Australian Life Scientist July/August 2013

PP100009448 ISSN 1448-9791

Clinical trials in

cellular

therapy

Biological therapies take on chronic inflammatory disorders. 18 Vol 10 Issue 4 • July/August 2013

G LO BA L H E A LT H | G E N E TI C S | PE P TID O M IM E TI C S | PE RSO N A L I S E D M E D I C IN E

Contents FACE TO FACE

14 Aligning immunology

with virology Director of the WHO Collaborating Centre for Reference and Research on Influenza, Anne Kelso, shares some insights into her accomplished research career in microbiology, immunology and virology.

CLINICAL TRIALS

18 Cellular therapy

A team at Royal Perth Hospital are

turning up promising results in their clinical trials using cellular therapy for people with chronic illnesses such as graft versus host disease and Crohn’s disease.

GENETICS

22 DNA – a silent witness

Forensic geneticist Dr Dennis McNevin predicts that sensitive DNA profiling techniques are about to transform forensic science with the use of DNA to predict a person's physical appearance - a molecular photofit from DNA found at a crime scene.

22 PEPTIDES

PERSONALISED MEDICINE

26 Mimicking a cure

30 A step towards

personalised medicine

A collaboration of Melbourne-based

scientists have developed a peptide

‘mimic’ that promotes myelination

of peripheral neurons. Their research

is shedding much-needed light on

how the body controls myelination

and may translate into better

treatments for neurodegenerative disease.

REGULARS

IN THE NEXT ISSUE OF ALS

06 10 12 33 41 42

• ComBio 2013 meeting preview • Plant biology • AusBiotech 2013 meeting preview

Movers and shakers GrantWatch AusBiotech New products Publish or perish Events

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Immunotherapy for autoimmune diseases is gaining momentum thanks to stem cells. A pilot study underway in Sydney and ongoing research in Adelaide are two examples of a more tailored approach that is being taken to treat autoimmune diseases.

Cover image: The manufacture of mesenchymal stromal cells for use in humans takes place in the licensed facility at Royal Perth Hospital.

Cover background image: © www.sxc.hu/gerard79_bloodcells

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L

in translation

ab-based research and practical clinical outcomes seem to almost go hand in hand when it comes to medical research these days. The distance between getting a research discovery out of the lab and into the clinic is getting shorter.

Translational research has become a bit of a buzzword - the integration of basic research into the clinical setting. Collaborations between clinical researchers and lab investigators in designing trials are important to a trial’s success.

Well-designed clinical trials are the way that we determine whether a new therapy is effective.

And trials are key to advancing medical knowledge and improving treatments.

One area that we cover in this issue of Australian Life Scientist is the manufacture of therapeutic products for use in humans. Considerable progress is being made in this area and some exciting research is underway in Australia on a number of fronts.

Research into mesenchymal stromal cells (MSCs) is one example of the growth happening in this field. We look at clinical trials that reveal some surprising successes of MSCs in treating chronic inflammatory disorders (page 18).

In the last few years there has been an extraordinarily rapid and extensive use of MSCs clinically. But as often happens in biological research, the cells remain somewhat elusive. When cultured, MSCs appear to behave differently to what they do in the body. Ongoing exploratory research is still needed to understand how MSCs mediate tissue regeneration and generate effective responses in patients.

In the area of stem cell research and immune-based therapies, another early phase trial is underway that is assessing whether umbilical cord blood can restore immune balance and prevent the development of type 1 diabetes in at-risk children (page 30).

These trials, even though they involve a small number of patients, will at least tell us how much of a treatment effect these cells are having and, in turn, help clinicians make treatment decisions.

Modulating the immune system to recreate the balance that is lost in autoimmune diseases is the focus of other research that is still in the lab in Adelaide (page 30). This work is looking at the transplantation of a type of immune cell, the regulatory T cells.

One of the challenges in getting these cells into the clinic has been generating enough of them for therapeutic use. Having overcome this feasibility issue, the researchers now want to ensure their lab data translates effectively into in vivo studies before moving into trials in patients.

Another article in this issue reports on some investigative neuroscience research in Melbourne that has turned up some unexpected advances in knowledge (page 26).

Researchers have developed a novel peptide mimetic of a neurotropic factor that promotes nerve myelination. At the same time as developing a compound that has potential in the treatment of neurological diseases, the work is providing insights into the basic mechanisms underlying the actions of these molecules.

This work reinforces that the outcomes of labbased studies are key to generating a rationale for the clinical application of biological materials.

Susan Williamson

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Geneworks 33 Interpath 13,39 Labtek 36 Laftech 02 Life Technologies 09 Lonza 4,25

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MOVERS & SHAKERS

Primitive fish fossil nods its head

Preserved muscle tissue on an ancient fish fossil may provide insights into the early evolution of vertebrates, according to a new study involving a collaboration between Australian and European scientists.

The first vertebrates were jawless fishes and included the cyclostomes (lampreys and hagfishes). Development of the varied jaw structures seen in vertebrates today required the reconfiguration of the muscular and skeletal systems of the vertebrate head and neck. Without fossilised musculature to prove otherwise, researchers have assumed that the earliest

jawed vertebrates - an extinct class of armoured fish called placoderms - had a primitive structure, much like that found in sharks.

Now, research by Dr Kate Trinajstic from Curtin University in Perth and colleagues, using the only known example of preserved three-dimensional musculature from a placoderm fossil (a 380-million-year-old Eastmanosteus fossil from the Upper Devonian Gogo Formation of Western Australia), shows something different.

By examining patterns in fossilised placoderm muscle tissue, the researchers found that the muscle architecture was distinct from that of sharks. This included the creation of a separate shoulder girdle with several distinct neck muscles and a notable dermal joint between the skull and shoulder girdle.

In contrast, sharks have a broadly flexible neck lacking these jointed elements.

The researchers also found musculature in the placoderm abdomen that closely resembled abdominal muscle tissue of tetrapods, the first four-limbed vertebrates.

Transverse abdominal muscles were thought to be a tetrapod innovation. However, this discovery suggests it arrived with jawed vertebrate, or at least this group of placoderms. This research was published online by the journal Science.

Australian biotech sector grew 7% in 2012

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keep pace with revenue growth. Australian public biotech companies spent US$636 million on R&D during 2012, up just 3% from 2011.

Globally, R&D spending increased 5% in 2012, well below the 9% growth rate from 2011. Here again, R&D spend by major biotechnology companies remained strong, while smaller, pre-commercial companies substantially reduced their rate of spending growth.

The report notes that R&D spending is a greater indicator of the health of the biotechnology industry than profitability.

Although the biotech industry became profitable in the wake of the 2008 financial crisis, this was a sign not of strength but of uncertainty and weakness in the sector, as companies were forced to dramatically cut costs to stay afloat.

In 2009, the sector’s R&D spending fell for the first time ever. While 2011 brought signs of recovery, Ernst & Young said the 2012 results show that the sector is not out of the woods yet. © iStockphoto.com/mohamed sadath

The Australian biotechnology industry grew 7% in 2012 when measured by revenues of publicly listed companies, according to Ernst & Young’s latest Biotechnology Industry Report.

Revenues for the sector edged past US$5.05 billion ($4.88 billion), with growth as usual driven by CSL (ASX:CSL).

But combined net income fell 9% to US$777 million and the total number of publicly traded biotech companies fell by four to 54. Across the world’s major biotechnology markets - Australia, North America and Europe - revenue from publicly traded companies grew 8% to nearly US$90 billion.

Net income increased 32% to US$5.2 billion.

But growth for the year was dominated by the 16 biotech companies with annual revenues of over $500 million. Revenue was either flat or declined among the vast majority of companies in the sector which fall below this revenue threshold. The report also shows that in Australia and internationally, R&D spending failed to

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© iStockphoto.com/susandaniels

MOVERS & SHAKERS

Prize-winning research on genetic markers

Superfast plant breeding Plant breeding projects that aim to increase food production traditionally depend on self-pollination for several generations because of the need to obtain ‘pure lines’ of plants - which can take a lot of time.

But this is about to change. A team of international researchers, including researchers from the University of Western Australia (UWA), has developed a new technique that enables up to eight generations of wheat and nine generations of barley to be produced a year.

Until recently, the fastest way to obtain ‘pure lines’ was to exploit differences in latitude or altitude, such as the ‘shuttle breeding’ technique developed by the ‘Father of the Green Revolution’, the late Nobel Laureate Dr Norman Borlaug. However, even this technique, which involves growing plants at different places, achieved only two or three generations a year.

The team - involving researchers from CSIRO, UWA and China - has perfected a method of embryo culture.

Although embryo culture has been used before, the team combined it with specially modified water, light, temperature, humidity and potting-mix management to achieve stunning results.

The study was just published in the international journal Euphytica.

Co-author Associate Professor Guijun Yan, from UWA’s School of Plant Biology and Institute of Agriculture, said a skilled technician in the team was able to dissect 60 plant embryos per hour from the developing grains.

“By dramatically shortening times required to obtain pure-line plant genotypes, our method could have wide applications in breeding and biological studies,” Associate Professor Yan said.

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Professor Matthew Brown from The University of Queensland Diamantina Institute has been awarded the $1.25 million Premier’s Science Fellowship to develop his work in the diagnosis and treatment of rheumatoid arthritis and tuberculosis.

UQ will match the state funding dollar for dollar to further support Brown’s research.

“Rheumatoid arthritis affects 2.5% of Queenslanders and more than 513,000 Australians,” Professor Brown said. “There are no treatments to prevent it, or to induce remission.”

Tuberculosis remains a concern around the world with a rise in cases of multidrug-resistant tuberculosis being reported in Papua New Guinea and the Torres Strait islands. The lack of useful treatments means this disease is often fatal.

Although rheumatoid arthritis is an autoimmune disease and tuberculosis a chronic infection, Brown said the two shared susceptibility factors, so research into the diseases was related.

“Immunity against TB is highly heritable so a person’s genetic make-up is a significant determinant of whether they develop or clear the infection,” he said.

Brown said the fellowship funding would enable his Translational Research Institute team - working in a tri-nation partnership with researchers from China and Norway - to spend the next five years identifying genes that underlie the causes of rheumatoid arthritis and tuberculosis.

“We expect to roll out affordable diagnostic tests within five years, paving the way for new treatments targeting the root cause of the diseases,” he said of the work, which shows how partnering between clinicians, scientists, academic institutes and industry can achieve productive outcomes.

In the past, Brown has taken out three patents for tests to diagnose ankylosing spondylitis - a severe type of arthritis affecting more than 80,000 Australians - and his genetic findings on that have led to treatment trials.

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MOVERS & SHAKERS

© iStockphoto.com/Yong Hian Lim

GrantWatch Grant writing, a waste of time? of 38 working days and a resubmitted proposal took an extra 28 working days.

This came to an overall average of 34 days spent on each grant proposal.

The study found that Australian scientists spend 550 working years preparing research-grant proposals, which translates into annual salary costs of $66 million.

Because just 20.5% of these applications are successful, the equivalent of some four centuries’ worth of effort returned no immediate benefit to researchers - the more time spent preparing a proposal did not increase the chances of success for lead

Funding boost for dementia research © iStockphoto.com/alexdans

Researching sexual health in Indigenous Australian men

The Clem Jones Centre for Ageing Dementia Research (CADR) at the University of Queensland (UQ) will receive $9 million in funding over the next 5 years from the Queensland Government.

Based at the Queensland Brain Institute (QBI) at UQ, CADR was established in 2011 and is dedicated to researching dementia and Alzheimer’s disease.

QBI Director Professor Perry Bartlett said the funding would support their research into advancing diagnostic tools for earlier and improved diagnosis of the disease, and exploring new therapeutic approaches. The centre was named after the late Lord Mayor Clem Jones, the longest serving Lord Mayor of Brisbane. The QBI secured $2 million in research funding from Jones’s estate late last year.

Alzheimer’s Australia CEO Victoria Beedle praised the announcement, stating that increased funding for dementia research was a priority for Alzheimer’s Australia. “The $9 million from the state government is a step in the right direction and it is my hope that Queensland has now set the scene for the rest of the country.”

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researchers or other researchers. Success rates for grant applications historically sit at around 20-25%. Thus, much of the time spent preparing applications has no immediate benefit to researchers or society, and there are large opportunity costs in lost research output.

The study authors suggest that the grant application process could be shortened so that only information relevant for peer review, not administration, is collected.

This would have little impact on the quality of peer review and would save time, which could be reinvested into research.

The paper was recently published online.

Andrology Australia has created a scholarship for Aboriginal and Torres Strait Islander students to undertake full-time research towards a PhD degree.

The aim of the project will be to focus on health service access and primary health care for Aboriginal and Torres Strait Islander men, including reproductive and sexual health. Raising awareness of reproductive health disorders in men and associated conditions in the Aboriginal and Torres Strait Islander community is one of the priority areas for Andrology Australia, the peak Australian body for male sexual health research.

As a group, Indigenous Australians experience poorer health outcomes than other Australians, including in the area of sexual health. The appointed PhD student will be involved in working on how the management of chronic disease may be improved with Aboriginal and Torres Strait Islander men.

The scholarship will be equivalent to the NHMRC Postgraduate Scholarship Stipend (about $30,500-$35,500 p.a) for three years full-time.

Adam’s study was published in the Medical Journal of Australia.

AU S T R A L I A N L I F E S C I E N T I S T

© iStockphoto.com/Philip Down

Most researchers are well aware that they spend considerable time preparing NHMRC Project Grant proposals.

A recent study published in BMJ Open quantified this by estimating the time researchers spent preparing grant proposals.

Funding success was then predicted by time spent preparing grants.

The study included 285 researchers who submitted one or more NHMRC Project Grant proposals in March 2012 - the largest funding scheme in 2012. The 285 researchers who participated in the study submitted 632 proposals and 21% were successful.

Preparing a new proposal took an average

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AUSBIOTECH | VIEWPOINT

Revised code to benefit ASX-listed life science companies

A

revised ‘Code of Best Practice for Reporting by Life Science Companies’ (Ed 2) has been released by AusBiotech, in collaboration with the Australian Securities Exchange (ASX).

The updated code was funded by the Victorian Government and was recently launched at the opening of the AusMedtech conference by the Victorian Minister for Technology, the Hon Gordon Rich-Phillips.

The code aims to support the directors of Australian ASX-listed life science companies to adopt international best practice in reporting events to investors, as life science firms have a unique business model.

Many Australian biotech firms are small and are loss-making for an extended period, making them less attractive for institutional and other investors who may not have specific knowledge of the life sciences.

One of the ways to address information quality and asymmetry, thus improving investor confidence, is to provide guidelines that are recognised by the ASX. This provides potential investors with a measure to see that companies are managing disclosure and reporting appropriately, as well as acting as

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educational support for potential investors and directors regarding clinical trials and other matters.

The ASX have a keen interest in ensuring best practice in investor reporting, and assisted AusBiotech and the Reference Group to update this code.

In particular, the ASX provided guidance in relation to adherence to Guidance Note 8 to the Listing Rules, which assists listed companies to understand and comply with their disclosure obligations.

Substantially revised over the past 12 months and re-released in May this year, Note 8 contains important information on the principles underlying the rule and interpretation.

The code was first launched in 2006 and the revision was undertaken by a multidisciplinary and cross-organisational reference group to ensure that the code reflected current practice. A broad consultation process was also undertaken

Glenn Cross, Acting CEO, AusBiotec h

with investors and CEOs.The updated code is dedicated to the memory of Dr Mike Hirshorn OAM (1950-2011), a significant leader in the Australian biotechnology industry.

Dr Hirshorn tirelessly led the conception and development of Edition 1 of the Code in 2005-06. His contribution to the industry was highly respected, as a founder and CEO of Cochlear, and a founding director of ResMed. In recent years he was director of Sydney-based Four Hats Capital. We mourn his passing and we remember him for his contribution to not only the revision of this code, but also to the biotechnology, science and business communities.

Copies of the code can be obtained by visiting the AusBiotech website at www.ausbiotech.org/reports or the ASX website www.asx.com.au.

Companies that have implemented the code may be recognised by the display of the web banner. ALS

This This life sciences company company has CODE OF OF has implemented implemen im entted d the the CODE BEST PRACTICE for Reporting Reporti r ng by

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Aligning

Š www.sxc.hu/gerard79

FACE TO FACE | ANNE KELSO

immunology and virology Susan Williamson

Running a lab, directing a CRC and now a WHO Centre, Anne Kelso discusses her accomplished research career in microbiology, immunology and virology.

Australian Life Scientist: How did you first become interested in science?

Professor Anne Kelso: I always loved science as a child and I was interested in microbiology quite early - partly because my mother showed me things under the microscope. She studied botany at the University of Melbourne and introduced me to that amazing microscopic world when I was about nine years old.

I just had in my mind that I would be a microbiologist. I majored in microbiology at the University of Melbourne and during those years I just loved it more and more. Then I did honours and a PhD with immunologist

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Bill Boyle. Bill was terrific and I became passionate about immunology through my thesis work with him.

My PhD was on cytotoxic T lymphocytes and trying to understand what they see and how they are regulated.

It seems very primitive looking back now compared with what we can do today but it was a time of tremendous growth in immunology. This was in the 1970s just after Peter Doherty and Rolf Zinkernagel had made their amazing discovery of MHC restriction.

I then went to Lausanne in Switzerland to do a postdoc with Teddy Brunner, JeanCharles Cerottini and Rob MacDonald.

AU S T R A L I A N L I F E S C I E N T I S T

Their lab had discovered cytotoxic T lymphocytes and developed the first assays for them, so it was a bit like going to Mecca for me at that stage of my career.

I spent three years there and had a fantastic time in the lab - that was when I felt most certain that I wanted to continue with research.

ALS: After three years in Switzerland, what brought you back to Australia?

AK: I heard Don Metcalf giving a talk in Europe and I was fascinated by his work on single haemopoietic stem cells in vitro, learning how to regulate their differentiation.

Don was visiting our institute in Lausanne and I nabbed him in the tea room and asked if I could come back to his lab. So I went to the Walter and Eliza Hall Institute (WEHI) as a QEII fellow in Don’s lab.

In Lausanne I had been looking at the production of cytokines by T cells, which was a pretty new field at that stage, so

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ANNE KELSO | FACE TO FACE

when I came back to Melbourne, Don suggested that I look at T cell production of the colony stimulating factors (CSFs).

I was there when the group cloned the granulocyte-macrophage colonystimulating factor gene and some other haemopoietic growth factor genes. It was an exciting time in the world of CSFs because now the sequences could be understood, the regulation of production could be examined in a different way which I proceeded to do in T cells - and large quantities of material could be produced. This meant that all sorts of experiments were possible - in the lab and in clinical trials in Melbourne. It was a brilliant time to be there and I learnt a lot.

After I had been in Don’s lab for four and a half years, I moved into Gus Nossal’s lab where I built up my own group as well as sharing some of the responsibility for looking after Gus’s students and postdocs. That was a great opportunity for me. Although clearly returning to the heartland of immunology, I developed a greater independence and widened my collaborative range.

I continued on the theme of trying to understand cytokine production by T cells - that core question of how T cells decide what to do in an immune response has been the main theme of all my personal research even up until now.

In the end I had 10 years at the WEHI. Don and Gus were wonderful people to work with - two men of very different personal and scientific styles - and I learnt a lot from them both.

ALS: What led you to develop a broader scientific career?

AK: In 1992, I moved to the Queensland Institute of Medical Research (QIMR).

It was a time of change in my career in many ways and provided me with big opportunities in my scientific development.

At QIMR I built up a group where we continued working on functional specialisation in T cells in a variety of mouse models. However, I’d started to be more involved in activities outside the lab when I was at WEHI - for example, with the Australasian Society for Immunology (ASI) and the Australian Research

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“Running a joint venture like a CRC is about trying to make the family work - encouraging collaborative links, making decisions that everybody will be happy with, because you don’t have line management control in the way you would in a company, for example.”

Council’s Biological Sciences Panel, which I was on for four years from 1991. Then in 1995 and 1996 I was President of the ASI. These forays outside the lab gave me a different type of engagement with the scientific community, in Australia in particular.

From 1998 I was Secretary-General of the International Union of Immunological Societies for three years. That was a great broadening experience. In particular it got me involved in some teaching courses in developing countries in Asia and Africa and engaging in a different way with the international community of immunologists beyond the ones we tended to collaborate with.

Then the opportunity came up to apply for the Directorship of the Cooperative Research Centre (CRC) for Vaccine Technology. I’d been a member of that CRC since its inception in 1993 Michael Good had been director from the beginning but in the year 2000 he resigned to become director of QIMR. So, in addition to having my research lab, my last six years at QIMR were also spent being director of the CRC for Vaccine Technology.

ALS: How did that work compare with being in a research lab?

AK: The role at the CRC gave me the opportunity to learn about research commercialisation, intellectual property, how to protect it and how to design experiments focused on the goal of making something useful.

It’s quite different from being director of an institute or the head of a lab. Running a joint venture like a CRC is about trying to make the family work - encouraging collaborative links, making decisions that everybody will

AU S T R A L I A N L I F E S C I E N T I S T

be happy with, because you don’t have line management control in the way you would in a company, for example.

It was absolutely the hardest job I’ve done. There were some difficult times but I wouldn’t have missed the experience for the world. It was a huge growth experience and we also had a lot of fun working together.

It was semi-commercial. Most of the partners in our CRC were basic research labs - QIMR and WEHI, Monash, Melbourne and La Trobe Universities, the Australian Red Cross Blood Service (ARCBS), the Livestock Industries Division of CSIRO and CSL as our commercial partner.

Most of the work was firmly laboratory based in the universities and research institutes of the CRC. It was focused on developing prototype vaccines or immunotherapies to deal with real problems in humans or animals, particularly livestock animals.

CSL was at the table to give advice on the work and how to develop projects to become commercialisable, and then to have first option to commercialise that research if they wished. We had contracts with CSL and a number with other international pharmaceutical companies as time went on.

ALS: And you are now director of one of the WHO collaborating centres, what led you into public health?

AK: It was another one of those transition points for me and the position as director of the WHO Collaborating Centre for Reference and Research on Influenza came up at the exactly the right time.

I moved back to Melbourne in February 2007 to start the job which I currently hold.

Again, it’s been a really big shift.

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FACE TO FACE | ANNE KELSO

In the CRC for Vaccine Technology I was learning a lot about commercialisation and a somewhat different way of doing science, whereas now I’m in an arena where the goals relate directly to global public health and are not in the profitmaking domain.

We have a terrific lab of people here who isolate and analyse flu viruses from around the Asia-Pacific region to monitor how flu viruses change throughout the year and from one year to the next.

We do a lot of gene sequencing and antigenic analyses to understand the relationship between the flu viruses circulating in humans right now and the viruses in current vaccines.

We identify viruses that are suitable representatives of the strains we expect to be circulating in the forthcoming season.

With the four other centres like ours in the Northern Hemisphere, we assist WHO in making recommendations on virus strains to go into influenza vaccines that are used worldwide, and then we make those strains available to all influenza vaccine manufacturers.

ALS: How mutable is the flu virus?

AK: This is what is so interesting about the flu virus - it has a very unstable RNA genome and mutates at quite a high rate.

The main selection force for the emergence of new variants is human population immunity.

It’s a very nice alignment of immunology and virology. The human immune response drives the emergence of new variants every season or so and

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this means we need to update at least one of the components of the flu vaccine most years. And in the case of antiviral drug resistance, in some cases it’s the use of the drugs themselves that drives the emergence of variance.

By monitoring the changes in influenza viruses, we can provide advice on the need to update vaccines and other important changes such as sensitivity to antiviral drugs like Tamiflu and Relenza. Then, if there is a spread of antiviral drug resistance we can alert public health authorities.

ALS: Do you maintain a hand in research?

AK: I am part of an NHMRC Program led by Peter Doherty at the University of Melbourne and I have a small lab there that continues to work on fundamental aspects of T cell immunology.

We also have quite a bit of research going on in our centre; for example, on antiviral drug resistance and in ferrets to try to understand how immunity develops in response to infection, how this can be modulated with vaccination, and how infection and vaccination can drive the emergence of new variants.

We also have a large number of collaborative links, within Australia and overseas, which allow us to spread our wings in the type of work that we do.

ALS: Can you describe what it was like to be involved in the flu pandemic in 2009?

AK: We were involved right at the beginning. The virus had apparently

AU S T R A L I A N L I F E S C I E N T I S T

emerged from swine somewhere in North America and was first confirmed as a new virus in the middle of April 2009. It was Anzac Day in 2009 when we first heard that there was a significant problem and WHO started to raise the alert about the potential for a pandemic.

We heard the next day from colleagues at the National Influenza Centre in Auckland that they had a group of high school students who had returned from a trip to North America and quite a number of them had flu-like symptoms. An alert GP there realised, given where these students had been, they might have picked up this new swine flu.

The laboratory in Auckland sent us some samples to test for the new virus.

At that stage there were no diagnostic tests so we went straight to gene sequencing, with some centre scientists working through the night to get the result as quickly as possible. It took about 24 hours from receiving the samples to getting the result and letting the New Zealand Ministry of Health know that they did have some H1N1 infected students, so it was really very early on in what turned out to be a pandemic.

We then prepared reference material from one of the Auckland viruses and supplied this to diagnostic labs around Australia, New Zealand and other countries in the Asia-Pacific region so that they could set up their own diagnostic tests. At that stage everybody was concerned that their flu cases might be due to this new virus and they needed to find

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ANNE KELSO | FACE TO FACE

that out quickly in case they needed to trigger pandemic plans in those areas.

It was a good test of our emergency response and the next time such an event occurs we will be better placed to be on top of it early.

I was also one of a group of people from around Australia who were on committees advising the chief medical officer and the Department of Health and Ageing on public health measures in response to the pandemic. Government, of course, makes the final decisions, but it was very interesting to be considering those issues and providing technical advice to support the policy decisions that needed to be made.

ALS: Are you concerned about the new strains of flu that are emerging?

AK: The H7N9 virus, a new one that has recently emerged in China, has been one of the most concerning events for quite a few years in the influenza world.

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Our centre needs to be ready to move if this strain becomes transmissible between humans. It is apparently circulating in poultry, and when humans have been infected many have become severely ill and the death rate has been quite high.

There’s been a wave of these infections in China, particularly in Shanghai and the surrounding provinces. More than 130 cases were confirmed over about an eightweek period. So far there haven’t been large numbers of subclinical infections reported, so it seems to be a difficult virus to catch but a nasty one. In that way it is like the other bird flu, H5N1, that’s been circulating since 2003.

The reason H7N9 is such a concern is that it has a number of features that suggest it can infect mammals relatively easily, much more easily than H5N1. So it may not have to undergo many more changes to become transmissible from human to human. We are watching and waiting to see whether it

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acquires those changes. If it does we’ll have a serious problem on our hands. Other WHO laboratories are working on modifying the virus so it is safe for the production of vaccine. This is well underway now so that vaccine producers will have something to work with if governments need an H7N9 vaccine at short notice.

ALS: Do you have plans for the future?

AK: I’m very happy in my role here and we have a very exciting prospect for the centre coming up. We’ll be moving into the Peter Doherty Institute for Immunity and Infection early next year with the Department of Microbiology and Immunology from the University of Melbourne and a number of other groups from Melbourne Health and the university. To be cohabiting with these other really excellent people is going to be terrific for the centre and I’m looking forward to that a lot. ALS

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CLINICAL TRIALS | CELLULAR THERAPY

Chronic inflammatory disease on trial

Susan Williamson

The results of clinical trial using cellular therapy in patients with Crohn’s disease were so encouraging the research was published before the trial had been completed.

I

t was about four years ago that Professor Geoff Forbes, a gastroenterologist at Royal Perth Hospital, had talks with Dr Marian Sturm about trialling a cellular therapy in patients with Crohn’s disease.

Sturm, Principal Scientist and Production Manager of Cell and Tissue Therapies at the Royal Perth Hospital, was involved in a study trialling the use of mesenchymal stromal cells (MSCs) in patients with steroid refractory graft versus host disease (GVHD).

The trial Sturm was involved in was producing positive results and she was aware that one of the symptoms in these patients was similar to that in patients with Crohn’s disease.

“The liver, skin and gut are commonly affected by this inflammatory condition,” said Sturm of GVHD. “Crohn’s disease is also an inflammatory disease characterised by chronic inflammation of the bowel.”

Forbes, who was well aware of the few treatment options available for end-stage Crohn’s patients, soon became the principal investigator of a clinical trial looking at the use of MSCs in patients with Crohn’s disease.

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REALISING BENEFITS

GVHD is a serious complication that develops in patients who receive bone marrow transplants for cancers that affect blood, bone marrow and lymph nodes, such as lymphoma and leukemia.

Because it is an immune-mediated disorder - the newly transplanted bone marrow cells attack the transplant recipient’s tissue - it is usually treated by suppressing the immune system with medications such as steroids. But when this treatment fails, there are few treatment options available.

The initial phase 1 study that Sturm was involved with looked at the safety of MSCs in patients with steroid-refractory GVHD. The study showed that many study participants benefited from the treatment.

“Together with Professor Richard Herrmann, who is medical director of our facility, we started manufacturing MSCs in 2007 and treated our first patient with GVHD that year,” Sturm recalled.

Sturm is currently involved in a phase 2 trial in GVHD, which is comparing patients who receive standard of care (treatment with steroids) with those receiving standard of care plus MSCs.

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CELLULAR THERAPY | CLINICAL TRIALS

“The MSC treatment in the phase 2 study in GVHD commences earlier in the disease - so patients are randomised as soon as they get GVHD, before they become refractory to steroids,” she added.

A CHRONIC INFLAMMATORY CONDITION

The experience Sturm’s group had in making MSCs and the success of the trial in patients with GVHD paved the way for Forbes to pursue a trial with Crohn’s patients.

Crohn’s disease most commonly affects the lower small intestine and the colon, but it can involve any part of the gastrointestinal tract from the mouth to anus. The inflammation extends from the mucosal lining through all the layers of the gut wall and causes pain, diarrhoea and a range of other debilitating symptoms.

“Getting these symptoms or the active disease under control is important, which is what we refer to as getting the disease into remission,” Forbes said. “Once the disease is in remission, we aim to maintain remission. Different treatments are often used to achieve these two aims.”

The activity of the disease is measured by the Crohn’s Disease Activity Index (CDAI), which takes into account factors such as number of bowel motions per day, abdominal pain and extraintestinal problems such as joint pain, skin rashes, eye problems and haemoglobin.

“If your CDAI value is less than 150 you are seen to be in clinical remission,” Forbes explained. “If it’s over 250 you have moderately active Crohn’s disease and if it is more than 350 you have severely active disease.”

The conventional treatment for Crohn’s disease is corticosteroids, such as prednisolone, to help induce disease remission.

Then, to maintain this remission, treatment with immunomodulators like azathioprine or methotrexate is used. If these drugs fail, patients can be given anti-tumour necrosis factor agents, such as infliximab and adalimumab, which are used for inducing and maintaining disease remission.

A TRIAL IN CROHN’S DISEASE

With funding sourced through Therapeutics Innovation Australia and the US-based Broad Medical Research Program, Forbes got the trial underway in 2010, with colleagues in Perth, Adelaide, Melbourne and Sydney. A phase 2 study, the aim was to look at the efficacy and safety of using MSCs in patients with moderate to severely active Crohn’s disease who had failed treatment with all conventional medical therapies and otherwise needed surgery.

Patients were required to have a CDAI greater than 250 points to be eligible to join the study and disease activity was confirmed with an endoscopy. Patients were then given MSCs intravenously once a week for four weeks and study outcomes were evaluated at six weeks.

The primary endpoint for the study was clinical response, which was measured by a reduction in CDAI of 100 points or more.

A number of secondary outcomes were measured, including clinical remission.

“For clinical remission to occur, the CDAI needs to have fallen to less than 150,” said Forbes “So if you start at 350 and drop to 100 you’ve had a clinical response and you’re in clinical remission.”

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Interim data for 14 patients became available towards the end of 2012. Of the 14 patients, a clinical response was obtained in 12 - that is their CDAI fell by 100 points or more - and eight patients went into clinical remission with their CDAI falling to less than 150 points.

“This was quite extraordinary because we powered the study so that if we got to the first 10 patients and none had a clinical response

“We have about six clinical trials underway here using MSCs ... Basically, we have a freezer of cells that are available on request for clinical trials.” we would then double the dose of the MSCs,” Forbes said. “And if we got to 20 patients and we had no response we were going to pull the study or if we got to 20 patients and we had one response we would continue through to 30 patients.”

“It is the clinical data that has been so encouraging,” Sturm concurred about the surprising efficacy of the MSC treatment in patients with refractory end-stage disease. The study is continuing and 19 patients have now joined the study. The plan is to recruit 30 patients.

Both researchers were keen to emphasise that the trial shows some evidence of efficacy in a small group of patients and that a multitude of questions remain to be answered.

“It remains to be seen whether MSCs are more effective in achieving disease remission, maintaining disease remission or for use in combination with current treatments to achieve better outcomes for patients,” said Forbes.

UNIVERSAL DONOR CELLS

MSCs are found in the bone marrow and connective tissue of the body. They have immunosuppressive and immunoregulatory activity and migrate to sites of affected tissue to control local inflammation.

One of the fascinating characteristics of MSCs is that they are not recognised by the immune system. This makes them a universal donor cell - anybody’s MSCs can be given to anybody else without concerns about rejection.

“One of the issues that we had to get our heads around was the concept that we could administer MSCs to an individual without tissue matching,” recalled Forbes. “They are considered to be immunologically privileged but how they act and what they do is unclear.”

Based on work in animal models and in vitro studies, MSCs appear to facilitate the function of Treg cells. Tregs are a type of immune cell that play a key role in modulating the immune system.

It is thought that patients with Crohn’s disease have an undersupply of Tregs or poorly functioning Tregs and that MSCs may exert their action by upregulating Treg function in some way.

“It is something we are looking at and evaluating in our patient cohort in biopsy samples and blood samples that have been taken,” said Forbes. “We’ve got some clinical data so far, but we don’t have the laboratory data yet.”

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CLINICAL TRIALS | CELLULAR THERAPY

The lab analysis will look at cytokines and cytokine gene expression to determine the effect of MSCs on key aspects of the immune system. This data will be important in increasing the understanding of how MSCs act and further understanding of the pathogenesis of Crohn’s disease.

In the lab, MSCs have the capacity to pass down differentiation pathways and form different cell types, such as adipocytes, osteoblasts and chondroblasts - fat-, bone- and cartilage-making cells.

In contrast, in vivo they appear to exert their anti-inflammatory effect and then ‘disappear’. An unsatisfactory explanation, said Forbes, adding that this largely reflects the lack of understanding of exactly how MSCs function.

“It’s intriguing that 50 or 60 years on from the early use of drugs such as prednisolone, azathrioprine or mercaptopurine in Crohn’s disease, we still don’t understand precisely how these drugs work,” continued Forbes. “We just know they work. As our understanding of the immunological network in the gut improves so will our understanding about how potentially even these drugs we’ve used for 50 years might work.”

Dr Marian Sturm (FFSc RCPA, PhD, MSc) is Principal Scientist and Production Manager of the Ray and Bill Dobney Cell and Tissue Therapies (CCTWA) facility at the Royal Perth Hospital. She is also Adjunct Senior Lecturer in Pathology and Laboratory Medicine with the Faculty of Medicine, Dentistry and Health Sciences at the University of Western Australia.

Sturm has been instrumental in the development, construction and commissioning of the CCTWA facility that services the public sector of Western Australia. She is on various advisory committees related to cellular therapies and has worked in medical research for more than 20 years. Her current research focus is in the development and delivery of cell- and tissue-based therapies across multiple disciplines, including mesenchymal stromal cells.

MAKING CELLS

Sturm is responsible for the laboratory manufacture of the cells and the regulatory mire associated with this. The licensed manufacturing facility that she runs, which is part of the Royal Perth Hospital, is the only one of its kind in the Australian public sector that makes MSCs. “We now have a licence to manufacture MSCs for clinical trials,” said Sturm, explaining that an assortment of mandatory tests need to be conducted on the cells to be compliant with Therapeutic Goods Administration requirements.

Over time, Sturm’s team has perfected the techniques involved in isolating and growing MSCs. In their expert hands they “grow very easily”.

“We have a very rigid manufacturing protocol and we’ve had quite good clinical responses to our cells,” Sturm said. “We have certain techniques we use that we think may make our cells better than many other centres.”

Professor Geoff Forbes (MB BS, MD, FRACP) is a senior staff specialist in gastroenterology at the Royal Perth Hospital and Clinical Professor and adjunct staff member in the School of Medicine and Pharmacology at the University of Western Australia. His interest in Crohn’s disease began during his training in Perth and London, England. He was appointed to RPH in 1995 and then has had a wide range of commitments centred on patient care, teaching and clinical research. He was departmental head between 2000 and 2008, and director of clinical governance for the hospital in 2007 and 2008. His other research interests have included gastrointestinal GVHD, Helicobacter pylori infection, colorectal cancer screening and aspects of endoscopic practice.

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Throughout the entire procedure, from getting the cells out of a marrow sample to expanding them and seeding new cultures to cryopreserving them for later use, the cells are tested for factors such as growth rate, phenotype and ability to differentiate into different tissue types. When they meet all the characterisation and manufacturing requirements they can be made available for infusion into patients.

“From about 10 mL of bone marrow from a donor we can derive about 80 doses of MSCs,” said Sturm explaining that bone marrow donors are relatives or friends of patients accrued to the trials.

“The cells are packaged in aliquots of 50 or 100 million cells and we administer around 2 million cells per kilo patient weight at each infusion.”

BECOMING ROUTINE

Sturm predicts that MSCs are going to be big - because of their ability to modulate the immune system and their potential to treat any of the numerous immune disorders. She sees the MSC infusion procedure becoming routine, like bone marrow transplants have become a regular procedure conducted in hospitals, although she is quick to point out that unlike bone marrow transplantation, chemotherapy drugs are not required prior to receiving MSCs. “We have about six clinical trials underway here using MSCs, including some in organ transplantation,” said Sturm. “Basically, we have a freezer of cells that are available on request for clinical trials.”

Two studies that are about to start recruiting involve chronic obstructive pulmonary disease and cranial reconstruction.

“This is our first foray into tissue regeneration,” Sturm said of the trials that will be looking at bone generation. “We will be using allogeneic MSCs that are attached to a ceramic carrier and placed within a polymer scaffold to rebuild tissue. It’s very early days yet, but the studies are ready to go.” ALS

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GENETICS AND GENOMICS | FORENSIC SCIENCE

DNA - a silent witness Forensic science is about to be transformed with the use of new technology to predict a person’s physical appearance - a molecular photofit derived from DNA found at a crime scene.

Graeme O’Neill

O

n New Year’s Day, 1999, a 92-yearold woman was bashed and raped in her home in the small NSW town of Wee Waa, population 1650.

Police suspected the rapist was a local and narrowed the search to a dozen suspects but could go no further. Finally, in April 2000, after 14 months of inconclusive investigation, they took the unprecedented step of asking all local males between the ages of 18 and 45 to voluntarily provide a saliva sample for DNA testing.

Ten days later, before the DNA analyses were complete, one of the donors - a 44-yearold farm labourer and father of three, Stephen James Boney - went into the Wee Waa police station and confessed, after realising he was certain to be found out.

The cost and logistical challenge involved in testing all males of a certain age makes such an approach impractical in large communities.

But what if, instead of interrogating a list of suspects, police could profile an anonymous criminal’s DNA, found at the scene, and within hours, produce a generalised description of the unknown

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culprit: their hair and eye colour, the length of their nose, the distance between their eyes and even their ancestry?

A MOLECULAR PHOTOFIT

University of Canberra forensic geneticist Dr Dennis McNevin is leading a three-year collaborative research project to develop one of Australia’s first DNA-based ‘photofit’ systems, based on single-nucleotide polymorphisms - the ultimate source of human individuality.

The Australian Research Councilfunded project involves researchers from the University of Canberra, the Queensland Institute of Medical Research, the Australian Federal Police and the Victoria Police. McNevin says both police forces are keen to apply the new technology when the project delivers the first working version of the genotyping platform in around three years’ time.

Many crimes go unwitnessed. McNevin says even if there are witnesses, their descriptions of the culprits are notoriously unreliable. Often, the only clue to the identity of the perpetrator may be traces of DNA

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from the scene. Standard DNA ‘fingerprints’, based on microsatellite DNA sequences scattered across the genome, can only identify a suspect if his or her DNA sample matches a signature held in a database of DNA signatures of convicted criminals. If the unidentified suspect has no criminal record, the DNA trail ends there.

But an anonymous DNA trace from a crime scene contains phenotypic information about a suspect - McNevin describes it as a ‘silent witness’ that can be interrogated to produce a generalised description of the perpetrator.

He and his colleagues hope to provide police with the tools to assemble a DNAbased photofit of a suspect who has left a trace of their DNA at the crime scene.

McNevin says that only in very restricted circumstances would a generalised DNA photofit directly identify the perpetrator of a crime.

“What we hope to do, a long way down the track, is identify a few hundred to perhaps one thousand SNPs from which we could put together an image of someone’s face, using physiological metrics,” he said.

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FORENSIC SCIENCE | GENETICS AND GENOMICS

“What we hope to do ... is identify a few hundred to perhaps one thousand SNPs from which we could put together an image of someone’s face, using physiological metrics.” be translated into a very objective way of representing the subject’s phenotype.

“We don’t care what the genes are - we’re interested only in the SNPs associated with each particular phenotype.”

MATCHING FACIAL METRICS

SHADES OF BLUE

The panel of SNPs will be used to produce primers for a PCR assay kit to detect corresponding SNPs in DNA traces recovered from the crime scene; special software will transform the pattern of hits into a DNA photofit of a suspect.

Various SNPs involved in melanogenesis (the genetic pathway that synthesises melanin) have a major influence on eye, hair and skin colour, although only the first two characters are useful for a DNA photofit because of the range of variance in skin hue with exposure to ultraviolet radiation.

Eye and hair colour also vary, and McNevin says the regression technique used to predict these traits from SNP variants will provide a categorical rather than a definitive prediction - for example, if the subject’s SNP profile confirms he or she has blue eyes, it may only be possible to categorise the hue as a pale or darker shade of blue.

“A regression is only as good as the data set, and the bigger the data set, the better,” he said. “The important thing is to devise a rigorous classification system so that when the computer makes a prediction, it can

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McNevin says magnetic resonance-imaging (MRI) scans of the size and shape of human skulls have led to the identification of a number of SNPs that strongly influence facial metrics like the distance between the eyes and the length of the nose.

The QIMR’s main contribution to the project thus far has been the discovery of a set of SNPs found in seven genes associated with male pattern baldness.

The QMIR research project has shown that X does not mark the bald spot - several of the genes are located on autosomes, scotching the popular misconception that maternally inherited genes on the X-chromosome are solely responsible for male susceptibility to pattern baldness.

“In an ideal situation, we might be able to collect DNA from a crime scene, genotype it for key SNPs and upload the resulting photofit to airports and border crossings,” McNevin says.

“Video cameras linked to face-scanning software could identify individuals whose facial metrics are a near match to the photofit and they would be taken aside and interviewed by police - that would be a great boon for forensic science.

McNevin says an SNP-based photofit can also be very useful in the inverse situation, where police need to narrow a large field of candidate suspects, by excluding those who do not match the basic set of physical characteristics inferred from a DNA sample.

He says the approach his group has adopted means that as more data are added to the original training set of SNPs, the phenotype predictions can be refined, leading to more accurate photofits.

Relatively few phenotypic traits are monogenic; most emerge from the concerted action of multiple genes.

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Given that phenotypes are also strongly influenced by environmental factors, McNevin says it is effectively impossible to predict how multiple alleles of multiple genes interact with environmental factors to produce highly variable phenotypic features like body weight.

Even a strongly heritable trait like a person’s height is influenced by multiple genes - although the dominant influence of one or two genes like the human growth hormone (HGH) and insulin-like growth factor (IGF-1) genes may permit a ballpark estimate of a suspect’s stature.

TRACING ANCESTRY THROUGH MITOCHONDRIAL DNA

In a paper published in the Australian Journal of Forensic Science (AJFS) in March 2011, McNevin and several colleagues from the University of Canberra, the Australian Federal Police and Victoria Police describe how genetic markers in maternally inherited, non-recombining mitochondrial DNA (mtDNA) can provide a detailed record of the ancestry of females and males.

McNevin says ancestry can be greatly refined with the additional use of the nonrecombining part of the Y chromosome (NRY) as well as autosomal markers.

The most celebrated project to use SNP markers from mtDNA and NRY DNA is the National Geographic Society’s Genographics Project. It has assembled a detailed record of the epic migrations of our ancestors as they moved out of Africa to colonise Europe, Asia, the Americas, Australia and the Pacific over the past ~150,000 years.

In the absence of recombination, mtDNA is transmitted down the female line as a stable haplotype, subject to occasional, random single-nucleotide mutations.

Over tens of thousands of years, mutations accumulate that, because of the genetic founder effect, effectively constitute a record of when and where an individual’s direct female ancestors lived.

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The earliest non-African mtDNA mutations reveal the deep ancestry and early dispersal of female ancestors from their locus of origin.

SHARING COMMON ANCESTORS

Lineages inferred from mtDNA and NRY haplogroups typically tell different tales.

McNevin et al say historical differences in male and female migration patterns mean that mtDNA lineages are not as well correlated geographically as NRY lineages, because of patrilocality - the tendency of males to remain within the locality of their male ancestors, while females disperse.

They note that ancestry profiling by mtDNA SNP genotyping is not definitive - correlating haplogroups with genealogy requires surveys to determine population-defining SNP patterns.

Researchers have already defined the major European haplogroups for individuals from populations in Austria, Spain, Italy and the USA, and the East Asian haplogroups for individuals from Japan, Korea, China, Taiwan, and Asian immigrants in Argentina.

In comparison, haplogroups for Australia and Oceania have been poorly documented. The AJFS paper outlines the results of their SNP genotyping survey of 145 individuals, who voluntarily provided details of their female ancestry.

The study yielded a multiplex SNP assay that differentiated between Asian, Caucasian and African populations within Australia. Haplogroups M, B and F predominate in individuals of selfdeclared Asian ancestry, haplogroup L predominates in those of African ancestry, whereas haplogroups H, T and U predominate in non-Indigenous Australians of European ancestry.

“The endpoint of the project is that we would like to be able to settle on a stable, high-throughput genotyping technology for use with the available data - we’ll be happy if we can accurately predict ancestry and physical characteristics such eye and hair colour, detached or attached ear lobes, and a cleft chin.

“But in five years’ time, as the data become available, we might be able to include many other phenotypes.”

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Dr Dennis McNevin began his professional life as an engineer and a high school science teacher. He then obtained a PhD in Biochemical Engineering from the University of Sydney. After a three-year postdoc in molecular plant physiology at the Australian National University, he was a lecturer in the Department of Chemical Engineering at the University of Sydney.

In 2003, he became interested in forensic science and became a postdoctoral fellow in the Australian Federal Police Forensic Services laboratory in Canberra. He has been Assistant Professor of Forensic Studies at the University of Canberra since 2007. His research is broadly focused on forensic genetics - using genetics to enhance the value of DNA as forensic evidence and the use of next generation sequencing for forensic purposes.

SNP PROFILING - A CASE HISTORY

The first successful application of SNP profiling to help identify the perpetrator of a serious crime was in Baton Rouge, Louisiana, in 2002. As much as it helped police to identify a culprit, it also demonstrated the technique’s power to eliminate false leads.

On 17 July, a group of workers walking along the Mississippi River west of Baton Rouge found the naked body of a young woman. Pam Kinamore had been abducted from her home in Baton Rouge three days earlier.

Traces of semen recovered from her body revealed she was the third victim of an unidentified serial rapist-murderer operating in the area, but investigators were unable match the murderer’s microsatellite DNA profile to any profile in the CODIS (Combined DNA Index System), a national database containing the coded microsatellite profiles of 5 million known criminals.

After Kinamore’s body was found, a female witness reported seeing a young white man driving a white pickup truck in the vicinity around the time of her disappearance, with what appeared to be the body of a woman slumped in the passenger seat. Police regarded it as a strong lead, because another woman had been picked up and raped by a man driving a white pickup truck two days after Kinamore was abducted.

Despite the lead, police had no prime suspect when the murderer struck again in December. Investigators obtained DNA samples from 1200 young white men from the area. But months of analysis at a cost of more than US$1 million failed to produce a match to the murderer’s DNA. On the increasing probability that coincidence and a flawed witness report had misled police investigating Kinamore’s murder, Dr Tony Frudakis, founder of a struggling biotechnology company, told police he could determine the killer’s race from a DNA sample, with 99% certainty.

Using a set of 175 SNPS from loci known to be informative about an individual’s racial background, Frudakis tested the killer’s DNA and informed police the killer was of Afro-Caribbean or Afro-American descent - not Caucasian. By then, the rapist had killed again. After almost a year pursuing a false lead, police switched focus to an imprisoned sex offender, previously excluded by his Afro-American ancestry.

Four days before Pam Kinamore was abducted, Derek Todd Lee, 34, had been interrupted as he attempted to rape another woman in her home; he fled, but the woman was able to provide an accurate description of her assailant.

Despite the almost identical circumstances to Kinamore’s rape and murder, police did not attempt to obtain a sample of her assailant’s DNA, much less test it for a match with the DNA profile of Kinamore’s killer, whom they believed to be white.

Police subpoenaed Lee in prison for a cheek swab, and tests confirmed his DNA profile matched that of Kinamore’s killer. ALS

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PEPTIDES | PEPTIDOMIMETICS

Mimicking a cure Fiona Wylie

A collaboration of Melbourne scientists who describe themselves as a “happy confluence” is shedding much-needed light on how the body controls nervous system myelination, and how their tiny peptide ‘mimic’ could help patients with neurodegenerative disease.

T

he team in question comprises University of Melbourne researchers Drs Simon Murray and Junhua Xiao from the Neurotrophin and Myelin Laboratory and Associate Professor Tony Hughes, who heads the Drug Design Laboratory … and they are even happier now, with the recent publication of their combined experimental efforts in the Journal of Neurochemistry. In a nutshell, this paper reports how a novel peptide mimetic (functional mimic) of a key neurotrophic factor promotes the myelination of peripheral neurons both in the laboratory culture dish and in rats.

These findings are important for the field of neuroscience in general because the work is revealing more than the researchers themselves ever expected about some of the basic mechanics of neurotrophin action, which have remained frustratingly elusive up to now. Importantly, the results are welcome news for those working to develop better treatments for neurological diseases and, of course, for people living with these debilitating diseases.

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MEETING THE PLAYERS

The neurotrophins are a family of secreted neurotrophic factors that drive and regulate the development, function and survival of vertebrate nervous systems. As soluble molecules with the ability to affect neurological function, neurotrophins quickly became the subject of intense clinical and pharmaceutical interest when first discovered in the 1980s.

Their potential for use as therapeutic agents for treating neurodegenerative disorders and nerve injury looked promising.

However, these very large proteins are cleared rapidly by the body before enough can get to the site of action to have the desired effect and so neurotrophins themselves do not actually make very good drugs.

Neurotrophins exert their action on nerve cells through two biologically distinct classes of cell-surface receptors - the tropomyosinrelated kinase (Trk) receptors and the p75 neurotrophin receptor (p75NTR). From there it just gets more complex and at times murky with activation of these different receptors by the same neurotrophin producing functionally different outcomes in different cells - much

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PEPTIDOMIMETICS | PEPTIDES

about the mechanics of neural regulation via these factors and their receptors remains unknown.

Of the four neurotrophins, brain-derived neurotrophic factor (BDNF) is the focus of this work. Although primarily a growth and survival factor, more recent evidence also indicates that BDNF has a crucial influence on nervous system myelination.

The importance of myelination to healthy nerve function is evidenced by the large proportion of neurological diseases involving defective myelin formation or demyelination including multiple sclerosis, leukodystrophy and Guillain-Barré syndrome.

THE CRUCIAL PARTNER

The myelin sheath around the axon of neurons is an essential structural feature that allows electrical transmission to occur with miraculous speed and, at times, over relatively huge distances. Myelination is accomplished by the outgrowth of adjacent glial cells, which are sort of like the ‘Robin’ of the nervous system to the ‘Batman’ neurons. In the peripheral nervous system (PNS), Schwann cells snuggle up to the neurons to wrap them in myelin, while oligodendrocytes do the business in the central nervous system (CNS).

ESTABLISHING THE CONFLUENCE

According to Hughes, teaming up with Murray and Xiao a few years back came about through a shared interest in the molecular mechanisms of myelination, and in producing a clinically useful outcome based on their collective expertise. Indeed, the original shared goal was to develop potential therapeutic agents to induce new myelin formation or ‘fix’ defective myelination by targeting specific neural pathways.

A pharmacologist with a background in pharmaceutical and medicinal chemistry, Hughes had worked for many years designing and making peptide-based molecules as potential drugs. But it was his postdoc in Germany in the early 1990s that sparked a particular focus on neurotrophins.

“At that time, neurotrophic factors were going into clinical trials for treating neurological disorders, but the issues with their use as drugs quickly became pretty clear. So, when I came back to Australia in 1995, it made sense to start making small-molecule mimetics for these factors.”

Soon after, Murray, who had also trained in one of the big neurotrophin labs as a postdoc, began to help with some functional studies of a few small-molecule peptides. Hughes and his team had designed these peptides to mimic different regions of the BDNF molecule.

According to Murray, “one of our motivating hypotheses was that activating one of the receptors bound by BDNF and not the other using such a molecule might produce a more unified biological response compared to the diverse outcomes incurred with binding of the entire BDNF protein.”

Hughes soon found that one of his mimetics, a tiny circular peptide called cyclo-DPAKKR (CP) designed to mimic the region of BDNF binding the p75 receptor, promoted survival in a cell culture model and was indeed functionally relevant.

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TEAM SUCCESS

Meanwhile, back in their lab, Murray and Xiao’s interest in the effects of BDNF on myelination was growing. They had begun to see that BDNF could exert contrasting effects on peripheral myelination, depending on which receptor it was binding.

They realised that the structural-based approach of Hughes would be valuable in gaining the functional selectivity needed to demonstrate a therapeutic benefit of myelination in the clinic. Thus, the trio formed to start looking at the CP peptide’s effect, specifically on myelination.

Early experiments with the CP mimetic using a co-culture of peripheral neurons and Schwann cells (to represent an in-vitro model of peripheral nerve myelination) showed a strong and unified pro-myelinating effect, regardless of the type of neuron. This was just the result they were looking for because BDNF itself is only promyelinating in some types of peripheral neuron - it has inhibitory effects in others, even within the same population of nerve cells.

“These data were very clear - the peptide mimic was having only the one effect,” said Murray. “We then spent a lot of time confirming this mechanism of CP action in vitro to prove quite clearly that the peptide was acting through p75NTR expressed by the target neurons.”

The icing on the cake then came with Xiao’s experiments in rodents, which provided strong evidence that CP actually promotes the myelination of peripheral neurons in vivo.

“Injection of CP adjacent to the sciatic nerve of newborn rats significantly up-regulated myelin protein expression and increased the proportion of myelinated axons,” added Xiao. “In contrast, injection of BDNF failed to exert a significant effect.

“We are now investigating whether CP also exerts a protective effect upon myelin using rodent models of peripheral demyelinating diseases such as Guillain-Barré syndrome, a step that is taking our research in a new direction.”

Murray also emphasised that not only was this mimetic now a clear potential tool for clinical application, it also provided important information about the biology of the system because of the p75NTR-specific effects they saw on myelination in peripheral neurons.

Vehicle

cyclo-DPAKKR

This electron microscope image of two different sciatic nerves in cross-section shows that cyclo-dPAKKR increases the number of myelinated axons in the developing rat sciatic nerve. Myelin appears as black ‘electron dense’ rings surrounding relatively translucent axons.

Compared to vehicle-injected animals, injection of cyclo-dPAKKR increased the number of axons that were myelinated in the sciatic nerve, indicating it exerted a pro-myelinating influence in vivo.

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PEPTIDES | PEPTIDOMIMETICS

Dr Simon Murray (centre) originally trained as a physiotherapist and returned to study, completing his PhD in 2000. He then spent three postdoctoral years at the New York School of Medicine working on neurotrophin signalling, before returning to work at Melbourne’s Florey Neuroscience Institutes. He moved his lab to the University of Melbourne in 2005. Dr Junhua Xiao (left) completed her clinical medical training in China in 2000, before moving to Melbourne to do a PhD in neurobiology at the University of Melbourne in 2002.

She took up a postdoc position in Murray’s Neurotrophin Signalling Laboratory in 2005, focusing on mechanisms that control myelination, particularly neurotrophins. She joined the Department of Anatomy and Neuroscience as a lecturer in 2013. After studying pharmacy, Associate Professor Tony Hughes (right) completed a Master’s Degree in molecular modeling and a PhD in peptide chemistry at the University of London. He then undertook a postdoc stint at the Max Planck Institute for Psychiatry in Martinsried, just outside Munich, where he shifted fields to look at the cellular and molecular biology of neurotophic factors. He returned to Australia in 1995 to take up a teaching and research position in the Department of Pharmacology at the University of Melbourne.

“And, by design luck more than intent, CP also proved to be very metabolically stable in vivo due to its cyclic structure,” added Hughes. “This is in contrast to other, non-circular peptides that tend to be more susceptible to cleavage by proteases in the body and thus have very short half lives in a patient’s circulation.”

Another direction that Xiao and Murray would like to take now is using the same drug-like, peptide-based approach to look at myelination in the central nervous system. Just to make things even more complex, it seems to be regulated differently from the process in the periphery, via the other neurotrophin receptor TRK. The trio are currently using another peptide designed by Hughes, which mimics the region on BDNF that binds to the TRK receptor, and investigating whether it can promote myelination in the CNS. Hughes is also excited about these next steps as he sees it feeding into further peptide design activities on his side of things (see below).

“I think the project is taking quite a nice leap at the moment, because we brought the project together specifically to do the myelination stuff and it is now spreading out again as we move into other areas,” he said. “The happy bit is that we all like working together and so as well as being successful scientifically, that makes it a lot of fun.”

SPREADING THE PEPTIDE WINGS

Murray and Xiao’s genetics studies clearly show that CP exerts its promyelinating influence through the p75NTR receptor, and now they want to know more about that binding.

“One of our main aims now is to look at the interaction kinetics,” Murray explained. “How is CP snuggled up to p75NTR, exactly which residues are involved and where they are - some of the more structural and molecular aspects of the interaction.”

One of the mysteries of the system is the specific signalling mechanism used by this receptor to bring about a cellular effect, which the researchers say is remarkable because p75NTR has been known as an entity for around 30 years.

“It is a source of both fascination and embarrassment that no-one can figure this out. It is not a classic receptor, and people are still waving their hands when they talk about how it works,” Murray described.

Part of the murkiness around p75NTR is because there is no real robust biochemical assay to test its activity. However, Murray thinks the co-culture systems they have developed to test the myelination effects together with the p75NTR-specific signalling might just fill that gap.

“I hope over the next couple of years we will start to tease all these aspects out and thus make a contribution to both myelination and perhaps potential therapies for remyelination, but also get to the bottom of what p75NTR does and how it does it … that is the greater plan.”

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GRANDER DESIGNS

The CP (cyclo-DPAKKR) peptide has yielded some interesting drug design offshoots for Hughes’ own group. The structure of this cyclic peptide has intrinsic and useful drug-like properties: it is quite low in molecular weight (~580 daltons), metabolically stable and easily transported across cell membranes. But the structure of this molecule is also quite intriguing, according to Hughes.

“By NMR spectroscopy we see that this tiny pentapeptide of five amino acid residues forms a very tight and strong circle that has just a single conformation in solution. It actually turns out to be quite a nice template for other chemical and functional groups.

We could potentially use it as a sort of starting point to make other compounds that are not peptides.”

“So, we are now using University of Melbourne Interdisciplinary Seed Funding to see if we can jump from this rather neat peptide and produce some other scaffolds that may give us the same sorts of biological activities, but get even better control over the pharmacokinetic properties of the molecule,” said Hughes. They have already started by using in silico techniques to nut out how to present the functional groups on the peptide in the same way, but without the peptide backbone as a scaffold, and will then go on to do some binding studies with the peptide target. ALS

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PERSONALISED MEDICINE | AUTOIMMUNE DISEASE

A step towards

personalising medicine Giving children their own cord blood to prevent them getting type 1 diabetes and the development of a new immunotherapy are part of a more tailored approach to treating autoimmune disease.

Susan Williamson PREVENTING DIABETES WITH THE RIGHT CELLS

RESTORING IMMUNOMODULATION

Having a first- or second-degree relative with type 1 diabetes increases your chance of getting this lifelong disease, which results from an imbalance in the immune system. This knowledge has led Associate Professor Maria Craig, a paediatric endocrinologist at the Children’s Hospital at Westmead, to investigate whether umbilical cord blood may restore immune balance and prevent or delay the development of type 1 diabetes in children in this high-risk group.

“There is an increased risk of developing type 1 diabetes if your father, mother or a sibling has the disease,” said Craig.

“In fact, the risk is higher if your father has the disease, although we do not know why. It is probably related to the immune protection provided by the mother during pregnancy.”

Craig is lead investigator on the national study, called cord blood reinfusion in diabetes (CoRD). The study’s aim is to determine whether immune cells present in cord blood can stop and potentially reverse the immune destruction of the insulinproducing beta cells in the pancreas.

“This is a new direction for stem cell research,” Craig said of the pilot study. “We will be looking at whether giving children their own cord blood can prevent them progressing from pre-type 1 diabetes to full-blown type 1 diabetes. If successful, it will also validate the storage of cord blood for those at risk.”

In type 1 diabetes the breakdown of immune tolerance to selfantigens, such as insulin, results in the expansion of autoreactive T cells and the consequent destruction of the beta cells of the pancreas. There is an average of two new cases of type 1 diabetes diagnosed per day in Australian children.

Although an intervention that successfully prevents type 1 diabetes remains elusive, increasing evidence suggests that immune therapies, particularly those based on restoration of peripheral immune tolerance, may help thwart this autoimmune disease.

Regulatory T cells (Tregs) play a key role in inducing and maintaining immune tolerance to self and non-self antigens - that is they make sure the body does not attack itself.

Abnormalities in the function and/or number of Tregs have been found in people with type 1 diabetes and infusion of Tregs in the non-obese diabetic mouse model of type 1 diabetes prevents the development of the disease.

Umbilical cord blood contains large numbers of highly functional Tregs, as well as other stem cells that can develop into the different blood cell types. Cord blood infusions have been given to children who have already developed type 1 diabetes, but no studies have examined whether cord blood may have

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AUTOIMMUNE DISEASE | PERSONALISED MEDICINE

therapeutic potential in preventing type 1 diabetes in high-risk children. This is where the CoRD study comes in.

AUTOANTIGENS

The presence of multiple islet autoantibodies in the blood is a known risk marker for type 1 diabetes. These autoantibodies indicate that an immune attack directed towards the beta cells of the pancreas has already begun.

“There are four markers that can predict with significant accuracy whether you are on the pathway to developing type 1 diabetes,” Craig explained. “If you have two or more of these then you have about a 50% risk of developing the disease.”

Phase one of the two-phase CoRD study will screen children for autoantibodies to the four islet antigens - insulin, glutamic acid decarboxylase, insulinoma-associated-2 and a beta-cellspecific zinc transporter called ZnT8. Those who test positive for two or more of these antigens will be eligible to take part in the second phase of the study, where they will be given treatment and follow-up.

Treatment will involve a single infusion of autologous (their own) cord blood. The children will be tested before and after treatment to make sure they don’t have diabetes. And, their Tregs will also be counted before and after treatment, using flow cytometry, to see whether their cord blood infusion has led to an increase in these cells.

“We expect to screen 600-800 children and of these we expect about 5% to have two or more of these autoantibodies,” said Craig. “These 20 children will be at high risk of developing type 1 diabetes over the next two years. The maths doesn’t quite add up because we expect about half of the 40 eligible to participate.”

The study is currently in the screening phase, having received ethics approval last year.

BALANCING THE IMMUNE SYSTEM

Cord blood contains a large proportion of Tregs compared to blood from adults or children. And children who develop diabetes seem to be deficient in these T cells.

“We will be looking at whether there are enough regulatory T cells in cord blood to prevent these children progressing to type 1 diabetes,” Craig explained. “There are other factors that generate immune benefits in cord blood, the immune tolerance of the mother during pregnancy indicates this.”

The study will use cord blood from Cell Care Australia, a large private cord blood bank based in Melbourne that has provided funding for the study.

“It can be difficult to use public cord blood banks,” Craig explained. “This is because it is an altruistic donation to the public bank and donors cannot get their cord blood back under the current legislation - it would be a concern if people started accessing cord blood for their child when it is used for transplant in childhood leukemia.”

Children, or the parents of children, who have their cord blood stored in private banks and who have first- or seconddegree relatives with type 1 diabetes will be invited to take part in the CoRD study.

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Associate Professor Maria Craig is a paediatric endocrinologist at the Children’s Hospital at Westmead and St George Hospitals and an NHMRC practitioner Fellow at the University of Sydney. She completed her Bachelor of Medicine and Bachelor of Surgery (MBBS) at the University of Melbourne in 1989 and subsequently specialised in paediatric endocrinology. Since completing a PhD investigating the association between enterovirus infection and onset of type 1 diabetes at the University of New South Wales, in 2002, her major research focus has been the link between viruses and childhood diabetes. She is principal investigator of the CoRD study and lead investigator for NSW on the ENDIA (Environmental Determinants of Islet Autoimmunity) study, which is looking into what contributes to the development of type 1 diabetes in early childhood.

Craig said the disproportionately higher rate of cord blood stored by people with relatives who have type 1 diabetes compared with the general population suggests there is awareness in the community that cord blood may be useful.

IMMUNOTHERAPY AS A TREATMENT

One of Associate Professor Simon Barry’s goals is to develop a routine procedure for the transplantation of regulatory T cells (Tregs) to induce immune tolerance.

Barry is based at the Women’s and Children’s Hospital in Adelaide, where he is chief hospital scientist and head of the Molecular Immunology Laboratory. He also works at the University of Adelaide and the Women’s and Children’s Health Research Institute, making him well placed to pursue this ambitious task.

Tregs are a subpopulation of immune cells that play a key role in modulating the immune system. They maintain a balance between the immune system’s tolerance of normal cells, tissues and organs (known as self-tolerance) and reactivity to harmful foreign bacteria and pathogens.

“Autoimmune diseases involve the breakdown of this immune balance and if there is too much reactivity and not enough tolerance, the system becomes reactive to self,” explained Barry. “In contrast, cancer involves too little immune reactivity and too much immune tolerance so the cancer cells are able to grow undetected.”

In type 1 diabetes, the immune system overreacts and breaks down the beta cells of the pancreas. Defects in Treg function are increasingly being linked to autoimmune diseases like type 1 diabetes and multiple sclerosis.

There are 25,000,000 people with autoimmune disease worldwide, 100,000 people per year receive an organ transplant and 30,000 people suffer from graft versus host disease (GVHD) because of bone marrow transplants each year. Thus, a therapy that helps restore tolerance would benefit many people.

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PERSONALISED MEDICINE | AUTOIMMUNE DISEASE

Associate Professor Simon Barry is currently chief hospital scientist and head of the Molecular Immunology Laboratory at the Women’s and Children’s Hospital in Adelaide. Barry is also leader of the Autoimmune Diseases Stream at the Children's Research Centre in the Discipline of Paediatrics at the University of Adelaide, and Senior Research Fellow at the Women’s and Children’s Health Research Institute. Barry completed his PhD at the National Institute for Medical Research Mill Hill in London before pursuing a postdoc at the Hanson Institute in Adelaide. He then continued his research career at the University of Washington before working with Immunex and Amgen. At the University of Adelaide since 2004, Barry’s work is focused on the behaviour of Tregs and the genes that drive their formation and function, with a view to identifying new and better ways of diagnosing and treating autoimmune disease.

BIOMARKERS FOR BAIT

White blood cells make up 10-30% of cells in adult blood and 1% of these are the Tregs. One of the challenges Barry faces in getting these cells into the clinic is generating enough of them for therapeutic use.

To put Tregs to use in clinical work, the researchers need to be able to identify the cells and extract them while retaining their function.

The forkhead transcription factor p3 (Foxp3), a key regulator in the development and function of Tregs, is a specific marker for these cells. Along with the cell surface markers CD4, CD25 and CD127, it is used in flow cytometry to detect Treg cells.

“But staining for Foxp3 kills the cells,” Barry explained.

“Therefore, we went on a search for biomarkers to use as bait to pull live cells out and enable us to purify them for use in humans.”

For the last five years, Barry’s team has been mining gene array data to identify genes that are essential to Treg function. Using diseased and non-diseased human cohorts, they have identified many of the genes necessary for immune tolerance and regulatory T cell function.

“By looking at genes that were turned on or off in these different cohorts, we could work out which genes were important in Treg function and confirm a role for these genes,” explained Barry.

Their search for genes expressing cell surface proteins resulted in the discovery of PI-16, now called CD359, which is highly expressed on the surface of Treg cells. In a partnership with the Cooperative Research Centre for Biomarker Translation, PI16 is showing promise as a novel human Treg biomarker. “We made an antibody to PI-16, patented it and then used it to purify cells from cord or peripheral blood,” said Barry.

Putting their new antibody to work, the researchers retrieved cells that were functional and expressed high levels of PI-16 and

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Foxp3. But they also found a population of Tregs that did not express PI-16. “We were able to distinguish two subsets of Treg cells,” said Barry. “Natural Tregs that expressed PI-16 and inducible Tregs that did not.”

APPLICATION IN THE CLINIC

In a normal healthy gut the ratio of Tregs to T helper (Th 17) cells is in balance. In diseased guts, such as those of people with ulcerative colitis and Crohn’s, there are more Th17 cells than Treg cells.

Th17 cells are thought to play a role in autoimmune diseases; however, they also serve an important function in antimicrobial immunity at epithelial mucosal barriers such as the lining of the gut.

Proinflammatory cytokines, such as interleukin 6 and interleukin-1 beta, are also elevated in the guts of patients with ulcerative colitis and Crohn’s disease.

“We looked at purified Tregs and their differences in function and found that in an environment high in inflammatory cytokines the iTregs lose function but the nTregs do not,” Barry explained. “So, if the two subsets of Treg cells are separated out there may be an increased therapeutic benefit.”

Thus, the aim is to use PI-16 to pull nTreg cells out of cord blood and adult blood and ultimately generate a scalable technique that can be taken into trials with hundreds of patients.

“Feasibility was an issue,” said Barry. “We didn’t know whether we could make enough of them to readily expand for treatment.”

But now that they have the PI-16 biomarker to apply in a technique that generates highly purified cells, and these cells demonstrate robust function in assays, Barry sees them able to generate enough cells needed for cellular therapy. Being part of the new $59 million Cooperative Research Centre for Cellular Therapy Manufacturing, which opened in July this year, will help facilitate this process.

But Barry is keen to generate more data in humans before taking the work into the clinic. Efficacy tests in mouse models of human GVHD are underway and clinical studies are ongoing to determine whether there is an imbalance in PI-16 Tregs in human diseases including type 1 diabetes and irritable bowel disease.

Tregs have broad applications in the clinical context such as correcting immune imbalances in people with autoimmune disease, eliminating the chance of rejection after organ transplant and negating the need for immunosuppressant drugs.

“Lab data is not always a good predictor of how a therapy will work in real patients and we want to be more sophisticated in our testing before we go into a clinical trial in humans, “ Barry said, adding that he expects the work to enter into feasibility studies in humans within three years. ALS

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centre of each well, leveraging static and dynamic fluid proper-

to operate. All Omnitronics controllers include a trending screen that allows the

ties to provide consistent incubation on all 12 spots with low

user to watch critical parameters on an ongoing basis if required.

sample volume. As a result, Ciraplex can offer sensitivity greater

With optional ethernet control, the user can connect the freeze dryer to the

than ELISA and high reproducibility in a multiplexing format.

network, monitoring and controlling the product from any other location. The

Cirareports summarises multiplate studies simply and quickly.

ethernet option also allows for the printout of batch reports.

Pacific Laboratory Products

Scitek Australia Pty Ltd

Contact info and more items like this at wf.net.au/U362

Contact info and more items like this at wf.net.au/U431

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NEW PRODUCTS @ www.labonline.com.au

Gene panels Life Technologies Corporation has launched two Ion AmpliSeq Community Panels: the Ion AmpliSeq BRCA1 and BRCA2 Panel design and the Ion AmpliSeq Colon and Lung Cancer Panel design. The panels require just 10 ng of DNA input per primer pool, about 25-fold less than alternative gene panel approaches.

The workflow is a simple PCR reaction coupled with rapid ion semiconductor sequencing, enabling researchers to go from DNA to results in less than 24 h.

The BRCA panel analyses the coding regions of the tumour suppressor genes BRCA1 and BRCA2, which have been implicated in hereditary breast and ovarian cancers. The colon and lung panel analyses more than 500 mutations in 22 associated genes. The panels can accurately call all variants of interest, including those within difficult sequence contexts.

The Ion AmpliSeq Colon and Lung Cancer Panel design analyses more than 500 mutations in a single-tube assay. The workflow can be completed in as few as 3.5 h turnaround time. The panel design permits the user to describe a cancer according to the molecular subpopulation that compose the lesion; something said to be impossible with previous methods.

Petri dish rack and coloured tags Don Whitley Scientific has launched a 10-plate petri dish rack made from stainless steel.

The robust, sturdy and autoclaveable racks are suitable for stacking, sorting and storing petri dishes in a Whitley Workstation.

As well as the racks, the company has developed a coloured label system, useful in organising plates and having the facility to be written on. Furthermore, if a rack is accidentally knocked over, plates secured by this system will not spill out.

Life Technologies Australia Pty Ltd Contact info and more items like this at wf.net.au/T618

The set of tags includes 20 of each of seven different colours: yellow, blue, red, green, white, orange and grey.

Don Whitley Scientific Pty Limited Contact info and more items like this at wf.net.au/U364

Fraction collector and sample pump for protein purification system GE Healthcare’s fraction collector and sample pump are specifically designed to enhance ÄKTA pure, the modular chromatography system for flexible, reliable and intuitive protein purification.

Designed to handle both air and liquid, the sample pump together with the sample inlet valve - provides fast, automated and complete loading of up to seven samples. The air sensor integrated into the valve detects the final drop of sample, automatically preventing the introduction of air whilst ensuring the complete sample reaches the column. The sample pump also has a robust wash program that removes any air introduced between samples.

In order to secure precious samples from dust and contaminants, the fraction collector is covered and supports flexible combinations of collection vessels, from deep-well plates to 250 mL bottles. In addition to spillage-free collection across the entire flow-rate range of Ă„KTA pure, the fraction collector has in-built automatic detection of the collection vessel type to ensure sample security.

GE Healthcare - Biosciences Contact info and more items like this at wf.net.au/T968

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NEW PRODUCTS @ www.labonline.com.au

Biological safety cabinet The LaboGene Mars Class II Biological Safety Cabinet is said to set new standards in operator comfort, ergonomics,

Microplate reader for antioxidant assay

power efficiency and noise. Its programmable auto start and

Everyday metabolism, as well

procedures and protocols.

auto stop during the working week helps improve laboratory

as stress and environmental

Two fully electronic adjustable EC fans and a H14 HEPA

pollutants, causes cells in the

filter mean lower fan running speeds, contributing to less

human body to produce mol-

noise (<49 dBA), heat production and energy consumption,

ecules that are collectively known

all while conforming and certified to Australian Standards.

as reactive oxygen species (ROS). ROS

The large glass sides, angled motorised front sash, large

can be either free radicals or can form free

72 cm high front and adjustable illumination offer optimal

radicals through their interaction with biological molecules (ie, proteins, DNA/RNA

user operation, viewing and supervision. ‘V’ profile air intake

and lipids). Too many free radicals are thought to play a role in cancer, ageing and

grills ensure optimal airflow, promote better user posture and

other degenerative diseases, such as cardiovascular, Alzheimer’s and Parkinson’s.

preclude the need to use obstructive armrests. All access is

A method was needed that could measure the antioxidant capacity of a sub-

via the front of the cabinet for ease of servicing and cleaning.

stance, either directly from the food or indirectly in the blood after being consumed.

The compact units will fit on most benches, with a full

The oxygen radical absorbance capacity (ORAC) assay is such a method, and

range of adjustable stands available. Other options include:

can be conducted using the PHERAstar FS microplate reader from BMG Labtech.

UV lights; heated and cooled worktops; pre-filters; built-in

It is suitable for food manufacturers wishing to market their products as high in

PC monitors; access ports; customised internal work heights

antioxidants.

and more.

BMG LabTech Pty Ltd

LabGear Australia

Contact info and more items like this at wf.net.au/U392

Contact info and more items like this at wf.net.au/U219

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NEW PRODUCTS @ www.labonline.com.au

Cell image analysis software Thermo Scientific HCS

HIV 1/2 confirmatory system

Studio Software is a suite of high-content imaging

Bio-Rad Laboratories has an-

and analysis applications

nounced the release of Geenius

offering researchers the ability to

HIV 1/2 Confirmatory for markets

easily and quickly interact with and analyse cell

outside the US.

images and corresponding data.

The product is a complete system

The software delivers a sophisticated analysis tool with built-in intelligence and enhanced usability to

that provides unitary HIV-1 and HIV-

analyse multiple parameters simultaneously. Simple-to-use tools, including icon-driven interfaces, step-

2 confirmation and differentiation

by-step workflows and a desktop navigator, enable researchers to gain more knowledge about their

in serum, plasma or whole blood

cells. The software applications are said to be simple enough for the high-content novice but powerful

along with automated reading and

enough for the advanced high-content user.

interpretation of the results - all in

It is claimed to be the first high-content software to feature an integrated, automated plate handling

less than 30 min.

capability, enabling the Thermo Scientific Orbitor RS Plate Mover to be used in conjunction with either

Key benefits include: unitary and

the Thermo Scientific ArrayScan XTI High Content Analysis (HCA) Reader or the Thermo Scientific Cel-

immediate testing; easy-to-use soft-

lInsight NXT High Content Screening (HCS) Platform. The simple and automated simple plate handling

ware that uses a validated algorithm;

capability delivers a solution for high-content users who do not need plate scheduling, but rather

full traceability.

straight-forward movement of plates from stacks to stage.

Millennium Science Pty Ltd Contact info and more items like this at wf.net.au/U030

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Bio-Rad Laboratories Pty Ltd Contact info and more items like this at wf.net.au/U070

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Stereomicroscopes Nikon Instruments has released the SMZ25 and SMZ18 stereomicroscopes. Suitable for all bioscience applications, the microscopes meet the increasing need of scientists for imaging systems capable of spanning spatial scales from single cells to whole organisms.

The stereomicroscopes features a breakthrough in zoom technology, the innovative Nikon Perfect Zoom System, which enables a large zoom ratio of 25:1, high resolution and very good fluorescence transmission capability.

Both the automated zoom model SMZ25 and more economic, manual-intelligent zoom model SMZ18, can be equipped with a variety of automated and intelligent accessories including z-focus, LED Dia light intensity control and epifluorescence filter cube selection.

Coherent Scientific Pty Ltd Contact info and more items like this at wf.net.au/U378

Hepatitis C seroconversion panels SeraCare Life Sciences has launched three Hepatitis C Accuvert seroconversion panels designed to help diagnostics manufacturers and clinical labs evaluate, monitor and troubleshoot test and staff performance. The panels provide manufacturers with material from a variety of donor genotypes and recent collection periods. Each panel demonstrates how a single donor responds in the presence of the disease during the collection period, with a graphical depiction of seroconversion. Product data sheets also offer comparative data that is valuable in reviewing test results from a number of assays.

The Hepatitis C Accuvert seroconversion panels include: HCV Seroconversion Panel PHV924, providing undiluted plasma samples from a single male donor of genotype 2b, drawn over an 88day period in 2008; HCV Seroconversion Panel PHV925, providing undiluted plasma samples from a single male donor of genotype 1a, drawn over a 27-day period in 2008; HCV Seroconversion Panel PHV926, providing undiluted plasma samples from a single male donor of genotype 3a, drawn over a 14-day period in 2009.

Abacus ALS Australia Contact info and more items like this at wf.net.au/U017

Bead-based DNA target enrichment reagent kit Roche has launched its SeqCap EZ Pure Capture Bead Kit for supporting target enrichment workflows prior to DNA sequencing. The kit is designed to provide sensitive and robust methods for the capture and purification of DNA sample libraries, enabling high-quality targeted sequencing results.

Roche provides a comprehensive set of reagents and probe pools for good target enrichment performance. The probe pools include exome libraries, gene panels and custom designs. The company has also launched a suite of SeqCap EZ reagent kits for applications in single or multiplex target enrichment experiments. With the recent addition, the set of capture reagents will allow a more streamlined target enrichment workflow and ensure good sequence capture efficiency.

Roche Diagnostic Systems Contact info and more items like this at wf.net.au/U179

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NEW PRODUCTS @ www.labonline.com.au

10 mL laboratory glass bottle Duran has produced a miniature size 10 mL bottle. This is a suitable size for the testing of products - it allows for a decrease in the quantities used in the sampling process which will then have an ongoing effect to reduce the cost of experimentation, as well as the amount of waste.

The 10 mL bottles are of the same quality as the companyâ&#x20AC;&#x2122;s existing bottles, featuring good chemical resistance, nearly inert behaviour and high usage temperature. The original Duran laboratory glass bottles are suitable for many applications in the laboratory including the storing of sensitive media and reference samples. They are also suitable for shipping small sample volumes as well as storing proteins and DNA. The bottles are easy to clean, durable and have the following advantages: glass type l/neutral in accordance with United States Pharmacopoeia/European Pharmacopoeia and Japanese Pharmacopoeia standards; single-handed opening for easy pipetting; sterilisable and autoclavable; also available in amber or with a plastic coating.

As the 10 mL bottles are part of the Duran range with an existing thread type of GL 25, the user can mix and match the caps to suit their needs. The company has a range of premium caps, PBT caps, PP screw caps and membrane caps.

Schott Australia Pty Ltd Contact info and more items like this at wf.net.au/U037

Kits for the detection of resistance mechanisms Rosco Diagnostica acknowledges the increasing need for detection of resistance mechanisms; therefore, a series of kits has been developed. The objective is to supply users with a conveniently assembled kit complete with easy-to-handle cartridges of disc diffusion tablets and a guide for usage.

The range of kits available includes: detection of KPC/MBL resistance mechanisms; detection of KPC/MBL and OXA-48 resistance mechanisms; detection of plasmid-mediated AmpC betalactamase; detection of ESBLs in isolates producing chromosomal AmpC; screening for ESBLs and AmpC; detection of plasmid-mediated ESBLs; screening for plasmid-mediated ESBLs; detection of MBL resistance mechanisms; detection of hGISA, GISA, VRE and Daptomycin susceptibility.

Dutec Diagnostics Pty Ltd Contact info and more items like this at wf.net.au/T825

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PUBLISH OR PERISH

PUBLISH OR PERISH

The return of our regular round-up of some of the best Australian research published each month in leading peer-reviewed journals.

Anwar F, Davenport MP, Ebrahimi D. UNSW Footprint of APOBEC3 on the genome of human retroelements.

J Virol 2013 May 22.

Bekkers JM, Suzuki N. JCSMR, ANU, Canberra Neurons and circuits for odor processing in the piriform cortex.

Trends Neurosci 2013 May 3

Blyth E, Clancy L, Simms R, Ma CK, Burgess J, Deo S, Byth K, Dubosq MC, Shaw PJ, Micklethwaite KP, Gottlieb DJ. Univ of Sydney Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation.

Blood 2013 May 2

Carilla-Latorre S, Annesley SJ, MuñozBraceras S, Fisher PR, Escalante R. La Trobe Univ, Vic Ndufaf5 deficiency in the Dictyostelium model: new roles in autophagy and development.

Mol Biol Cell 2013 May 24

Davis FM, Azimi I, Faville RA, Peters AA, Jalink K, Putney JW Jr, Goodhill GJ, Thompson EW, Roberts-Thomson SJ, Monteith GR. Univ of Qld, Brisbane Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent.

Oncogene 2013 May 20

Engwerda CR, Kumar R. QIMR, Brisbane Mast cells fuel the fire of malaria immunopathology.

Nat Med 2013 Jun

Godler DE, Inaba Y, Shi EZ, Skinner C, Bui QM, Francis D, Amor DJ, Hopper JL, Loesch DZ, Hagerman RJ, Schwartz CE, Slater HR. Murdoch Child Res Inst, Royal Children’s Hosp, Vic Relationships between age and epi-genotype of the FMR1 exon 1/intron 1 boundary are consistent with non-random X-chromosome inactivation in FM individuals, with the selection for the unmethylated state being most significant between birth and puberty.

Hum Mol Genet 2013 Apr 15

Godwin JW, Pinto AR, Rosenthal NA. Monash Univ, Vic Macrophages are required for adult salamander limb regeneration.

Proc Natl Acad Sci USA 2013 Jun 4

Grkovic S, O’Reilly VC, Han S, Hong M, Baxter RC, Firth SM. Kolling Inst of Med Res, NSW IGFBP-3 binds GRP78, stimulates autophagy and promotes the survival of breast cancer cells exposed to adverse microenvironments.

Oncogene 2013 May 9

Henderson LA, Peck CC, Petersen ET, Rae CD, Youssef AM, Reeves JM, Wilcox SL, Akhter R, Murray GM, Gustin SM. Univ of Sydney Chronic pain: lost inhibition?

J Neurosci 2013 Apr 24

Holliday EG. Hunter Med Res Inst and Univ of Newcastle, NSW Hints of unique genetic effects for type 2 diabetes in India.

Diabetes 2013 May

Jackson CJ, Liu JW, Carr PD, Younus F, Coppin C, Meirelles T, Lethier M, Pandey G, Ollis DL, Russell RJ, Weik M, Oakeshott JG. ANU, Canberra Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance.

Proc Natl Acad Sci USA 2013 Jun 18

Jergic S, Horan NP, Elshenawy MM, Mason CE, Urathamakul T, Ozawa K, Robinson A, Goudsmits JM, Wang Y, Pan X, Beck JL, van Oijen AM, Huber T, Hamdan SM, Dixon NE. Univ of Wollongong, NSW A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

EMBO J 2013 May 2

Lee P, Swarbrick MM, Ho KK. Univ of Qld, Princess Alex Hosp, Brisbane Brown adipose tissue in adult humans: a metabolic renaissance.

Endocr Rev 2013 Jun

Levesque JP, Winkler IG. Mater Res & Univ of Qld It takes nerves to recover from chemotherapy.

Nat Med 2013 Jun

Lott WB, Doran MR. QUT, Brisbane Do RNA viruses require genome cyclisation for replication?

Trends Biochem Sci 2013 Jun 12

Metcalf D, Ng AP, Baldwin TM, Di Rago L, Mifsud S. WEHI, Vic Concordant mast cell and basophil production by individual hematopoietic blast colony-forming cells.

Proc Natl Acad Sci USA 2013 May 28

Rea SL, Walsh JP, Layfield R, Ratajczak T, Xu J. WAIMR, Univ of WA and Sir Charles Gairdner Hosp, Perth New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of Paget’s disease of bone.

Endocr Rev 2013 Apr 23

Regev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X, Coleman BM, Rug M, Bursac D, Angrisano F, Gee M, Hill AF, Baum J, Cowman AF. WEHI and Univ of Melb Cell-Cell Communication between MalariaInfected Red Blood Cells via Exosome-like Vesicles.

Cell 2013 May 23

Rist MJ, Theodossis A, Croft NP, Neller MA, Welland A, Chen Z, Sullivan LC, Burrows JM, Miles JJ, Brennan RM, Gras S, Khanna R, Brooks AG, McCluskey J, Purcell AW, Rossjohn J, Burrows SR. QIMR, Brisbane HLA peptide length preferences control CD8+ T cell responses.

J Immunol 2013 Jun 7

Sparrow DB, McInerney-Leo A, Gucev ZS, Gardiner B, Marshall M, Leo PJ, Chapman DL, Tasic V, Shishko A, Brown MA, Duncan EL, Dunwoodie SL. Victor Chang Cardiac Res Inst, Sydney Autosomal dominant spondylocostal dysostosis is caused by mutation in TBX6.

Hum Mol Genet 2013 Apr 15

Trinajstic K, Sanchez S, Dupret V, Tafforeau P, Long J, Young G, Senden T, Boisvert C, Power N, Ahlberg PE. Curtin Univ, Perth Fossil musculature of the most primitive jawed vertebrates.

Science 2013 Jun 13

Wikstrom ME, Fleming P, Comerford I, McColl SR, Andoniou CE, Degli-Esposti MA. Univ of WA A chemokine-like viral protein enhances IFN-α production by plasmacytoid dendritic cells but delays CD8+ T cell activation and impairs viral clearance.

J Virol 2013 May 8

Nguyen LS, Kim HG, Rosenfeld JA, Shen Y, Gusella JF, Lacassie Y, Layman LC, Shaffer LG, Gécz J. Univ of Adelaide, SA Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders.

Wu MC, Brennan FH, Lynch JP, Mantovani S, Phipps S, Wetsel RA, Ruitenberg MJ, Taylor SM, Woodruff TM. Univ of Qld, Brisbane The receptor for complement component C3a mediates protection from intestinal ischemiareperfusion injuries by inhibiting neutrophil mobilization.

Hum Mol Genet 2013 May 1

Proc Natl Acad Sci USA 2013 Jun 4

Pera M, Trounson A. Univ of Melb Cloning debate: Stem-cell researchers must stay engaged.

Wyatt AR, Yerbury JJ, Ecroyd H, Wilson MR. Univ of Wollongong, NSW Extracellular chaperones and proteostasis.

Nature 2013 Jun 13

Annu Rev Biochem 2013 Jun 2

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EVENTS

DATES FOR THE LIFE SCIENCES CALENDAR © iStockphoto.com/spanteldotru

The coming year is packed with exciting local and international events. Here’s a taste.

Australian Society for Infectious Diseases Gram-Negative ‘Superbugs’ Meeting

August 2-3, Gold Coast The two-day Gram-negative ‘Superbugs’ Meeting, hosted by the Australasian Society for Infectious Diseases, will bring together clinicians, microbiologists, pharmacists, infection control nurses and researchers from Asia, Australia and New Zealand to provide updates on the latest developments in the issues around gram-negative superbugs. This new group of superbugs, which is on the rise, is particularly dangerous because they develop resistance very quickly. International and local speakers will review novel therapeutic approaches, the importance of the microbiology laboratory, dosing optimisation and the use of combination therapy and innovative approaches to keep pace with drugresistance and maintain infection control.

www.asid.net.au/gramnegative

International Marine Biotechnology Conference November 11-15, Brisbane

www.imbc2013australia.com/

Laboratory Management & Design Conference November 18-20, Brisbane

www.labmanagers.org.au/

HPLC 2013 - 40th International Symposium on High-Performance-Liquid-Phase Separations and Related Techniques November 18-21, Hobart

www.hplc2013-hobart.org Research Australia: Philanthropy in Health & Medical Research August 20–21, Sydney

www.researchaustralia.org/events/category/ conference Familial Aspects of Cancer Meeting August 25-28, Cairns

www.meeting-makers.com/fac

International Society for Gastrointestinal and Hereditary Tumours August 28-31, Cairns

www.insight-group.org

Tech Transfer Summit Australia 2013 September 3-4, Melbourne

www.ausbiotech.org

Joint Meeting of the Australian Vascular Biology Society and the Australia and New Zealand Microcirculation Society September 5-8, Barossa Valley

www.avbs.org/?

ComBio2013 September 29-October 3, Perth

www.asbmb.org.au

Australasian College for Infection Prevention and Control Conference September 30-October 2, Gold Coast

www.acipcconference.com.au

2nd Annual NHMRC Research Translation Faculty Symposium - from Bench to Bourke: improving practice, policy and commercialisation October 2-3, Sydney

www.nhmrc.gov.au/media/ events/2013/2nd-annual-nhmrc-symposiumresearch-translation 42

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Annual Bioprocessing Network Conference October 22-24, Gold Coast

www.bioprocessingnetwork.com.au/ Conferences.html

The Australasian Bioenergy and Bioproducts Symposium 2013 October 25, Brisbane

www.tabbs.com.au

5th Asia-Pacific NMR Symposium and 9th Australian & New Zealand Society for Magnetic Resonance October 27-30, Brisbane

http://apnmr2013.org

15th World Conference on Lung Cancer October 27-31, Sydney

www.2013worldlungcancer.org Australia Biotech Invest October 28-29, Melbourne

http://ausbiotechinvest.com/ Association of Biosafety for Australia and New Zealand 3rd Annual Conference October 29-November 1, Auckland, New Zealand

www.absanz.org.au/Conference 2013.html AusBiotech 2013 October 29-November 1, Brisbane

www.ausbiotech.org

9th World Sponge Conference November 4-8, Freemantle, Western Australia

www.aims.gov.au/web/sponge/home

Australian Society for Biophysics Annual Meeting November 24-27, Melbourne

www.biophysics.org.au/Meetings/2013/ index.html

International Symposium on Computational Models for Life Sciences November 27-29, Sydney

http://cmls-conf.org/2013/

Australasian Flow Cytometry Group 36th Annual Meeting November 28-30, Wellington, New Zealand

www.malaghan.org.nz/afcg2013

5th Congress of the International Society for Applied Phycology 2014 June 22-27, 2014, Sydney

www.isap2014.com

2014 International Biophysics Congress August 3-7, 2014, Brisbane

www.iupab2014.org

15th International Conference on Systems Biology September 13-19, 2014, Melbourne

www.emblaustralia.org

ComBio2014 September 28-October 2, 2014, Canberra

www.asbmb.org.au

AusBiotech 2014 October 28-31, 2014, Gold Coast

www.ausbiotech.org

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