequently present with overlapping clinical signs of ASD/ADHD, and it is likely that the epilepsy-ASD-ADHD phenotype has a complex and heterogeneous pathogenesis. This research programme will objectively study the behaviour of dogs with epilepsy to identify ASD and ADHD phenotypes, and identify neuroanatomical and electrophysiological processes associated with these phenotypes. We will study a cohort of pet owned Border Collies, recruited through liaison with veterinary surgeons, in a case-control study with half affected by epilepsy, and half healthy controls. Detailed seizure diaries and behavioural/training histories will be taken to explore their influence upon behaviour and the relationship between ASD/ADHD and drug response. Objective behavioural tests combined with owner questionnaires will be conducted to quantify ASD/ADHD-like behaviours and explore whether dogs with epilepsy show increased levels in comparison to healthy controls. Once a behavioural phenotype is established, we will investigate whether it is associated with neuroanatomical and/or neurophysiological markers. Awake and sedated EEG methods will be used to quantify interictal neurophysiological profiles, and magnetic resonance images will be collected to quantify brain volumetrics, to detect differences in brain activity and/or anatomy associated with ASD/ADHD, as are seen in humans with these disorders. Cognitive (judgement) bias tests will be used to measure the effect of ASD/ADHD on the affective state of dogs, and whether they have a negative impact on canine welfare. A validated ASD/ADHD questionnaire tool will be created and deployed as a cross-sectional prevalence survey to estimate how common ASD/ADHD are in the general canine population. This tool could ultimately be used by veterinary surgeons to screen for comorbidities. This approach has the potential to deepen our understanding of the underlying pathophysiology of epilepsy and neurodevelopmental disorders, establish the presence of new co-morbidities in dogs with epilepsy that require further attention (including treatment development) to protect canine welfare, and strengthen the dog as a spontaneously occurring model of epilepsy and its comorbidities.
Agency: GTR | Branch: MRC | Program: | Phase: Research Grant | Award Amount: 234.01K | Year: 2016
Contagious pleuropneumonia is a severe acute disease that kills many growing pigs and causes lifelong damage to the lungs of those that survive. This impacts on the profitability of the production system. Pig farms regularly use antibiotics to control this disease because there is very little that can otherwise be used. Before the advising veterinary surgeon on a pig unit can reduce the use of antibiotics, we need alternatives for them to use. Apart from improving husbandry practices such as increasing ventilation and reducing the number of animals held together, there is little that can be offered. This is a problem throughout the world and is one of the primary reasons for using prescription antibiotics in pigs throughout Europe. In the UK, there is no effective vaccine because these have failed to protect pigs from disease, or the vaccine was itself toxic. Efforts to make on-farm vaccines (emergency vaccines) have not solved the problem because they are not efficacious and antibiotics, in feed, in water and by injection, are used in an attempt to avoid catastrophic losses. The disease is caused by a bacterium, Actinobacillus pleuroneumoniae, which produces two of three different protein toxins (ApxI, II and III). Production of these toxins by the pathogen is key to the disease process. Pigs that recover from disease have antibodies which neutralize the toxins and evidence suggests this is crucial in protecting pigs from the disease. It needs to be replicated in a successful vaccine. However, simply using the toxin(s) as a vaccine does not protect pigs. Despite stimulating production of antibodies these are not toxin-neutralizing antibodies. It appears that these bacteria have evolved to synthesise toxin molecules with irrelevant but highly immunogenic regions to distract the immune response to the wrong part of the toxin so that the antibody response is ineffective allowing the bacteria to spread and the disease continue. In this project we will eliminate those parts of the toxin that are distracting the immune response and which appear to be causing failure of the toxin molecules to generate a neutralizing response when used as a vaccine. These small fragments will be joined to a carrier protein, modified diphtheria toxin. This will enhance the immune response and make the antigen large enough to be recognised by the pigs immune system as a vaccine antigen. We will immunize pigs with these modified toxins and measure the immune response and the neutralizing effect against the active toxins. To improve the method of testing the toxins and the effect of neutralizing antibody, we will develop and test a pig model of dermal oedema. This will be used to indicate the best vaccine antigens for use in the pig model of pleuropneumonia. We will then proceed to immunize pigs and test the efficacy of the vaccination by experimental challenge of the pigs with the virulent pathogen. If this hypothesis is correct, and the immune response to the toxin fragment is effective, this could be the step needed for production of an effective vaccination against pleuropneumonia and the opportunity, finally, to offer the pig industry an alternative to antibiotics which would markedly reduce the quantity of these drugs used in controlling pig respiratory disease.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 304.22K | Year: 2017
Epilepsy is a complex brain disease, in which individuals are pre-disposed to show spontaneous seizures. Idiopathic epilepsy (IE) is classified as epilepsy of predominantly genetic or presumed genetic origin and in which there are no gross abnormalities of the structure of the brain nor other relevant underlying diseases causing seizure activity. IE is the most common chronic neurological condition in domestic dogs, estimated to affect 0.6% of dogs, but markedly higher in some breeds e.g. 17-33% in the Belgian Shepherd. Quality-of-life is also limited by side-effects of the currently used anti-epileptic drugs (AEDs), and quantity-of-life is potentially reduced due to an increased risk of premature death secondary to epilepsy. Further challenges faced by veterinarians treating dogs with epilepsy, and owners of affected dogs ARE (i) drug-resistance: a lack of response to currently available AEDs affecting up to 60-86% of treated dogs, and (ii) neurobehavioural changes comorbid with IE, which are poorly understood in dogs but highly prevalent in people with epilepsy. Our own studies have previously found that as few as 14% of dogs become seizure free on treatment, and increases in fear/anxiety and defensive aggression are seen following the onset of IE. These common features make the dog an ideal translational model for spontaneously occurring drug resistant epilepsy. It is clear that there is a need to gain a deeper understanding of IE in the dog to identify risk factors for (i) the development of IE, (ii) the lack of response to available AED therapies, (iii) the development of behavioural changes, and (iv) the interplay between these factors, so that future efforts to treat or prevent IE are targeted and effective. Genetic markers of both epilepsy and AED response have had limited success thus far, may be hard to interpret and account for only a limited proportion of susceptibility. In this study we instead investigate biochemical by-products of metabolic pathways (the metabolome) and the microorganisms living in association with the body (the microbiome) which reflects the interaction between an organisms genome and its environment and are a better potential indicator of observed characteristics, which can be potentially modified as treatment strategy. Although metabolomic markers of IE have not yet been found, profiles of anxiety have been identified in humans and mice. The microbiome has not yet been studied in IE development, but is involved in the metabolism of AEDs, and changes have been associated with anxiety levels through brain-gut interactions. This research programme will characterise types of disease presentation, the variation in behavioural characteristics, variation in metabolites in the metabolome, and differences in micro-organism populations in the microbiome in dogs with and without IE to identify novel biological markers of both the disease, drug response and behavioural signs (and associations between these factors) that could provide new perspectives on the underlying disease biology and provide new treatment targets. We will study these novel measures in two stages: firstly, a case-control study of breed and age-matched dogs with and without IE recruited from our hospital populations to directly compare profiles; secondly, a prospective cohort study of 5000 puppies from the South of England to identify physical and behavioural profiles measured before seizure onset that act as risk factors for IE development. Urine and faecal samples will be collected for metabolomic and microbiomic analysis. Behavioural testing will characterise aspects of the dogs underlying affective state, to reveal whether IE and drug-resistance are associated with an underlying characteristic that predisposes individuals to perform anxiety-related behaviours. This novel and comprehensive approach is needed to unravel the mechanisms underlying IE, the occurrence of drug-resistance and behavioural abnormalities.
Agency: GTR | Branch: BBSRC | Program: | Phase: Research Grant | Award Amount: 362.06K | Year: 2016
This proposal aims to validate two innovative biomarkers of brain resilience to ageing. The biomarkers use two well-known and widely available imaging technologies, magnetic resonance imaging (MRI) and positron emission tomography (PET) that are presently used both in animals and humans. The quest for biomarkers of ageing is a difficult one as their necessary requirement is prediction power - ideally, when studying an intervention aimed at modulating the ageing process, we would like to know its effect on the ageing trajectory as soon as possible without having to wait for the ageing process to eventually happen. In the case of brain, this can be achieved if we can measure its resilience - and the one relevant biological parameter determining the ability to withhold cellular pathology is its metabolic reserve. Why metabolism? In the average adult human, the brain represents about 2% of the total body weight yet it accounts for 20% of all the energy consumed, 10 times that predicted by its weight alone. Note that this very high rate of energy consumption is required only by its resting state while the additional energy associated with any mental activity is remarkably small, often less than 5% of the baseline. In this highly energetic organ, metabolic reserve is the amount of energy reserve that brain cells can still use to fight all those pathological phenomena that we call ageing. Big metabolic reserve will then mean great resilience to ageing, poor or no reserve means little or no resilience. When measuring energy reserve we wish to target the functioning of small cellular organelles called mitochondria. There are two key observations about mitochondria. 1)They are the fundamental elements of the cellular machine performing aerobic respiration e.g. using oxygen to oxidise the products of glucose and convert them into energy. 2) The process of mitochondria maintenance is highly dynamic with a constant turnover of these components particularly the older and damaged ones. We have focused on these two key aspects of mitochondrial function and devised two measures. 1) MEASURE 1 - We wish to measure mitochondrial respiratory reserve capacity by measuring oxygen cellular metabolism with MRI before and after a challenge with Methylene Blue (MB). MB is a compound, already authorized for human use, that induces substantial increases (>40%) in cell respiration. Using MB we can expand mitochondrial capacity to its limit, far more than using any mental task that requires very little incremental energy and is far less controllable in an experimental or clinical setting. 2) MEASURE 2 - We wish to measure the level of expression of one particular mitochondrial protein, called the 18Kd translocator protein or TSPO. TSPO regulates mitochondrial turnover and acts as a quality controller driving the process that removes defective mitochondria following damage or stress. Importantly TSPO levels can be measured in-vivo by PET in both animals and humans. We will validate these measures by following through time two groups of rats. One group of rats will be submitted to an intervention which is known to benefit the brain ageing process and mitochondria function in particular. The treatment will consist in environmental enrichment and dietary restriction by which we will mimic human healthy lifestyle. During their lifetime, we will continuously monitor their cognitive ability using appropriate tests and we will verify whether our measures, taken early on, predict their mental decline. Once validated, these measures will be extremely powerful tools for ageing research. They will shorten timings in experimental settings and will allow an optimal use of animals as they do not require animal sacrifice. They will be immediately translated to humans as both imaging technologies and the pharmacological challenge used have been separately validated and authorized for human use.