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Background Hepatitis B virus (HBV) is a major health problem with almost 300 million people affected worldwide. Long-term infection may be established in the liver, especially if exposure occurs early in life, and this increases the risk of scar tissue formation, cancer and liver failure. Current treatments (such as tenofovir, entecavir) are well tolerated, available in tablet form and highly effective at suppressing viral replication; however, traces of the virus persist and life-threatening inflammation may occur if treatment is stopped prematurely. As new treatments become available over the next few years, it is increasingly important to develop blood tests that closely reflect the burden of virus in the liver and activity of the immune population. Ultimately, this will allow individuals to be matched with the most appropriate treatments and thus minimise side effects. Aim(s) of the research Our research project will explore the distribution of hepatitis B virus in the liver and identify markers in the bloodstream that reflect the burden of infection (as well as the immune response). Design and methods used The research will be conducted across three sites, namely King's College Hospital (London), Francis Crick Institute (London) and the Nuffield Department of Medicine (Oxford). In the first part of the project, individuals with chronic hepatitis B infection who have tissue samples stored in our research biobank, will be identified. Using techniques called "in-situ hybridisation" and "multiplex antibody staining", we will map the distribution of viral activity (genetic material, proteins) within the liver and the layout of local immune cells. In parallel, we will measure a panel of molecules in the bloodstream and explore how closely they reflect viral activity within the liver. In the second part of the study, a cohort of patients (n=100) will be approached at King's College Hospital and extra blood tests will be taken at the time of clinical review. After isolating immune cells from the bloodstream, a series of experiments will be conducted to assess their function and ability to replicate; the extent to which these cells are "exhausted" and unable to carry out their normal protective functions will be correlated with molecules in the serum (including Tim3 and sPD-1). Potential applications and benefits The research project has the potential to fundamentally change our understanding of HBV replication in the liver. From a clinical perspective, blood tests that closely reflect viral activity will help clinicians to decide which patients should be started on treatment, the duration of therapy, and suitability for withdrawal. In addition, they may help to identify those at greatest risk of developing liver cancer, in whom a more stringent surveillance programme should be adopted. Communication plan We envisage that our results will have a significant impact on the way patients with chronic hepatitis B infection are managed in the future, and we want to maximise visibility amongst other experts in the field. To achieve this, the data will be presented at international conferences and published in medical journals. In addition, we recognise the importance of updating patients on the progress of the research, and this will be achieved through educational programmes that we run regularly at King's College Hospital, as well as newsletters, websites and local networks.

Hepatocyte (Liver cell) transplantation is an alternative to liver transplantation for certain conditions. It involves the transplantation of the cells into the patient's liver, usually via the main vein supplying the liver, in a minimally invasive way. Patients for whom this treatment is especially suitable are those who are missing a particular enzyme which is made in the liver cell. This leads to buildup of toxic waste products and consequent irreversible brain injury. Two such conditions: Crigler-Najjar syndrome (where bilirubin cannot be broken down), and the urea cycle defects (where ammonia is not eliminated), cause profound brain injury due to accumulation of these toxic compounds. Replacing the whole liver, to replace the missing enzyme, is an option but comes at a high price as this involves major surgery, often with complications, a prolonged hospital stay, and leaves no fallback if the transplant fails. Furthermore, the donor organ pool is particularly small for children, who will need part of an adult liver. Thus the wait for a suitable organ may be years for children who have an extremely poor quality of life and with the potential to develop a metabolic 'crisis' at any point in time. In addition, they only actually need a fraction of liver in order to correct the deficiency. There is thus a redundancy in replacing a whole liver, particularly in view of the restricted resource and large demand. Hepatocytes are isolated from livers that don't meet the criteria for organ transplantation, and so they are an excellent use of an otherwise unusable resource. They can be frozen and stored and thus are available off the shelf. Hepatocyte transplantation also allows the patients own liver to be preserved while providing 10-15% of the liver function, i.e. sufficient function from the otherwise deficient enzyme to correct the disease. We know that, at time of infusion into the portal vein, the cells encounter attack from the innate immune system: blood begins to clot, proteins are released, attracting the defence cells of the body, and hepatocytes are destroyed before they can reach the liver to engraft and settle. Conventional immuno-suppression including steroids doesn't allow to overcome this and so, more than ninety percent of cells are destroyed before they have the opportunity to engraft. Alpha-1 antitrypsin (AAT) is a protein made in liver cells and has a range of effects which can block a lot of the adverse effects of inflammation without the side effects of most conventional immunosuppressants, which leave the body vulnerable to attack from infection and cause poor wound healing. AAT is used in clinical trials world-wide, in a variety of conditions which are influenced by inflammation such as type 1 diabetes and ischaemic heart disease. Considerable success of AAT as an immune system modifier has also been seen in the field of islet transplantation in patients with diabetes, where these insulin producing cells are also transplanted into the liver. We wish to test the use of AAT in the field of hepatocyte transplantation as we believe that it will lead to improved engraftment of cells in the liver and thus, better medium- to long-term function of the cells, making a significant difference to those treated. We predict that the mechanism through which this occurs will be the suppression of the innate (immediate) immune system and we will measure both the function of the cells in terms of supplying the missing enzyme as well as the effect that AAT has on the immediate immune response once these cells are infused. Though hepatocyte transplantation is a small field, it can result in life-changing treatment for children with devastating liver-based disease. Should we demonstrate the success of this therapy using AAT, this will give further credence to the use of AAT as an effective and safe immuno-modulator, in the context of transplantation in general, and in other inflammatory conditions.

A key contributor to outcome following a stroke or a traumatic brain insult is the development of secondary brain injury. This is a process of more damage to brain tissue that occurs over a number of hours and days after the original event. Currently, there is little we can do to restore normal working in brain regions that have been damaged beyond repair by the initial trauma or stroke. However, we might be able to prevent secondary brain injury if we were able to detect this as it progresses. After an injury, a region of vulnerable but still potentially viable brain tissue is established around the injury core. This is the region that is susceptible to secondary brain injury and is called the 'penumbra'. We now know that electrical brain waves, called "spreading depolarisations" (SD), frequently occur in the penumbra after an injury. We believe a special class of these SDs to be very harmful and to contribute significantly to the process of secondary injury. However, there is another group of SDs that bring with them an extra supply of oxygen and glucose. This can be very beneficial to the neurons in the penumbra in helping them to recover. As such, whilst we would like to prevent SDs that are harmful, we do not want to prevent those that might be favourable. The problem is, we currently have no way of telling the difference between them. One way we might be able to tell the difference though is to examine the effect they have on the health of the neurons. Over the last few years I have worked to develop a method to probe neuronal health and to monitor changes in the viability of the neurons in real-time. Now I would like to use this method to characterise the changes that indicate declining neuronal viability. This would enable us to see secondary brain injury happening live at the patient's bedside, giving us a much better opportunity to intervene before it is too late. I would also like to use this method to identify which SDs are causing harm to the neurons and which ones are potentially helping them recover. This would mean that we could personalise treatments for patients experiencing harmful SDs. Furthermore, because even just one harmful SD will likely damage or kill neurons in the penumbra we would ideally like to prevent them from happening at all. This would mean though, that we would have to identify the changes that make neurons vulnerable only to harmful SDs. Luckily, we know from pre-clinical experiments that there are some very specific changes in the tissue environment that predispose neurons to harmful SD. Importantly, these changes are not seen before those that might be beneficial. As a part of the work I have done in developing a method to probe neuronal health, I have also established a way of assessing changes in the tissue environment that predispose neurons to harmful SDs. I would like to use this method to see if I can predict which patients are likely to begin having harmful SDs. This would mean that in the future, instead of waiting for them to start we could give pre-emptive treatments to susceptible patients. Finally, in order to actively prevent secondary brain injury, we need to know both when neuronal health is in decline and also what treatments are effective in improving this. By continuously tracking neuronal health status in real-time I aim to provide physicians with an online bedside tool that will both identify declining health and provide immediate feedback on the effectiveness of therapeutic interventions. The implementation of such a tool into clinical practice has the potential to limit the amount of secondary brain damage that occurs after an injury and thereby to significantly improve the outcome of the patients.

Acute liver failure (ALF) is a medical emergency that carries mortality of 80-90% in patients who fulfill liver transplant criteria. Liver transplantation depends on the timely availability of a suitable donor organ and requires life-long immunosuppression with life-threatening complications. Alternatively, the liver has a tremendous regenerative potential: if the failing liver could be supported until regeneration occurs, organ replacement and its associated complications can be avoided. Currently there is no proven liver support device available that can bridge the patient to native liver recovery or to transplant. It has been demonstrated that partial replacement of a patient's liver with a healthy donor liver (called an auxiliary transplantation) can allow clinical stability during native liver regeneration, eventually making the transplanted liver redundant, in up to 70% of the patients. For this, only a small mass of liver tissue is necessary to support the patient. Auxiliary transplant still requires availability of a suitably sized organ, major surgery, and immunosuppression, however. Transplantation of hepatocytes (cells rather than an organ) has been shown to improve synthetic and detoxification function in small animal models with subsequent human application in patients with ALF. The advantages of hepatocyte transplantation in this context are considerable. For example, (i) hepatocytes may be derived from livers which are unsuitable for transplantation; (ii) isolated cells can be frozen for years and used off-the-shelf, something that is impossible with entire organs, thereby eliminating the wait for an appropriate organ; (iii) cells isolated from one liver could treat more than one patient, thereby reducing the need for donor organs; and (iv) this provides options to infants and small children for whom the wait for an appropriately sized organ may be extremely prolonged. Earlier clinical experiences with human hepatocytes in ALF have been only partly successful when cells were injected either in the liver, or the peritoneal cavity, mainly because of rejection and use of immunosuppression in extremely sick patients, which increases the risk of infections. We have developed a technique using liver cells encapsulated in a bio-compatible gel (hepatocyte microbeads - HMB001) that can be infused temporarily in the peritoneal cavity of the patient, to replace the failing liver until regeneration. Importantly, the gel protects the cells from the immune system. The patients therefore do not require immunosuppression. We have previously treated 8 children and infants with HMB001 on a named patient basis (compassionate use). The technique proved to be safe and, importantly, displayed some efficacy: though all children met eligibility for organ transplantation, 4 children recovered with the treatment while awaiting transplant thus entirely avoiding the need for liver transplantation and are still well, up to 8 years after the procedure. We have refined our prototype of hepatocyte microbeads, which now involves multiple cell types and an improved gel that better supports the cell function (HMB002). The new microbeads have shown superior function and longevity in vitro as well as in vivo, in preclinical studies. The aim of this project is to run a clinical trial to test these new microbeads.