BJA/RCoA Non-clinical PhD Studentship

Background
Sepsis (severe infection) is the main cause of death on intensive care units. Mitochondria are tiny structures inside cells where energy is made and if they are damaged they are unable to make enough energy. Such damage to mitochondria can alter the way the cell works, and contributes to the inflammatory pathways which are activated in cells during sepsis. Melatonin, a substance which is produced naturally in the body but can also be chemically manufactured, can protect these mitochondria in the laboratory under conditions which mimic sepsis and is being used in a trial in patients with sepsis. However, although it appears to be non-toxic, the way in which melatonin treatment may affect inflammatory pathways in cells needs clarifying.

Aim
The proposed PhD studies will determine the effects of melatonin and its major metabolites on several key process in cells which contribute to the inflammatory pathways activated in sepsis.

Study design
The student will use three different types of cell lines (these are cells which originate from human donors and can be grown indefinitely in the laboratory) and also cells isolated from the blood of patients with sepsis, plus healthy control subjects of a similar age.

Cells will be treated with a variety of substances to create conditions similar to those seen in sepsis along with a range of different doses of melatonin or its metabolites. The effects on the proteins involved in several inflammatory pathways in cells will be determined. The role of these pathways to explain the effects of melatonin and its metabolites will also be further clarified by seeing what happens when the pathways are inhibited or augmented.

The student will gain expertise in a range of laboratory techniques and technologies, will learn how to culture several types of cells 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 in patients with sepsis.

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. Despite the large number of ongoing studies the effects of treatment of cells with melatonin or relevant metabolites are not fully clear and the effects under conditions of sepsis are very patchy. This project will identify the pathways which are affected by melatonin and key metabolites, determine whether these effects are similar in different cell types and whether different activating substances are important. Such work may also allow identification of targets for the development of more specific drugs in the future.

Systemic inflammatory response syndrome

The 'systemic inflammatory response syndrome' (SIRS) is a clinical term used to describe patients that develop a form of whole body inflammation due to various causes, including severe infection (sepsis), major trauma and severe burn injuries. In SIRS, there is an intense spread of inflammation from the infected or injured part of the body to other healthy tissues/organs via the bloodstream. If such inflammation becomes severe, it can substantively impair essential functions of various organs across the body, leading to life-threatening conditions and occasionally death despite active treatment in the intensive care unit. Up until now, attempts to prevent SIRS and multi-organ failure by reducing the systemic inflammatory response have been unsuccessful and in some cases have exacerbated the disease. These disappointing results have led to major reassessments of previous treatment strategies with the proposal of alternative approaches.

Microvesicles

Microvesicles are extremely small membrane particles released from cells in response to stress or infection, carrying numerous molecules related to inflammation. Because they keep such molecules within their membranous sacks, they are able to transfer inflammatory signals to other cells over a very long distances. We and others have shown that microvesicle levels increase in the bloodstream of patients with SIRS, suggesting that they may be involved in the spread of inflammation. Microvesicles are already known to produce inflammation in isolated cells in the laboratory, but little is understood about the interaction of microvesicles with cells within the bloodstream, specifically those attached to, or lining the walls of blood vessels in organs. We have investigated microvesicle behaviour in the blood circulation of mice during systemic inflammation, and found that instead of their normal removal by cells within the liver, they bind to and are taken up by an immune cell known as a 'monocyte' within the lung vessels. These monocytes within organ vessels are important in commencing organ inflammation, as they, when activated, release inflammatory mediators that have potent effects on other cells within that organ. Microvesicles may be attractive therapeutic targets for limiting the propagation of inflammation in SIRS, especially as selective blocking of their remote effects in healthy organs may avoid impairment of beneficial immune and healing responses within other local tissues/organs.

Aims and objectives

Our primary aim is to elucidate the interactions of microvesicles with their target cells as it occurs during SIRS. Using cells from healthy volunteers we will determine the following: (1) which cells within the bloodstream interact with microvesicles, (2) how these cells bind to and process microvesicles, and (3) the inflammatory effects of microvesicles in their target cells. These results produced with laboratory generated microvesicles, will then be compared with microvesicles isolated from SIRS patients' blood to verify our main findings and evaluate any relationship between patient illness and microvesicle inflammatory activity. The identification of clinically relevant microvesicle effects and the molecular mechanisms responsible can hopefully be used to inform future design of therapies for prevention of SIRS related illness.