rupert leong 04_hires

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Associate Professor Rupert Leong is using laser light to probe the intestinal lining, a pioneering technique which can provide both structural and functional details of gastrointestinal diseases To begin, could you provide a broad outline of your recent project and your reasons for venturing into this area of research? Confocal endomicroscopy allows endoscopists to view the cell structure of the lining of the bowel. The main function of the bowel is to protect the rest of the body from the bacteria inside the bowel ‘lumen’ – the cavity where the faeces reside. Using confocal endomicroscopy, we are now validating the characteristics of the bowel wall barrier. This is really exciting because this has never been seen before. With confocal endomicroscopy technology, we can see things that conventional microscopy misses because we are viewing living tissue. For instance, the functional effect of the bowel lining is not seen when taking forceps biopsies, because removing the cells from the bowel wall kills the cells and they are no longer functional. In vivo in situ visualisation of the intestinal lining cells gives us not only the information of their structure and architecture, but their adequacy of barrier function as well. Further to this, how are you integrating your dual research directions of confocal endomicroscopy and proteomics? In combination, proteomics and confocal endomicroscopy allows us to evaluate both the structure and the barrier function of the bowel, and this is a new way of assessing the disease process. Confocal endomicroscopy can visualise and measure the severity of the breakdown in the barrier of the bowel lining. Plasma proteomics then measures the bacteria products that escape into the bloodstream to quantitate the aftermath of the breakdown. In addition to the diagnosis and assessment of the severity of bowel inflammation, I also hope to be able to individualise treatment for patients. Some patients need minimal medication to reconstitute the barrier function, but others require the ‘big-guns’ of treatment called biological agents. This concept of personalised medicine works well using confocal endomicroscopy and proteomics. Have you been able to explore certain features of inflammatory bowel diseases that other procedures are unable to identify? Yes, the functional effect of the bowel lining – the barrier function. This is the barrier that stops bacteria and other organisms from entering the bloodstream. At present, the concept behind inflammatory bowel disease is that this barrier may be broken and leak bacterial products into the intestinal wall, resulting in chronic inflammation in the bowel. Taking conventional forceps biopsies kills the cells and they no longer function so you cannot evaluate the barrier function. When an intravenous contrast is introduced at the time of the confocal endomicroscopy procedure, the contrast leaks out of the bowel into the gut. This is significantly more common in inflammatory bowel diseases than in age- matched controls, ie. people who do not have inflammatory bowel diseases. What we are noticing may be the reason why patients have inflammation in their bowels – the gut lining may no longer block the entry of foreign matter into the bowel wall. This could then trigger the inflammation cascade that results in ulcers forming in the inner lining of the gut. In severe cases, the intravenous contrast floods through into the bowel very rapidly and in multiple areas. This way we can measure the severity of the leakage. What advantages have you gained by fitting the flexible endoscope with a confocal laser microscope, and where do you go next with the research? Our ongoing research is to be able to objectively measure the dysfunction of the gastrointestinal tract and to see if medications can reverse this breakdown in the barrier. The microscope allows us to visualise living tissue of the bowel and we can then evaluate the endoscopy in the standard fashion, plus obtain functional information on the bowel wall lining. We have already obtained good evidence that drugs used to treat inflammatory bowel disease can all reverse the barrier dysfunction. In particular, biological agents are the best drugs to repair the barrier breakdown of the bowel lining. The technology is constantly improving and we have moved on to looking at other diseases that include detecting Barrett’s mucosa pre-cancerous changes of the oesophagus and gastric intestinal metaplasia, which may also be pre-cancerous changes of the stomach. And lastly, what have been your proudest achievements during your work so far? My research has attracted a level two (the highest) clinical Career Development Fellowship from the National Health and Medical Research Council of Australia because the research materials are novel and the topic is high in impact. Only five of these fellowships were awarded nationally at this level for clinical research when I applied. In addition, I enjoy seeing my previous trainees and fellows doing well in their careers, and some have even followed my footsteps into gastroenterology research. Making internal biopsies redundant 64 INTERNATIONAL INNOVATION ASSOCIATE PROFESSOR RUPERT LEONG

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Page 1: Rupert leong 04_hires

Associate Professor Rupert Leong is using laser light to probe the intestinal lining, a pioneering technique which can provide both structural and functional details of gastrointestinal diseases

To begin, could you provide a broad outline of your recent project and your reasons for venturing into this area of research?

Confocal endomicroscopy allows endoscopists to view the cell structure of the lining of the bowel. The main function of the bowel is to protect the rest of the body from the bacteria inside the bowel ‘lumen’ – the cavity where the faeces reside. Using confocal endomicroscopy, we are now validating the characteristics of the bowel wall barrier. This is really exciting because this has never been seen before. With confocal endomicroscopy technology, we can see things that conventional microscopy misses because we are viewing living tissue. For instance, the functional effect of the bowel lining is not seen when taking forceps biopsies, because removing the cells from the bowel wall kills the cells and they are no longer functional. In vivo in situ visualisation of the intestinal lining cells gives us not only the information of their structure and architecture, but their adequacy of barrier function as well.

Further to this, how are you integrating your dual research directions of confocal endomicroscopy and proteomics?

In combination, proteomics and confocal endomicroscopy allows us to evaluate both the structure and the barrier function of the bowel, and this is a new way of assessing the disease process. Confocal endomicroscopy can visualise and measure the severity of the breakdown in the barrier of the bowel lining. Plasma proteomics then measures the bacteria products that escape into the bloodstream to quantitate the aftermath of the breakdown.

In addition to the diagnosis and assessment of the severity of bowel inflammation, I also hope to be able to individualise treatment

for patients. Some patients need minimal medication to reconstitute the barrier function, but others require the ‘big-guns’ of treatment called biological agents. This concept of personalised medicine works well using confocal endomicroscopy and proteomics.

Have you been able to explore certain features of inflammatory bowel diseases that other procedures are unable to identify?

Yes, the functional effect of the bowel lining – the barrier function. This is the barrier that stops bacteria and other organisms from entering the bloodstream. At present, the concept behind inflammatory bowel disease is that this barrier may be broken and leak bacterial products into the intestinal wall, resulting in chronic inflammation in the bowel. Taking conventional forceps biopsies kills the cells and they no longer function so you cannot evaluate the barrier function.

When an intravenous contrast is introduced at the time of the confocal endomicroscopy procedure, the contrast leaks out of the bowel into the gut. This is significantly more common in inflammatory bowel diseases than in age-matched controls, ie. people who do not have inflammatory bowel diseases. What we are noticing may be the reason why patients have inflammation in their bowels – the gut lining may no longer block the entry of foreign matter into the bowel wall. This could then trigger the inflammation cascade that results in ulcers forming in the inner lining of the gut. In severe cases, the intravenous contrast floods through into the bowel very rapidly and in multiple areas. This way we can measure the severity of the leakage.

What advantages have you gained by fitting the flexible endoscope with a confocal laser microscope, and where do you go next with the research?

Our ongoing research is to be able to objectively measure the dysfunction of the gastrointestinal tract and to see if medications can reverse this breakdown in the

barrier. The microscope allows us to visualise living tissue of the bowel and we can then evaluate the endoscopy in the standard fashion, plus obtain functional information on the bowel wall lining. We have already obtained good evidence that drugs used to treat inflammatory bowel disease can all reverse the barrier dysfunction. In particular, biological agents are the best drugs to repair the barrier breakdown of the bowel lining.

The technology is constantly improving and we have moved on to looking at other diseases that include detecting Barrett’s mucosa pre-cancerous changes of the oesophagus and gastric intestinal metaplasia, which may also be pre-cancerous changes of the stomach.

And lastly, what have been your proudest achievements during your work so far?

My research has attracted a level two (the highest) clinical Career Development Fellowship from the National Health and Medical Research Council of Australia because the research materials are novel and the topic is high in impact. Only five of these fellowships were awarded nationally at this level for clinical research when I applied. In addition, I enjoy seeing my previous trainees and fellows doing well in their careers, and some have even followed my footsteps into gastroenterology research.

Making internal biopsies redundant

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Illuminating inflammatory bowel disease

A new imaging technique pioneered in Australia is revolutionising our understanding of bowel diseases by bringing together laboratory and clinical research

to analyse the microscopic structure of the gut

ENDOSCOPY IS A medical technique in widespread use for the diagnosis and treatment of bowel complaints. It consists of a camera mounted on the end of a flexible tube to allow direct visualisation of the internal gastrointestinal lining (the mucosa). A new technique called confocal endomicroscopy (CEM) is a revolutionary improvement in endoscopic imaging as it incorporates a confocal laser microscope into the tip of the endoscope.

Associate Professor Rupert Leong from The University of New South Wales is pioneering this technique. His research focuses on the microscopic cellular architecture of the bowel wall which is invisible to conventional endoscopes. CEM instantly diagnoses neoplastic growth in the gastrointestinal tract through the irregular arrangement, size and shape of cells. The microscopic hair-like projections of the small bowel (called villi) can be instantly recognised using CEM and

coeliac disease – a genetically determined wheat allergy disease – can be diagnosed immediately.

Typically, analysis of the cellular architecture (histology) requires biopsies to be taken by forceps from the wall of the gastrointestinal tract. However, the forceps used in biopsies often disrupt the structure of the sample as well as removing the tissue from its living environment, resulting in artefacts that may lead to misdiagnosis. Furthermore, biopsies may result in false negative results from non-representative specimens or failure to orientate tissue samples. In addition, using forceps biopsies carries the rare risk of bleeding and bowel perforation. The advantage of CEM is that it allows for the analysis of the gastrointestinal mucosa in its natural living environment.

PIONEERING RESEARCH

In Australia, Leong has been the top contributing research gastroenterologist for two consecutive years at their annual national society conference and has achieved the highest level clinical Career Development Fellowship from the Australian National Health and Medical Research Council – only five such fellowships were awarded nationally in the year of Leong’s application. This reflects both the novelty and impact of his research.

As an Associate Professor at The University of New South Wales, and as Head of the Inflammatory Bowel Diseases Services at Concord and Bankstown Hospitals, Sydney, Australia, Leong is ideally positioned to bring together laboratory and clinical research. Previously, he has shown CEM to be as least as accurate as forceps biopsies in the diagnosis

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ASSOCIATE PROFESSOR RUPERT LEONG

Image of confocal endomicroscopy of the bowel show-ing leakage of intravenous contrast into the lumen.

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INTELLIGENCEPROTEOMICS AND CONFOCAL ENDOMICROSCOPY IN THE EVALUATION OF INFLAMMATORY BOWEL DISEASES

OBJECTIVES

To measure the integrity of the intestinal mucosal barrier. Confocal laser endomicroscopy visualises leakage across the intestinal lining. Evaluation of intestinal micro-organisms contaminating the bloodsteam is performed with a sensitive tool called proteomics. Together, they support the theory that the bowel wall barrier is broken in inflammatory bowel diseases.

KEY PARTNERS

Bankstown-Lidcombe Hospital

Concord Repatriation General Hospital

NSW Health

University of New South Wales

FUNDING

National Health and Medical Research Council of Australia

CONTACT

Associate Professor Rupert Leong MBBS, MD, FRACP, AGAF Director of Endoscopy, Concord and Bankstown Hospitals Conjoint Associate Professor of Medicine

Bankstown Hospital Eldridge Road Bankstown, NSW 2200 Australia

T +61 2 9767 6111 F +61 2 9767 6767 E [email protected]

RUPERT LEONG is a consultant Senior Staff Specialist gastroenterologist at Bankstown, Concord and Macquarie University Hospitals, and Associate Professor of Medicine at the University of New South Wales and Macquarie University, Sydney, Australia. His expertise includes interventional endoscopy and management of inflammatory bowel diseases. Leong is committed to individualised, high-quality management, and fostering novel research.

of coeliac disease, and has reviewed the safety of the intravenous contrast agent fluorescein that can be used to stain cellular structures for visualisation during CEM. Recently, Leong has turned his expert eye towards CEM in the diagnosis and evaluation of inflammatory bowel diseases.

INFLAMMATORY BOWEL DISEASES

Currently, little is known of the causes of inflammatory bowel diseases, which include Crohn’s disease and ulcerative colitis. Both genetic and environmental factors have been recognised. With no known causes, it follows that there are no known cures for these diseases; current medication focuses on controlling the symptoms and disease progression.

A major symptom is the development of ulcers in the gastrointestinal tract. Ulcers are typically the cause of bleeding from the bowel, severe abdominal pain, weight loss and diarrhoea. In addition, patients often become iron deficient and hence anaemic, afflicting them with additional symptoms such as fatigue. In extreme cases, patients may develop cancer or require extensive surgery to remove whole sections of the bowel. Up to three in 1,000 people suffer from inflammatory bowel diseases. Unfortunately, many patients are in the formative years of their lives and the debilitating impact of Crohn’s or ulcerative colitis can be highly inconvenient.

Normally, inflammatory bowel diseases are diagnosed by a colonoscopy to look for inflammation and ulcers, followed by biopsies for further study by pathologists. Leong instead has been using CEM not only to diagnose and evaluate inflammatory bowel diseases at Bankstown Hospital – his instrument is the only one in the state of New South Wales – but now to also assess the barrier function of the bowel lining. From these clinical investigations, he has shown that CEM is superior to biopsies and has furthermore revealed that the lining of the gastrointestinal tract of inflammatory bowel disease patients becomes ‘leaky’.

DISCOVERING A ‘LEAKY’ LINING

CEM has allowed Leong to view the function as well as the structure of the gut lining. Biopsies disrupt the natural environment of and kill the extracted cells, resulting in a loss of function. Therefore, biopsies can only provide information on the structure of tissue. In contrast, CEM visualises living tissue, providing information on both structure and function.

When examining the mucosa of inflammatory bowel disease patients, Leong noticed that the fluorescein contrast agent given intravenously was leaking through the gut lining and into the lumen of the intestine. One of the most important functions of the mucosa is as a barrier to separate the contents of the lumen, including digestive enzymes, bacteria and faecal waste, from the rest of the body. Therefore, a ‘leaky’ lining, seen to a significantly higher degree in inflammatory bowel disease patients relative to age-matched controls, represents a loss of this barrier function.

With this discovery, Leong is focusing his research on using CEM to study the integrity of the intestinal mucosa in the pathogenesis of inflammatory bowel disease: “We are now validating the features of a ‘leaky gut’ - this is really exciting because it has never been seen before”. He theorises that invasion of the gut wall by foreign matter from the intestinal lumen may trigger the inflammatory cascade, resulting in ulceration of the mucosa.

PROTEOMICS

To explore the effects of the leaky gut, Leong is searching for the presence of bacterial proteins in the blood of inflammatory bowel disease patients. And in order to do this, he is using proteomics. Proteomics is the identification of the constituent proteins of a system from protein fragments known as peptides. Peptides, or combinations of peptides, are unique to their parent proteins. As different organisms (and indeed different cells) are composed of different sets of proteins, proteomics can be used to detect the presence of foreign bodies. If the leaky mucosa of inflammatory bowel disease patients is indeed allowing gut bacteria and other foreign matter to cross the gut wall, then there will be higher concentrations of gut bacterial proteins in the blood of these patients relative to healthy individuals.

Leong hopes to use the presence of these proteins as a measure of leakiness and to help confirm the findings of the impaired barrier function of the bowel. By combining CEM and proteomics, both the structure and function of the mucosa can be evaluated, and the result of the loss of bowel integrity can be measured. Reversal of the abnormal changes and normalising the protein constitutent of the blood may be an end point of treatment. He has already extended this combined approach to other diseases such as pre-cancerous changes of Barrett’s oesophagus, gastric intestinal metaplasia and pre-cancerous changes of the stomach.

By combining confocal endomicroscopy and proteomics, both the

structure and function of the mucosa can be evaluated, giving a more

in-depth analysis of the pathogenesis of inflammatory bowel disease

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