BJA/RCoA Non-Clinical PhD Studentships

Patients in hospitals are at risk of acquiring infections, and one of the commonest causes is the bacteria Staphylococcus aureus. A significant proportion of these infections involve antibiotic resistant strains, including methicillin resistant Staphylococcus aureus (MRSA). These infections can affect the lungs and sites of previous surgery, as well as several other body areas, and lead to increases in death and prolonged stay in hospital. Patients who are admitted to Intensive Care are particularly at risk of such infections, partly because their white blood cells do not function well.

We have previously shown that a major cause of impaired function in a key white blood cell, the neutrophil, is a molecule called C5a. C5a is found in high concentrations the blood of severely ill patients, and patients with this C5a-driven defect in neutrophil function are at high risk of infections from organisms such as Staphylococcus aureus.

Our group has developed several models of Staphylococcus aureus infection using blood from healthy donors, which can help us examine how the body fights this bug and how this fails when white blood cells are exposed to high concentrations of C5a. This has demonstrated that C5a impairs both the 'eating' (phagocytosis) and killing of Staphylococcus aureus by neutrophils. We have also developed a new method to extract the signalling proteins from inside neutrophils, allowing us to resolve signalling networks at a hitherto undescribed resolution. Using this approach, we have identified two new pathways which are involved in neutrophil 'eating' of heat-killed bacteria that are impaired by C5a. One of the pathways identified involves the assembly of the intra-cellular machinery required to kill bugs. The second pathway involves changes in the nucleus of the cell, and we believe the cell does this in order to change shape as it eats the bacteria. At present the consequences of this second pathway being disordered are unclear and present an entirely novel pathway which has not previously been described in this setting.

The aim of this project is to extend these findings into the eating and killing of live bacteria and to use our high-resolution signalling mapping to give us insight into the changes over time in the pathways we have identified. We will also use the same technique to map what happens when C5a impairs these processes. We will then go on to manipulate these pathways in cells, using a combination of drugs and prevention of protein expression by gene silencing. Use of high resolution live cell microscopy will allow us to observe cells eat bacteria in real time, and see how our manipulations of the pathways alter this. We anticipate that this project will yield new insights into how the neutrophil fights infection with Staphylococcus aureus, and how this is impaired in severely ill patients. This will allow us to identify new targets for therapies which can boost the patient's own defences against this bacterium and provide non-antibiotic treatments which can help limit the spread of antibiotic resistance.

Background
Melatonin is well known for its effects of the sleep-wake cycle and for jet lag. However it also has a multitude of other beneficial effects and is being used in clinical trials for a variety of conditions. There is some evidence that melatonin may also act as pain killer, but the way in which it does this is not clear. The body makes its own pain killers- called endorphins- and we think that melatonin may increase production of these. Our studies in the laboratory have shown that melatonin can indeed increase endorphin release in some types of cells. Both brain and immune cells not only make endorphins, contributing to pain relief, but can also make their own melatonin. We don't know if there is interaction between regulation of melatonin and endorphin release and we don't know if adding extra melatonin can increase endorphin release, or whether adding extra endorphin affects melatonin release. We also don't know if giving melatonin to patients with chronic pain affects their endorphin profile.

Aim
The proposed PhD studies will clarify how melatonin and endorphin release interact and determine the effects of melatonin on the body's own analgesia as a possible mechanism for its pain killing properties.

Study design
The student will use immune (macrophage) cells and brain (pituitary) cells and will treat them with a variety of substances which lead to release of endorphins and melatonin along with different doses of extra melatonin and a range of related substances and inhibitors. The effects on the release of several different types of endorphins will be determined. The way in which melatonin does this will also be further clarified by seeing what happens by using inhibitors, extra endorphins or more potent forms of melatonin. We also have ongoing trials where patients are receiving melatonin treatment; this will enable the student to investigate the endorphin profiles in people being given melatonin treatment. The student will gain expertise in a range of laboratory techniques, will learn about cell isolation and culture and will also gain experience in recruitment of patients and conduct of a clinical study. He or she will join an established group which is actively working on melatonin as a therapy and the role of macrophages in immune function.

Impact
The potential benefit of melatonin to human health is enormous, with an ever expanding
accumulation of evidence suggesting its benefit in a huge number of disease conditions. Pain is a common denominator across all medical specialities and identification of possible mechanisms for the effect of melatonin on pain will be invaluable in identifying new therapeutic approaches.

More than 1.6 million individuals aged >70 years undergo surgery each year in the UK, yet many struggle to return to independent function suffering increased complications after surgery, including wound infections, reduced kidney function, difficulty with their mobility. These common complications, which occur in more than 25% of those >70 years old, lead to distressing and prolonged stays in hospital. For older individuals who leave hospital, complications after surgery are associated with much shorter life expectancy. The reasons for why postoperative complications in older people lead to delayed recovery and/or early death remain unclear.

Inflammation is a key factor in causing long-term health problems. When older individuals suffer from complications after surgery, the immune system becomes dysfunctional. This leads to higher levels of inflammation and long-term health problems, including frailty. Alterations in white blood cell count because of increased inflammation are a well-recognised feature of ageing; however, it is often thought to be irrelevant. We don't think this is the case, we believe that a low WBC count is a major cause of complication following surgery. Indeed, we think that higher levels of inflammation that occur during ageing cause the loss of white blood count. We know that surgery causes the energy production of a particularly type of white blood cell called a T-lymphocyte to be reduced to levels that render the cells dysfunctional and prone to dying. We also know that T-lymphocytes from older people display the same reduced energy level, what we would like to find out is whether older people with lower white blood cell counts display these energy changes delaying, full recovery and independent function.

In this study, we will measure the amount of inflammation in patients with low white cell counts before and after surgery. We will also look at changes in the energy usage in T lymphocytes in people with low white blood counts and see if T-lymphocyte insufficiency contributes to incomplete recovery after surgery. If we can show that T-lymphocytes are damaged in patients with low white cell counts, there are a number of potential treatments that may reverse- or even prevent- these changes happening after surgery. If we can help restore the energy function of the immune system we hope to improve the chances recovering fully after major surgery.

Problem
Sepsis is a dominant global illness in the 21st Century that indiscriminately affects all age groups, from new-borns to centenarians. One in three adult patients admitted to intensive care units in England with sepsis die in hospital. For the patients that survive, their immune system is compromised significantly and they have an increased risk of infection, reduced quality of life and increased longer-term risk of death.

Knowledge gap
Despite nearly 250 clinical trials of various medications, there is still no cure to sepsis. Treatment is limited to antibiotics, fluids and supportive care. However, altering how the immune system responds to the infection is considered to be key to restoring health in sepsis patients. Our knowledge gap in there is limited understanding of how immune system alterations change over time and whether there are common patterns that could be identified. We have shown previously that sepsis leads to a rapid loss of specific types of blood cells called lymphocytes. This happens as early as the day of intensive care unit admission. However, neither this loss nor their recovery is uniform or predictable. What determines these differences and how to promote immune system recovery remain unanswered questions.

Importance
Thus, it is vital that research is undertaken to decipher how sepsis affects the immune system, specifically to identify major effects that could be modified to improve lives of sepsis patients admitted to hospital and sepsis survivors.

Project goals
In this context, we propose to study the immune system in sepsis patients from admission through to discharge with repeated measurements, which to date has not been done.

Novelty of our project
In this project, we plan to overcome many of the limitations in previous studies including studying few immune cell markers without the optimal control groups and often without confirming the reliability of the cellular markers.

We address these issues by
- Using state of the art technology with high reliability to characterise the immune cell populations using markers recommended by an international group of immunology experts.
- Exploring how these changes on immune cells are related to protein molecules in blood to generate a comprehensive picture of immune function patterns.

- Measuring at four clinically important timepoints from admission to discharge to analyse how the immune system recovers in some people and why it does not in others.

Overall, we believe that by studying patterns in the immune response over time in sepsis we can better inform treatments of future patients.