BJA/RCoA Project Grants

Background
Sepsis is defined as a massive inflammatory response caused by infection and is the main cause of death on intensive care units in the UK. The inflammatory process in patients with sepsis is essentially out of control and can eventually result in organ damage and, in about half of patients, death. Inflammation occurs when parts of the cell walls of infecting bacteria activate processes within cells. These processes are designed to clear the infection, but when they get out of control, they can damage cells and organs. One of the main processes involves a protein called nuclear factor kappa B - or NFĸB - which acts to 'switch on' other processes. The body usually has an 'off switch' for NFĸB. We have found a way of pushing the 'off switch' using an antibody, which should enable us to control the activation of NFĸB and so also control inflammation.

Aim
To determine if switching off NFĸB in cells cultured under conditions mimicking sepsis, can reduce inflammatory responses. This ultimately may lead to the development of a new therapy for sepsis.

Study design
In this study we will culture different type of cells in the presence of bacterial cell wall proteins to mimic what happens in sepsis. We already know this results in NFĸB being switched on, causing the same inflammatory responses seen in patients with sepsis. We will then use our 'off switch' antibody and determine if this will reduce NFĸB activation and decrease the inflammation. We will measure the inflammatory responses in several ways including analysing levels of key proteins released by cells, analysing their ability to perform certain cellular functions, and their ability to activate other types of cells. This study will enable us to determine the potential of our antibody to control inflammation under conditions of sepsis and evaluate its potential as a therapeutic strategy in the future. In addition, the 'off switch' antibody may also be important in controlling disease processes involving chronic inflammation, such as types of inflammatory arthritis and therefore the results of this study may have a broader impact beyond sepsis.

There were 14 million new cases of cancer worldwide in 2012 and this is expected to increase to 22 million by 2030. Whilst there may be other options, surgery remains the best chance of long term survival for many cancers. Paradoxically, there is evidence that surgery itself may be associated with the spread of cancer. Surgery can disrupt the tumour and/or the blood vessels supplying it, leading to the spread of tumour cells into the bloodstream. Stress during surgery leads to depression of patients' immune system which may otherwise prevent the implantation of circulating cancer cells. This combination of potential cancer seeding and a poor immune response makes patients undergoing cancer surgery susceptible to the spread of their cancer. This has led to increasing interest in the period around surgery and its impact on cancer progression.

In the UK, doctors use inhaled drugs for anaesthesia for 90% of operations and the intravenous drug propofol for about 10%. Test tube studies, including in our laboratory, have suggested that inhaled anaesthetic agents may promote cancer cell survival. There is also data suggesting that propofol has the opposite effect.

We recently studied over 7,000 patients who had undergone cancer surgery at the Royal Marsden hospital from 2010 to 2013. We found after matching patients for risk factors, that patients receiving inhaled anaesthetics had a chance of dying almost 1.5 times higher than those who received propofol anaesthesia. Although we tried to compare similar groups, this data may be biased as patients were not randomly assigned to one anaesthetic or the other. We do not feel that a clinical trial comparing one anaesthetic to the other should be done yet as we don't understand the biological reasons why one might be better than the other. All cancers behave differently and thus understanding why one type of anaesthetic agent may differ from another in affecting tumour growth and/or progression or even the body's response to cancer surgery, would allow us to design a large trial recruiting those patients who may benefit to answer the question definitively.

This proposal aims to build on our preliminary data and perform some experiments in (the minimum possible number of) laboratory mice that will allow us to study our early observations in models that mimic tumour growth in cancer patients. Alongside this, we will perform a small randomised trial of patients undergoing cancer surgery. In the patient trial, we aim to take blood samples before and after surgery and a sample from the tumour just as surgery is completed. All of these investigations complement each other and will allow us to try and answer this question robustly and translate findings from the laboratory towards patient benefit.

There are millions of people undergoing cancer surgery worldwide every year. Intravenous propofol is slightly more expensive than inhaled anaesthetic drugs but this reflects only a small fraction of the cost of most cancer drugs. If we found a difference, this could easily and rapidly change practice across the NHS and beyond.

Inflammation of peripheral tissues is one of the most frequent pathological conditions associated with ongoing (prolonged) pain. Currently available drugs very often fail to reduce prolonged pain during tissue inflammation. However, prolonged pain induces unnecessary suffering, may interfere with physical and emotional recovery, ruins the quality of life and generates financial loss for the patient, the health system, the economy and ultimately the society. Therefore, the development of effective drugs to reduce prolonged pain is of paramount importance, which necessitates better understanding of cellular and molecular mechanisms involved in the development of prolonged pain. Recently we have made a major discovery in this area; we have found that the enzymes mitogen- and stress-activated kinases 1 and 2 (MSK1/2) are necessary for the development of burning pain, one of the main types of pain we experience during tissue inflammation. While evidence indicates that MSK1/2 promotes the development of burning pain through altering the expression of a set of genes, the molecular mechanisms responsible for those alterations as well as the identity of the genes whose expression is altered through MSK1/2 are not known. Though MSK1/2 may induce changes in gene expression in various neurons involved in the development of prolonged pain, our preliminary findings show that inflammation of peripheral tissues initiates MSK1/2-dependent signalling in pain-sensing (nociceptive) primary sensory neurons that carry information about painful events in tissues to the brain and are pivotal for the development and maintenance of prolonged pain. Hence, our aim in the present study is to elucidate how MSK1/2 activation leads to transcriptional changes and to establish the identity of the affected genes in nociceptive primary sensory neurons.

We will use an animal model of inflammatory prolonged pain in wild type mice and mice lacking MSK1/2. Comparison of changes in the two types of animals will reveal processes by MSK1/2. We will assess the activation of molecules implicated in mediating MSK1/2's effect on gene transcription by identifying those molecules using standard techniques. We will then use state-of-the-art methodology (nucleic acid sequencing and bioinformatics) to establish the identity of genes affected by MSK1/2. Findings of this study will significantly improve our understanding how MSK1/2, in nociceptive primary sensory neurons, contributes to the development of burning pain in inflammation. In addition, this study will also generate data which could be used in further grant application.

Patients who are seriously ill in Intensive Care Units often receive a lot of fluid (water and salts) as part of their life-saving treatment. The lining of blood vessels is damaged when patients are seriously ill and therefore fluid leaks from blood vessels into surrounding soft tissues. This is often visible as swelling in the hands and feet, but also occurs in the lungs and other vital organs, where it may reduce the function of these organs. Our theory is that a conservative approach to fluid involving giving less fluid and using drugs or kidney dialysis to remove fluid (deresuscitation) helps organs to work better and thus patients to have better outcomes. However, there are possible risks to this, including a reduction in oxygen supply to organs, particularly the brain. After a critical illness, one of the biggest problems faced by many patients is difficulty with memory and thought-processing abilities: one small study suggested that this was worse with conservative fluid and deresuscitation, although another study found the opposite.

We plan to carry out a pilot trial (RADAR-2) comparing patients who are randomly put into one of two groups: one group will receive a conservative/deresuscitative fluid strategy is feasible and safe, and can understand the ways this therapy might work, we will undertake a much larger study to find out if this approach might improve patient outcomes.

We are requesting funding to study, as part of this trial, some of the ways by which the two different approaches to fluid could affect organ function. Our hypotheses are:

• That conservative/deresuscitative fluid strategy will result in improved heart function and recovery of damage to the lining of blood vessels compared to the usual care group. To test this, we will use echocardiography (ultrasound scanning of the heart) and measurement of substances in the blood which indicate damage to the lining of blood vessels.
  
• That oxygen supply to the brain will be similar between the two groups of patients, and that patients in the two groups will be similarly affected with memory and mental processing problems. We will measure brain oxygen supply for 72 hours through a soft adhesive strip attached to the patients'' forehead and will contact patients by telephone 3 and 12 months after they enter the trial to test their memory and thought-processing ability. We will compare brain oxygen levels, memory and thought-processing ability between the two groups.

This grant will give us a better understanding of the effects of different fluid strategies on the function of the heart, brain and blood vessels, and will help us to design future studies in a way most likely to be beneficial.

Mechanical ventilation is a vital tool in modern medicine. It enables otherwise impossible surgical procedures to take place, and keeps patients with fatal lung disorders such as acute respiratory distress syndrome (ARDS) alive. In the last 20 years however it has become clear that mechanical ventilation can be very damaging. Specifically, if the amount of gas delivered with each breath (the tidal volume) is too high, areas within the lung become over-stretched leading to inflammation and further damage, a phenomenon known as ventilator-induced lung injury (VILI). This extra injury caused by ventilation is a major factor determining whether or not patients survive their intensive care stay. Unfortunately, especially in patients with underlying disorders such as ARDS, some development of VILI is inevitable whenever patients are ventilated. It is therefore vitally important to develop therapeutic strategies to reduce the impact of VILI.

In recent years many studies have explored the biological, inflammatory basis of VILI. However, no treatment strategies have translated into patient improvements apart from simply ventilating more gently, suggesting that there are still important gaps in our knowledge. To try to fill these gaps, we considered an alternative approach. Specifically, based on clinical observations we hypothesised that obese/overweight patients may be protected from developing VILI. We further hypothesised that if we understood why this was the case, it could point us towards new therapies.

To investigate this we induced obesity in mice and discovered that fat-fed animals were indeed protected from VILI. While exploring the reasons behind this, we found that a particular protein called CD147, which is normally increased in the lungs during VILI was not increased in protected fat-fed mice. CD147 is the cell surface receptor for a protein called cyclophilin A (CypA), which is secreted from activated cells. CypA binding to CD147 can lead to many cellular consequences, and the CypA:CD147 axis has become a target of interest in a number of diseases including cardiovascular disease, rheumatoid arthritis and cancer. Very little is known about CypA:CD147 in the lungs apart from observations that the axis seems to be involved in lung inflammation, through mechanisms we do not understand.

Based on our previous findings and preliminary studies showing that VILI induces a dramatic increase in CypA secretion in the lungs, we believe it is likely that the CypA:CD147 axis plays important, as yet unexplored roles during VILI. We will initially investigate the basic biology of CypA:CD147 in lung cells using in vitro cell culture models. These will begin with studies looking at which cells secrete CypA in response to stretch and other stimuli, and what happens when CypA is added to cells. We will then extend this to explore the nature of CypA:CD147 signalling using animal models, followed eventually by experiments to investigate whether blocking the CypA:CD147 axis is indeed able to prevent VILI. These studies will greatly increase our understanding of the CypA:CD147 axis during lung inflammation and injury, and may potentially point towards the axis as a novel therapeutic target for ventilated patients.