Bioscience Today 10

SCIENCETODAY

BIO

MAYJUNE2017

hope as research unlocks secrets of heart disease cell line development • alzheimers • three parent babies • drug delivery • PDRN • pancreatic cancer cells

cover story:

cardiovascular disease

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| welcome |

Welcome

Tackling the effects of an older population John Dean

Editor in chief

Editor John Dean john.dean@distinctivepublishing.co.uk

Design Distinctive Publishing, Unit 6b, Floor B, Milburn House, Dean Street, Newcastle Upon Tyne NE1 1LE Tel: 0845 884 2385 www.distinctivepublishing.co.uk

Contributors John Dean & Francis Griss john.dean@distinctivepublishing.co.uk

Welcome to the latest edition of the Bioscience Today magazine and we are delighted that former UK Prime Minister David Cameron has written a piece outlining his support for dementia research.

Every so often an illness emerges that affects us all in some way. In days gone by, it was the likes of polio and tuberculosis, more recently it has been cancer.

Today, the big emerging concern is dementia and there is an increasing realisation that we need to get to grips with it and do so quickly.

David Cameron agrees, which is why he has become the new President of Alzheimer’s Research UK, the UK’s leading dementia research charity.

Mr Cameron made dementia a focus of his time in office, launching the Prime Minister’s Dementia Challenge in 2012 and starting a drive to deliver improvements in care and research.

Now he is continuing his work with his support for the charity.

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The growth in dementia is a direct result of us living longer and it casts a long shadow. Finding ways of halting its progress are to be welcomed.

Distinctive Publishing, Unit 6b, Floor B, Milburn House, Dean Street, Newcastle Upon Tyne NE1 1LE Tel: 07813 874 970 email: john.neilson@distinctivegroup.co.uk www.distinctivepublishing.co.uk

Having seen what dementia did to my father in his final years, slowly stripping him of his dignity, I can only applaud the initiatives under way to unravel the mysteries of this cruel condition.

The edition also looks at work to tackle cardiovascular disease, another one of those conditions that kills millions and touches just about every family in some way.

Distinctive Publishing or BioScience Today cannot be held responsible for any inaccuracies that may occur, individual products or services advertised or late entries. No part of this publication may be reproduced or scanned without prior written permission of the publishers and BioScience Today.

Cardiovascular disease is a leading cause of death and disability worldwide and in an effort to turn the tide the research community in the UK is highly focused on finding new treatments.

One of the organisations working hard is the National Institute for Health Research Clinical Research Network.

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An example of such research is the International Study of Comparative Health Effectiveness with Medical and Invasive Approaches trial, known as the ISCHEMIA trial.

Every year tens of thousands of people in the UK with stable angina have one or more stents inserted in a blood vessel to aid healing or relieve an obstruction in their arteries.

However, except in emergency cases, there is no evidence that treating angina by inserting stents, followed by medical and lifestyle interventions, is better than medicine and lifestyle changes alone.

Now a research team at Northwick Park Hospital, part of the London North West Healthcare NHS Trust, has been leading the way in finding out which treatment is better and safer. We examine the work in this edition of the magazine.

We also examine developments in cell line technology and improvements in drug delivery, all targeted towards the emerging idea that medical treatment is not a one-size-fits-all process but one that has to be much more targeted.

A drug that works on one patient may be less effective on another and the move towards more targeted health therapies seeks to take advantage of the fact.

One common theme links all these stories and dominate medical thinking. Helping us live longer is all very well but not if those later years are of poor quality.

Creaking limbs are one thing, an old age stalked by serious illness is another and we should applaud all those scientists who are playing their part to ensure that we get through our twilight years in decent shape.

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Anti inflammatory property of PDRN - An in Vitro study cultured macrophages

Features

22 34 Hope as research unlocks the secret of heart disease

‘Three-parent babies’ a step closer after regulators grant approval

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Contents 3

Introduction/Foreword

4-5

Contents

6-7

Biodigestables

8-14

UK News

16-19

World News

22-23

World News

24-29

Cardivascular Disease

30-31

Cell Line Development

32-33

Alzheimers

34-37

Three Parent Babies

38-40

Drug Delivery

42-47

48 5

48-54

Food Poisining risks identified

Research leads to ban

Grants are awarded

Hope as research unlocks the secret of heart disease

Cell line technology provides optimism for researchers

Former Prime Minister takes on key dementia research role

Three-parent babies a step closer after regulators grant approval

Drug delivery market witnesses significant growth

PDRN

Anti Inflammatory property of PDRN - An in vitro study cultured macrophages

Pancreatic Cancer Cells

Evaluation of pancreatic cancer cell migration with multiple parameters in vitro by using an optical real-time cell mobility assay device Evaluation of pancreatic cancer cell migration with multiple parameters in vitro by using an optical real-time cell mobility assay device

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BIODIGESTABLES

Marker could help cancer Taking a collaborative treatment approach

Research programme is launched

Researchers have discovered that a marker found on aggressive prostate cancer cells could also be used as a way to guide treatments to the cancer.

A new bioeconomy research programme has been announced by Colombia and the UK.

The molecule, called NAALADL2, is already measured to see if prostate cancer is likely to return but the new study has shown that it can also help direct treatment to the cancer.

The team, based at UCL, had already found that prostate cancer cells have more of the NAALADL2 molecule on their surface compared to cells from healthy tissue.

Supplements backed Taking omega-3 supplements could improve muscle function in older women, potentially increasing their quality of life into old age and preventing unnecessary falls and loss of independence, according to researchers.

In a study led by the Universities of Glasgow and Aberdeen, scientists found that supplementing the diet of older women with 3g of fish oil results in greater increases, when compared to a 3g of safflower oil placebo, in their muscle function when combined with resistance exercise training over 18 weeks.

A partnership between Liverpool University and NHS trust the Royal Liverpool and Broadgreen and Alder hase secured £3.4m for translational medicine research.

Translational medicine is a rapidly growing discipline in biomedical research and aims to accelerate the discovery of new diagnostic tools and treatments by taking a collaborative approach.

The funding, provided by the National Institute for Health Research (NIHR), will support the infrastructure costs for early translational research.

Researchers have discovered that blocking a molecule could bypass bowel cancer’s defence against the drug cetuximab.

Cetuximab is used to treat advanced bowel cancer and just under half of patients are given the drug.

While it helps many patients, there are some for whom it doesn’t work and for others it loses effectiveness.

Scientists at Queen’s University Belfast, who treated bowel cancer cells in the lab with cetuximab, found .that some cells survived the treatment by increasing the activity of the protein ADAM17. If they gave a drug that blocked ADAM17 at the same time as cetuximab, the cancer cells died.

Signalling mechanism identifed

Arthritis research partnership

Researchers from the European Cancer Stem Cell Research Institute have identified a new signalling mechanism responsible for clearing damaged cells from healthy epithelial tissues, which line the surfaces and cavities of organs).

A new partnership has been formed between the Universities of Oxford and Birmingham to speed up the development of treatments for arthritis, supported by a £7 million investment from the Kennedy Trust for Rheumatology Research.

The partnership will be based at the Kennedy Institute of Rheumatology at Oxford and the Institute of Translational Medicine in Birmingham.

Prior research has shown that epithelial cells carrying oncogenic Ras mutations (RasV12 cells) are detected by ‘normal’ cells. As a result, the mutant cells are often expelled from the epithelial tissues, potentially preventing cancer initiation.

How fish resist pollution Molecule breakthrough

The £20 million Colombia Bio programme, which builds on the strong relationship between the countries, has been established to develop new drugs, medicines, biofertilisers and pollutiontackling products. The programme is supported through The Newton-Caldas fund.

However, the way in which normal cells identify the mutant cells and trigger the elimination process has remained elusive. The new research, led by Dr Catherine Hogan, identified differential EphA2 signalling as the mechanism which underlies the process. The research was funded

by Cardiff University and Amser Justin Time.

A report has identified the genetic mechanism responsible for evolutionary adaptation to toxic pollution observed in wild Atlantic killifish populations.

Drug receives approval

The Atlantic killifish is renowned for its ability to tolerate large fluctuations in temperature, salinity and oxygen levels.

French-American company Sanofi has received U.S. Food and Drug Administration approval for Soliqua for the treatment of adults with type 2 diabetes.

A team of research institutions including the University of Birmingham analysed the genomes of four wild populations compared with four nontolerant populations, to identify the mechanism behind the adaptation. They found that the genes responsible for the trait were those involved in the aryl hydrocarbon receptor (AHR) signalling pathway, which combined with observations of desensitisation of this pathway in tolerant populations, led them to conclude that the AHR pathway is a key target of natural selection.

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The decision followed a trial which showed an encouraging drop in blood sugar levels within 55% of the 736 patients.

Elias Zerhouni, M.D., President, Global R&D, Sanofi, said: “Sanofi continues to be a pioneer in developing diabetes therapies and in bringing forward new treatment options for the approximately 50 percent of patients whose blood sugar levels remain uncontrolled on daily basal insulin.”

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BIODIGESTABLES

Centres to be established Grant awarded

Collaboration launched

The University of Queensland in Australia has secured $13.95 million over five years from the National Health and Medical Research Council (NHMRC) to help tackle some of the world’s biggest health challenges.

A new collaboration between Cohen Veterans Bioscience and Stanford University School of Medicine in the United States will investigate clinical biomarkers as a way to match post-traumatic stress disorder patients to the best treatment.

Drug use ‘could be halved’ Patients with a chronic type of leukaemia could safely reduce the side-effects of life-long treatment by cutting their dose, according to a University of Liverpool-led study.

Drugs known as tyrosine kinase inhibitors (TKI) have drastically improved the prognosis for patients with chronic myeloid leukaemia (CML), essentially turning what was a deadly cancer into a chronic disease that can be managed with a daily pill. However, their side effects and cost have led some patients and their doctors to question whether it would be feasible to discontinue use after a patient achieves consistently-negative leukaemia test results.

The new study suggest that many CML patients may be able to safely reduce TKI side effects by cutting their dose in half.

Mark Throsby, Chief Scientific Officer of Merus, said: “We believe a clinically successful Biclonics® that effectively modulates tumor immunity has the potential to significantly improve the treatment paradigm in lung cancer and other solid tumor indications.”

New plant Italian company Bio-is to begin producing its own biopolymers at a new plant to be completed this year with an expected investment of €15 million Euro.

Entering the special bioplastics production sector adds to the Milan company’s existing research and development of 100% biodegradable biopolymers and the production licensing which it has developed since its foundation in 2007.

The PHAs bioplastics developed by Bio-on are made from renewable plant sources such as sugar beet and sugar cane production waste.

Cancer research begins

Anti-microbial programme is launched

American company Riptide Bioscience has announced a research agreement with the National Cancer Institute to investigate how Riptide proprietary Peptide RP-182 can treat pancreatic cancer.

A £13 million programme to help combat antimicrobial resistance has been launched imvolving the UK Research Councils and India’s Department for Biotechnology (DBT).

Pancreatic cancer is among the most lethal of all diseases; 90% of patients die within one year of diagnosis. Even following surgical removal of the primary tumor, long-term survival rates are poor, with tumours recurring in virtually all patients.

The announcement of the Newton Bhabha programme was made by Jo Johnson, Minister of State for Universities, Science, Research and Innovation, during a visit to New Delhi to mark a £13 million collaborative investment in research between the UK and India.

Riptide believes that RP-182 will prove to be complementary to both conventional chemotherapies and emerging immunotherapies.

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Amit Etkin, MD, PhD, Associate Professor in the Department of Psychiatry and Behavioral Sciences at Stanford University and at the Palo Alto VA, and colleagues, supported by a grant from Cohen Veterans Bioscience, will look at 160 veterans who are receiving treatment in the Mental Health Clinic at the Palo Alto VA and Albuquerque VA. Patients will be evaluated before treatment to measure a biomarker that predicts whether or not that individual will respond to therapy.

Vaccine to be rolled out The World Health Organisation has pre-qualified Pfizer’s new pneumococcal vaccine Prevenar 13® vaccine, based on results of a study conducted by the Medical Research Council in Gambia.

Pneumococcal disease is one of the biggest preventable killers of children and the majority of deaths occur in developing countries. The WHO decision allows for the global roll-out of the vaccine, by United Nations agencies.

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Send them to our Editor, John Dean, at john.dean@distinctivepublishing.co.uk

BIO

SCIENCETODAY

Almost $5 million will be used to establish two NHMRC Centres for Research Excellence, one with a focus on cerebral palsy and the other on reducing stillbirths.

Merus N.V. has received a €1.5 million EUREKA Eurostars Grant with Aquila BioMedical Ltd to jointly develop immunological assays for identification of immunomodulatory bispecific antibodies which can be used in the treatment of cancer.

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Food poisoning risks identified Bioscientists have revealed that just a small amount of damage could massively stimulate the presence of the food poisoning bug Salmonella in ready-prepared salad leaves.

Microbiologists from the University of Leicester have discovered that juices released from damaged leaves also had the effect of enhancing the virulence of the pathogen, potentially increasing its ability to cause infection in the consumer.

The research was led by Dr Primrose Freestone of the university’s Department of Infection, Immunity and Inflammation and PhD student Giannis Koukkidis, who has been funded by a Biotechnology and Biological Sciences Research Council (BBSRC) i-case Studentship.

They investigated methods of preventing food poisoning pathogens from attaching to the surface of salad leaves to help producers improve food safety. The findings suggested that juices from damaged leaves in bagged spinach and mixed salad increased Salmonella pathogen growth 2,400fold over a control group.

Dr Freestone said: “Salad leaves are cut during harvesting and we found that even microliters of the juices (less than 1/200th of a teaspoon) which leach from the cut-ends of the leaves enabled Salmonella to grow in water, even when it was refrigerated. These juices also helped the Salmonella to attach itself to the salad leaves so strongly that vigorous washing could not remove the bacteria and even enabled the pathogen to attach to the salad bag container.

“This strongly emphasises the need for salad leaf growers to maintain high food safety standards as even a few Salmonella cells in a salad bag at the time of purchase could be become many thousands by the time a bag of salad leaves reaches its use by date, even if kept refrigerated. Even small traces of juices released from damaged leaves can make the pathogen grow better and become more able to cause disease.

“It also serves as a reminder to consume a bagged salad as soon as possible after it is opened. We found that once opened, the bacteria naturally on the leaves also grew much faster even when kept cold in the fridge.”

The research did not look for evidence of salmonella in bagged salads. Instead, it examined how Salmonella grows on salad leaves when they are damaged.

Giannis Koukkidis said: “Anything which enhances adherence of food-borne pathogens to leaf surfaces also increases their persistence and ability to resist removal, such as during salad washing procedures. Even more worrying for those who might eat a Salmonella-contaminated salad was the finding that proteins required for the virulence of the bacteria were increased when the Salmonella came into contact with the salad leaf juices.

“Preventing enteric pathogen contamination of fresh salad produce would not only reassure consumers but will also benefit the economy due to fewer days lost through food poisoning. We are now working hard to find ways of preventing salad-based infections.”

Professor Melanie Welham, Chief Executive, BBSRC, said: “Food-borne pathogens like Salmonella are serious bacterial threats that affect our health which is why BBSRC invests in research to understand and combat food poisoning.”

Research published recently by the Food Standards Agency reported that annually there are more than 500,000 cases of food poisoning in the UK. While poultry meat was the most common source of infection, 48,000 of food poisoning cases were from fresh produce: vegetables, fruit, nuts and sprouting seeds. Salmonella was the pathogen that caused the greatest number of hospital admissions – about 2,500 per year.

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THE FINDINGS SUGGESTED THAT JUICES FROM DAMAGED LEAVES IN BAGGED SPINACH AND MIXED SALAD INCREASED SALMONELLA PATHOGEN GROWTH 2,400-FOLD OVER A CONTROL GROUP.

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Centre is opened The Queen and The Duke of Edinburgh, accompanied by The Duke of York, have opened the £650 million Francis Crick Institute in London.

The Crick is the biggest biomedical research institute under one roof in Europe and is investigating the fundamental biology underlying human health and disease.

During a tour of the new facility, The Queen started the sequencing of institute director Sir Paul Nurse’s genome – all three billion letters in his DNA code.

The Crick is bringing scientists together from across disciplines to tackle the pressing health concerns of the 21st Century. It will be home to 1,250 scientists and a further 250 support staff at full capacity in 2017.

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Its founding partners are the Medical Research Council, Cancer Research UK, Wellcome, UCL (University College London), Imperial College London and King’s College London.

During their tour of the building, which is next door to St Pancras and the British Library, the royal party saw some of the facilities for research, including the advanced sequencing and peptide chemistry laboratories.

Construction of the new building for the Crick was completed in August 2016.

As a world-leading centre of biomedical research, the Crick’s aim is to find new ways to prevent, diagnose and treat conditions such as cancer, heart disease and stroke, infections and neurodegenerative conditions like motor neurone disease.

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Team targets personalised cancer treatment A new personalised breast cancer programme has been launched to map patients’ DNA and RNA to tailor treatment.

The Cancer Research UK project, which was launched with £1.1 million from Addenbrooke’s Charitable Trust, (ACT), will analyse the genome and all expressed genes of tumour cells from 250 breast cancer patients.

Researchers say that finding out what genes have become faulty in breast cancer cells will help them better understand more about how cancer develops and spreads.

Breast cancer patients are treated based on the broad types of cancer, for example, those that are likely to respond to hormone therapies, but it can be difficult to predict how individual patients will respond to treatment.

The researchers hope to find out how personalised diagnosis and treatment programme could be implemented in the National Health Service’s (NHS) breast cancer unit in Cambridge and hope that one day this will extend around the UK. Professor Richard Gilbertson, director of the Cancer Research UK Major Cancer Centre at Cambridge University, said: “The Personalised Breast Cancer Project is truly ground-breaking.

“By sequencing the entire tumour genome of women with breast cancer in our clinic and integrating this extensive data with other biological and clinical observations, we will assign patients to optimal therapy, changing the way we treat breast cancer forever.”

Professor Carlos Caldas, project lead at the Cancer Research UK Cambridge Institute, said: “We already know that there are about ten different types of breast cancer and these respond differently to the available treatments.

“We’re looking at ways to predict this response, ensuring individual patients get the best treatment for them. We hope that this project will accelerate progress in developing personalised treatment for breast cancer patients.”

Sir Harpal Kumar, Cancer Research UK’s chief executive officer, said: “Today eight in ten women with breast cancer survive their disease for at least ten years. The ability to tailor treatment to individual patients will help ensure this number continues to rise and should help reduce

side effects. This project will bring us closer to making personalised medicine a reality in the NHS and beyond.”

• A combination of two drugs could help some breast cancer patients with advanced cancer live longer, according to a small clinical trial.

The treatment delayed the time it took for the disease to get worse in women with advanced breast cancer compared to those given just one of the drugs, according to the results presented at the San Antonio Breast Cancer Symposium in the US. Dr Noah Kornblum, from Albert Einstein College of Medicine in the US and one of the researchers behind the study, urged caution until larger studies confirm the results but said the combination could offer an alternative treatment approach for some women with breast cancer in the future.

The study looked at 130 postmenopausal women who had a type of breast cancer called ‘HR-positive’ and had advanced disease, meaning their cancer had spread and stopped responding to a type of hormone treatment called an aromatase inhibitor.

In the trial, the researchers tested a different hormone therapy – called fulvestrant (Faslodex) – either with a placebo or in combination with another cancer drug called everolimus (Afinitor).

The researchers reported that, on average, it took twice as long for tumours to start growing again in those who were given the combination of cancer drugs, compared to those given fulvestrant with a placebo. Women given both drugs also experienced more side effects than those given just one.

Professor Arnie Purushotham,, Cancer Research UK’s senior clinical adviser, said: “Finding a group of breast cancer patients who may benefit from everolimus is promising but the study was in only 100 patients so larger trials are needed to confirm the findings. We need new treatments for women with this form of breast cancer and, if larger trials are successful, we’ll need to find ways to reduce the drug’s side-effects.”

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IN THE TRIAL, THE RESEARCHERS TESTED A DIFFERENT HORMONE THERAPY – CALLED FULVESTRANT (FASLODEX) – EITHER WITH A PLACEBO OR IN COMBINATION WITH ANOTHER CANCER DRUG CALLED EVEROLIMUS (AFINITOR).

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Research funding is agreed A total of £112 million has been agreed to support clinical research and trials.

The money is being given to 23 NHS organisations and will pay for specialist research nurses and technical staff, as well as providing facilities to support clinical research and trials.

Awarded by the National Institute for Health Research (NIHR), the funding will be provided over the next five years.

Minister for Public Health and Innovation Nicola Blackwood said: “UK researchers lead the world and our investment in this area so far has led to a variety of breakthroughs, including the first new asthma treatment in a decade and a promising treatment for peanut allergies in children.

“We know that such groundbreaking research simply would not happen without the support of these clinical research facilities.”

Previous funding for clinical research facilities has led to medical breakthroughs, including: • identifying an effective treatment for peanut allergies in children • trialling of the ‘bionic eye’ in retinitis pigmentosa (RP) –

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the first ever study to combine artificial and natural vision in humans • establishing an innovative and standardised approach to test treatment for cystic fibrosis • developing the first new therapeutic asthma treatment for a decade, reducing the severity and duration of lifethreatening asthma attacks.

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Research centres are confirmed Two Cambridge institutes have been confirmed as major research centres by biomedical research charity Wellcome.

The Wellcome/CRUK Gurdon Institute and Wellcome/MRC Cambridge Stem Cell Institute have been named as two of 14 Wellcome Centres which aim to advance understanding of health and disease by bringing together fundamental and social sciences, clinical research and engineering.

Stem cells give rise to the multitude of cell types that make up our bodies, and their dysfunction underlies numerous diseases including many current global health challenges.

Stem cells also provide unique tools for modelling disease and for generating novel cell-based therapies.

They will be co-funded by Cancer Research UK (CRUK) and the Medical Research Council (MRC) respectively.

In 2018, the researchers will come together in a new purpose-built building embedded within the Cambridge Biomedical Campus, near other research institutes and adjacent to Addenbrooke’s and Papworth hospitals.

One of them is the Gurdon Institute, a world-leading centre for research bringing together developmental biology and cancer biology, using model systems ranging from yeast to human organoids.

Across the Institute’s 25-year history the research has led to major insights into the molecular and cellular defects that give rise to cancer and other diseases of ageing, and several findings have been translated into drug discovery through spin-out companies.

Professor Daniel St Johnston, Director of the Wellcome/ CRUK Gurdon Institute, said: “We are delighted that the Wellcome Trust and Cancer Research UK have decided to renew the Centre funding for the Gurdon Institute, which will allow us to continue our ground-breaking research on the links between developmental biology and cancer.”

Also being supported is the Stem Cell Institute, which was established in 2012 and is a world-leading centre for stem cell research.

Professor Tony Green, Director of the Wellcome/MRC Cambridge Stem Cell Institute, said: “Stem cell research offers unrivalled opportunities for developing new approaches to the management of disease and I am delighted that both the Wellcome Trust and the Medical Research Council will continue to support our pioneering research at this exciting time.”

Wellcome’s Director, Dr Jeremy Farrar, said: “By creating places where researchers can flourish we can catalyse world-leading research and translation, and amplify its influence and impact.

“At Wellcome we believe in long term support for discoverydriven science, and Wellcome Centres are an outstanding environment for researchers to further our understanding of fundamental biology, accelerate translation to clinical practice, and explore the social and cultural context of medicine.”

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“BY CREATING PLACES WHERE RESEARCHERS CAN FLOURISH WE CAN CATALYSE WORLDLEADING RESEARCH AND TRANSLATION, AND AMPLIFY ITS INFLUENCE AND IMPACT.”

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CRC celebrates 50 years in cleanroom design and build Clean Room Construction is celebrating 50 years of cleanroom design and build success in 2017.

The Kent-based company has grown to become one of the UK’s leading cleanroom design and build specialists for blue chip clients right across the science and technology sectors.

Managing Director Steve Lawton said: “Our company’s success over the last 50 years has been built on the experience and expertise we have under one roof which makes us the first port of call for many companies needing cleanroom facilities. Experience really does matter when you’re designing and building highly complex and critical

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facilities for world-class universities and hospitals, leadingedge technology companies and major manufacturing, healthcare and pharmaceutical clients.

“As we celebrate 50 years in business we pledge to continue to invest and innovate to ensure we are in the best possible position to engineer first class solutions which enable our clients to achieve their project goals.

“We would also like to thank everyone we have worked with over the decades and look forward to building on those partnerships and establishing new relationships over the next 50 years.” www.crc-ltd.co.uk

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Researchers identify way to protect wheat

A mechanism has been identified which could lead to healthier wheat crops worldwide.

A team at UK-based Rothamsted Research set out to find out why some plants develop resistance to Septoria leaf blotch, a highly damaging disease in wheat.

Most studies have looked directly at the interaction between wheat and Septoria but scientists at Rothamsted Research, funded by the BBSRC, looked at how plant species that do not get infected by Septoria achieve their resistance.

Using tobacco plants and advanced molecular techniques, the scientists identified several genes linked to protection against infection.

They found that genes for many of the secreted proteins,

called ‘effectors’, used by Septoria to manipulate defences of its host plant wheat, were also expressed when the fungus was inoculated onto the tobacco. When the fungal effectors were expressed in tobacco leaves, they were recognised by the plant, stimulating defence reactions.

Gene-silencing methods allowed the researchers to demonstrate that two tobacco genes in particular were involved in triggering plant responses following recognition of effectors secreted by the pathogen.

Dr Kostya Kanyuka, a senior scientist at Rothamsted Research and co-author of the paper, said: “This interesting finding suggests that non-host plants may possess specific cell surface immune receptors recognising Septoria effectors and this opens up avenues for the development of new methods to protect susceptible wheat plants from Septoria.”

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National recognition for bio-refinery company A collaborative bio-refinery project, TeeGene Biotech, has received national recognition from a prestigious funding body.

The Teesside University spin-out focuses on the viability of removing phosphate contamination from waste water while producing viable quantities of high value lipids and biochar using a suitable microalgae strain. It has been selected as a case study by the Biotechnology and Biological Sciences Research Council (BBSRC).

BBSRC, which is funded by the Government’s Department for Business, Energy and Industrial Strategy (BEIS), is one of seven Research Councils that work together as Research Councils UK (RCUK).

NATIONAL HORIZONS CENTRE (NHC) Plans for the Darlington-based National Horizons Centre (NHC) are well underway, with an architect-led design team now in place.

The state-of-the-art centre for skills, leadership and innovation in emerging technology sectors is scheduled to open its doors in March 2019, with significant support from the Local Growth Fund.

Led and shaped by industry demand, the NHC will play a key role in developing the industries set to transform the UK economy, including biologics, industrial biotechnology and other advanced manufacturing sectors.

In 2015/16 it invested £473m in world-class bioscience, people and research infrastructure, and it currently supports around 1,600 scientists and 2,000 research students in universities and institutes across the UK. TeeGene’s project, led by Teesside University academics, was funded by a Business Interaction grant awarded through the High Value Chemicals from Plants Network, a BBSRC-funded Network in Industrial Biotechnology and Bioenergy, coordinated by the University of York and John Innes Centre.

It has established the viability of an integrated biorefinery based on a hydrothermal enabling technology.

TeeGene’s director Dr Pattanathu Rahman, based in the University’s School of Science & Engineering, said: ‘Microalgae are a promising source of biofuel, high value chemicals and nutraceuticals but there are major technological challenges which currently limit the extent to which they can be used. Our research sought to overcome these challenges.

‘We are delighted and very proud that our work has been highlighted as a case study.’

‘BBSRC has identified industrial biotechnology and bioenergy as high-level priority areas in its Strategic Plan 2016-2020. The support from BBSRC plays a major role in the development of next generation biological systems and novel bio-processes towards improved manufacturing in industries.

A COLLABORATIVE BIO-REFINERY PROJECT, TEEGENE BIOTECH, HAS RECEIVED NATIONAL RECOGNITION FROM A PRESTIGIOUS FUNDING BODY.

“Our long term aspiration is to integrate TeeGene’s core technologies and business aspirations with the hydrothermal processing expertise at Teesside University to create a central enabling technology for bio-refinery development in the North East of England.”

NEW LABORATORY SCIENTIST DEGREE APPRENTICESHIP Teesside University’s Laboratory Scientist Degree Apprenticeship began with its first cohort of students in September 2016, in response to the skills demands of two large North East employers, the Centre for Process Innovation (CPI) and Fujifilm Diosynth Biotechnologies.

The four strands to the Laboratory Scientist programme (analytical science, chemical science, research & development and life sciences) were adapted to fit the workbased learning needs of the two companies.

Higher and degree apprenticeships are a government initiative aimed at helping employers develop the talent and skills of new and existing staff, combining on-the-job training with study for a higher education qualification.

Teesside University is working directly with employers on a wide range of apprenticeships, in health, business, computing, science and engineering.

Contact us to see how we can help you develop the skills your business needs.

In 2016 TeeGene Biotech developed a number of collaborative working opportunities in New Zealand after being chosen to take part in a British research and development delegation. The intention was to create agriculture and plant and food research connections with New Zealand’s Crown Research Institutes, leading universities and specialist biotech firms.

Contact us Tees.ac.uk/theforge 01642 384068 theforge@tees.ac.uk

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Research leads to ban Research carried out by a UK-China collaboration played a key role in the introduction of a ban on the use of the antibiotic colistin as a feed additive for animals in China.

The Chinese Ministry of Agriculture’s ban was supported by the work of a team led by Professor Jianzhong Shen, of the Beijing Advanced Innovation Center for Food Nutrition and Human Health at China Agricultural University, Beijing, in collaboration with Professor Timothy Walsh, of the School of Medicine at Cardiff University, and Dr Jian-Hua Liu of South China Agricultural University in Guangzhou, China.

In 2015, the team identified a gene called MCR-1 that allowed bacteria to survive colistin treatment in animals and humans in China. MCR-1 is a ‘mobile gene’, meaning that it can be easily transferred to other bacteria, making them resistant too, and the team identified the gene in a strain of bacteria called Escherichia coli found in pigs.

Colistin is an important ‘last resort’ antibiotic, used to treat serious bacterial infections in humans resistant to other antibiotics. It is also used in animal feeds to help rear healthy animals but widespread use of the antibiotic encourages the development of resistance genes.

Following their discovery, the team worked with the Chinese Government to investigate the impact of MCR-1 on colistin use in animals and humans.

Newton funding helped them to continue the work and the immediate implication was the withdrawal of more than 8,000 tonnes of colistin as a growth promoter from the Chinese veterinary sector, which will be replaced by other non-human antibiotics, supplemented by traditional Chinese medicines.

The European Medicines Agency has also taken steps to update its advice on reducing the use of colistin in European veterinary practices.

Professor Jianzhong Shen said: “Antibiotic usage in food animals is becoming a global issue associated with food safety and public health. All countries in the world should use antibiotics in animals more prudently and rationally.

“On the basis of the evaluation of risk assessments of such antibiotics, the Chinese Government worked promptly to remove colistin in the list of feed additives for the purpose of growth promotion.”

Professor Timothy Walsh said: “This is a remarkable example of how scientific discoveries can positively impact on animal and human populations.”

The work was mainly funded by the National Key Basic Research Program of China and the National Natural Science Foundation of China, with additional support from the UK Medical Research Council (MRC).

Dr Jonathan Pearce, Head of Infections and Immunity at the MRC, said: “Antimicrobial resistance (AMR) poses a global challenge to healthcare and agriculture. Our response to this challenge must span nations and disciplines.

“By pooling our research efforts, we can develop a deeper understanding of the drivers of AMR. Through collaboration with society, industry and policy makers, we can apply this knowledge to turn the tide against the AMR threat.”

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“ON THE BASIS OF THE EVALUATION OF RISK ASSESSMENTS OF SUCH ANTIBIOTICS, THE CHINESE GOVERNMENT WORKED PROMPTLY TO REMOVE COLISTIN IN THE LIST OF FEED ADDITIVES FOR THE PURPOSE OF GROWTH PROMOTION.”

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Team target resistant infections Researchers in America have created a new way to identify drugs and drug combinations that may be useful in combating infections that are resistant to many different antibiotics.

Scientists at the National Institutes of Health’s National Center for Advancing Translational Sciences (NCATS) and the Clinical Center and National Institute of Allergy and Infectious Diseases (NIAID) developed a test to rapidly screen thousands of drugs to determine how effective they were against a resistant bacteria.

The method provides a potential new approach to repurpose known drugs and compounds to help deal with powerful, hospital-borne infections, as well as emerging infectious diseases.

NCATS scientist Wei Zheng, Ph.D., and NIH colleagues Peter Williamson, M.D., Ph.D., of NIAID, and Karen Frank, M.D.,

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Ph.D. of the Clinical Center, used the test to screen 4,000 approved drugs and other biologically active compounds, identifying 25 that suppressed the growth of two drugresistant strains of Klebsiella pneumoniae that have become resistant to most major types of antibiotics. Drug-resistant Klebsiella has been a source of fatal infections in many hospitals across the country.

The researchers also used the screening test to gauge the effectiveness of combinations of drugs against antibiotic-resistant bacteria. They found three three-drug combinations that were effective against 10 common strains of multi-drug-resistant bacteria.

Wei Zheng said: “The results are very promising, and we think that the test can eventually help repurpose approved drugs and other compounds and find clinically relevant drug combinations that can be approved for to use in different ways that we have never used before. We’re hoping this approach will lead to approvable, effective ways to combat dangerous infections by drug-resistant bacteria.”

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Research into Parkinson’s Disease turns focus on the gut

Research carried out by a team of American researchers has suggested that gut microbes may play a critical role in the development of Parkinson’s Disease (PD).

The findings could lead to new treatments that can slow, stop or even prevent the development of the condition.

Researchers based at the California Institute of Technology studied mice with a small genetic change that causes them to produce too much of the protein alpha-synuclein.

As the mice aged, they naturally developed clumps of alpha-synuclein inside brain areas involved in controlling movement similar to those experienced by people with Parkinson’s.

Mice raised in germ-free cages had almost normal mobility and much reduced build-up of protein clumps in their brains.

Parkinson’s affects ten million people worldwide, making it the second most common neurodegenerative disease, and 75% of sufferers have gastrointestinal (GI) abnormalities, primarily constipation.

The research was carried out in the laboratory of Sarkis Mazmanian, the Luis B. and Nelly Soux Professor of Microbiology and Heritage Medical Research Institute Investigator.

Sarkis said: “The gut is a permanent home to a diverse community of beneficial and sometimes harmful bacteria, known as the microbiome, that is important for the development and function of the immune and nervous systems.

“Remarkably, 70% of all neurons in the peripheral nervous system—that is, not the brain or spinal cord—are in the intestines, and the gut’s nervous system is directly connected to the central nervous system through the vagus nerve. B “Because GI problems often precede the motor symptoms by many years, and because most PD cases are caused by environmental factors, we hypothesised that bacteria in the gut may contribute to PD.” The researchers used mice that over-produce αSyn and display symptoms of Parkinson’s. One group of mice had a complex consortium of gut bacteria and the others, called germ-free mice, were bred in a completely sterile environment at Caltech and thus lacked gut bacteria.

The researchers had both groups of mice perform several tasks to measure their motor skills, such as running on

treadmills, crossing a beam, and descending from a pole.

The germ-free mice performed significantly better than the mice with a complete microbiome.

Timothy Sampson, a postdoctoral scholar in biology and biological engineering and first author on the paper, said: “This was the ‘eureka’ moment. The mice were genetically identical; both groups were making too much αSyn. The only difference was the presence or absence of gut microbiota.

Once you remove the microbiome, the mice have normal motor skills even with the overproduction of αSyn.” “All three of the hallmark traits of Parkinson’s were gone in the germ-free models. Now we were quite confident that gut bacteria regulate, and are even required for, the symptoms of PD.” In a final set of experiments, Sarkis Mazmanian and his group collaborated with Ali Keshavarzian, a gastroenterologist at Rush University in Chicago, to obtain fecal samples from patients with PD and from healthy controls. The human microbiome samples were transplanted into germ-free mice, which then began to exhibit symptoms of PD. Sarkis said; “The data suggest that changes to the gut microbiome are likely more than just a consequence of PD. It’s a provocative finding that needs to be further studied, but the fact that you can transplant the microbiome from humans to mice and transfer symptoms suggests that bacteria are a major contributor to disease.

“For many neurological conditions, the conventional treatment approach is to get a drug into the brain. However, if PD is indeed not solely caused by changes in the brain but instead by changes in the microbiome, then you may just have to get drugs into the gut to help patients, which is much easier to do.”

Dr Arthur Roach, Director of Research at Parkinson’s UK, said: “In recent years, evidence has been growing that Parkinson’s may begin in the gut but the chain of events involved has so far remained a mystery.

“This work opens an exciting new avenue of study on the gut-brain connection in Parkinson’s. There are still many questions to answer but we hope this will trigger more research that will ultimately revolutionise treatment options for Parkinson’s.”

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“THE DATA SUGGEST THAT CHANGES TO THE GUT MICROBIOME ARE LIKELY MORE THAN JUST A CONSEQUENCE OF PD.

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Zika vaccine trial begins The first of five early stage clinical trials to test the safety and ability of a Zika vaccine candidate has begun in America.

The Zika Purified Inactivated Virus (ZPIV) vaccine, which is designed to generate an immune system response, is being tested at the Walter Reed Army Institute of Research (WRAIR) Clinical Trial Center in Silver Spring, Maryland.

Scientists with WRAIR, part of the US Department of Defense, developed the vaccine and the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health is co-funding the Phase 1 clinical trial with WRAIR. The experimental ZPIV vaccine is based on the same technology WRAIR used in 2009 to successfully develop a vaccine for another flavivirus called Japanese encephalitis.

The ZPIV vaccine contains whole Zika virus particles that have been inactivated, meaning that the virus cannot replicate and cause disease in humans.

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NIAID Director Anthony S. Fauci, M.D. said: “We urgently need a safe and effective vaccine to protect people from Zika virus infection as the virus continues to spread and cause serious public health consequences, particularly for pregnant women and their babies.�

Led by WRAIR principal investigator Maj. Leyi Lin, M.D., the new study aims to enroll 75 people ages 18 to 49 years with no prior flavivirus infection. Flaviviruses include Zika virus, yellow fever virus, dengue virus, Japanese encephalitis virus and West Nile virus.

A sub-group of 30 of the participants who receive the twodose ZPIV regimen will receive a third dose one year later.

All participants in the trial will receive the same ZPIV dose at each injection (5 micrograms).

Four additional Phase 1 studies to evaluate the ZPIV investigational vaccine are expected to launch in the coming months.

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Integrated Wearable Drug Delivery Systems – Choosing the right Partner SMARTDOSE PLATFORM CURRENTLY INCORPORATES A POLYMER-BASED DRUG CONTAINER

(made from Daikyo Crystal Zenith® cyclic olefin polymer) with a drug delivery device that can be pre-programmed to deliver high volumes of viscous or sensitive drug products over time, making it easier for patients to self-administer medication outside of the clinical setting.

DELIVERY SYSTEM DESIGN For patients with chronic conditions, the use of selfinjectable biologic therapies is on the rise, and one of the most promising options for administering these treatments is wearable drug delivery technologies.

Wearable systems combine the drug and its primary containment system in a patient-friendly system that delivers the prescribed dose electronically. A truly successful wearable drug delivery system must consider both the design and the needs of the end-user during the different stages of a patient’s journey.

West Pharmaceutical Services’ SmartDose® platform, for example, was designed to easily integrate into a patient’s lifestyle. The platform is a single-use, electronic wearable injector that adheres to the patient’s body, usually on the abdomen. Discreet, intuitive and designed to minimize discomfort, SmartDose technology platform currently incorporates a polymer-based drug container

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In order to design a drug delivery system that helps to address the needs of both the drug and the patient, pharmaceutical manufacturers must consider the interface between the drug, container, delivery device and patient.

For the development of the SmartDose platform, the following considerations were key: • Primary container format: Vials may be necessary for initial use, but a syringe or cartridge system may provide a desirable solution for the patient when the system reaches the market.

• Drug/container compatibility: While glass is suitable for many pharmaceutical products, high pH drugs or otherwise sensitive products may require vials or syringes made from alternative materials such as cyclic olefin polymers.

• Container/delivery system interface: Dimensional

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tolerances and functionality should be tested to ensure proper activation and glide force.

• Patient interaction: Simply designing a device that patients/users “can” use is no longer sufficient—devices should be designed for affinity.

UNDERSTANDING PATIENT NEEDS The SmartDose platform was developed with extensive human factors testing to address potential obstacles to compliance: • Ease of use: The platform currently allows for the patient to load the cartridge containing the drug. A user-friendly activation button on the front of the device and LED indicator lets the patient know that the dose delivery is in progress.

• Discreteness: Special consideration was taken to ensure that the technology is easily concealed to avoid calling undue attention, creating distractions to others or creating feelings of stigmatization.

• Dose notification: The SmartDose technology platform is equipped with a microprocessor designed to offer immediate feedback via a dose confirmation window and audible cues indicating whether the prescribed medication was delivered.

• Pain: was designed to minimize discomfort throughout the duration of the dosage and to be easily removed once the dosage was complete.

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CHOOSING THE RIGHT PARTNER The role drug packaging and delivery systems play in the drug development process as well as how they fit into the regulatory and quality environment is critical to success. By working with a partner with an intimate knowledge of the complexity of design, testing, container selection and closure integrity associates with large molecules and sensitive drugs, regulatory and quality requirements, manufacturing at scale, and speed to market allows drug manufacturers to make more informed decisions about the partner of choice.

For example, West worked closely with Amgen to ensure compatibility between the SmartDose technology platform and the monthly single dose administration of Amgen’s Repatha®--the first combination product that incorporates SmartDose® technology platform for use in the United States and is FDA approved. The combination of Amgen’s innovative treatment with West’s technology platform exemplifies the close collaboration West has with its pharmaceutical and biotechnology partners to deliver advanced, integrated solutions for drug delivery and containment.

Partnering with a company like West that can provide expertise in the field of drug packaging and delivery systems should be an important part of the launch plan for any biologic to be delivered in a wearable system. Looking ahead, it’s imperative that the pharmaceutical industry remains focused on better understanding the interaction between patients, their medication and the drug delivery system, as this relationship may have a substantial impact on patient outcomes.

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THE ROLE DRUG PACKAGING AND DELIVERY SYSTEMS PLAY IN THE DRUG DEVELOPMENT PROCESS AS WELL AS HOW THEY FIT INTO THE REGULATORY AND QUALITY ENVIRONMENT IS CRITICAL TO SUCCESS.

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Grants are awarded

Four teams of scientists at Rice University and other Gulf Coast Consortia (GCC) institutions in America have secured research grants from the John S. Dunn Collaborative Research Awards.

GCC member institutions include Baylor College of Medicine, Rice University, the University of Houston, the University of Texas Health Science Center at Houston, the University of Texas Medical Branch at Galveston, the Institute of Biosciences and Technology of the Texas A&M Health Science Center and the University of Texas MD Anderson Cancer Center and the research grants are for $98,000 each.

Gang Bao of Rice and William Lagor, of Baylor College of Medicine, plan to improve the safety of genome editing by generating viruses that deliver CRISPR/Cas9 machinery to edit a disease-causing gene then self-destruct when the job is done.

They will test the self-deleting CRISPR/Cas9 system’s impact on a rare lipid disorder to determine if the system can successfully edit a therapeutic target gene then promote its own removal from the liver.

Angel Martí, of Rice, and Steven Curley, of Baylor, will develop a way to measure the temperature of microscopic domains where standard thermometers do not work.

Their probe will sense temperatures via luminescence and the team eventually hope to measure the temperature distribution of cancer cells at the subcellular level with and without the exposure to radio frequencies used in non-invasive therapeutic hyperthermia.

José Onuchic, of Rice, and Yun Huang, of the Institute of Biosciences and Technology (IBT) at Texas A&M Health Science Center, will combine expertise in computational modeling of biological systems and cancer epigenetics to understand how hematopoietic stem progenitor cells develop into all types of blood cells.

These stem cells are primarily found in bone marrow and depend on a complex regulatory network of genes to differentiate. The Rice and IBT labs hope to unveil the network’s operating principles and predict how the stem cells make decisions under normal and diseased conditions. Such models could help them understand cancer-associated epigenetic mutations and open a path to novel therapies.

Tomasz Tkaczyk, of Rice, and Kimberley Tolias, of Baylor, plan to observe the dynamic

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behaviors of living neural cells through genetically encoded fluorescent probes. Their aim is to develop an understanding of brain circuitry and function, as well as the nature of brain diseases.

Their work will require the development of fluorescent contrast agents and a spectrometer able to capture wide images of multiple probes at high speeds in living tissue.

Current imaging techniques, they say, are akin to ‘watching a football game with the ability to see only one or two players per game’. Their new technique will deliver a more complete picture of neural dynamics and allow them to observe interactions between signaling pathways, monitor multiple steps in a signaling cascade in a single cell, rapidly screen cellular responses to stimuli whose receptors are unknown and study events in the context of other events in living cells.

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XBiotech announces research collaboration XBiotech’s True Human product IL-1 alpha is to be used in researching treatment for Inflammatory Bowel Disease (IBD) US company XBiotech Inc, a developer of True Human(TM) therapeutic antibodies, has started a collaboration designed to find treatments for IBD. The company is working with a research team at Case Western Reserve University (CWRU) School of Medicine headed by leading gastroenterologist, Fabio Cominelli, M.D., Ph.D. Dr. Cominelli and his research team will conduct preclinical studies to help develop new treatments using the company’s approach to using natural human antibody therapy to neutralise harmful inflammation.

Dr. Cominelli is a world-leading expert in inflammatory bowel disease and his group was the first to report that specific blockade of interleukin-1 (IL-1) was effective in reducing disease severity in colitis and that deregulated inflammation is a cause of auto-inflammatory diseases, including IBD.

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The doctor, Chief of the Division of Gastroenterology and Liver Disease and Director of the Digestive Health Institute at CWRU School of Medicine in Cleveland and Chief Scientific Officer of the Digestive Health Institute at University Hospitals Cleveland Medical Center, said: “I have researched IL-1’s role in disease severity in colitis for many years, and am excited about the opportunity to be able to selectively inhibit IL-1 alpha to better define its role in IBD. These results will help shape the design of future clinical trials as we look for new and better treatments for this widespread condition.”

John Simard, the company’s President and Chief Executive Officer, said: “We are hopeful that this collaboration will provide for further advances in treatment.”

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www.thebiosciencejournal.co.uk

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heart d Hope as research unlocks the secrets of

Frances Griss

Since the beginning of the 1960s, deaths from heart disease in the UK have halved so that today only 26% of people have a cause of death attributable to the condition, compared with more than half of deaths half a century ago.

The centre says that older patients, particularly those that are frail who present with acute coronary syndrome (heart attack or unstable angina), have much poorer outcomes compared to their younger counterparts.

Huge as that achievement is, it still means that a quarter of the population are dying from a disease that has been proved to be often preventable and highly treatable.

Despite this, the team says, there is very little research data available on older patients with acute coronary syndrome; more than half of all trials between 1996 and 2000 did not enrol patients over the age of 75 years, and over a third actively excluded them. ICON1 has been designed to investigate patients over the age of 75 years who are at a higher risk following an acute coronary syndrome, by performing a comprehensive evaluation of their cardiovascular disease burden.

Given the prevalence of cardiovascular disease, it is no surprise that a great deal of money and effort is directed towards research in the area, both on lifestyle and environmental factors and into pharmaceutical and surgical interventions.

One of the most controversial treatments is the prescription of statins to apparently healthy people as well as those with known disease, but the benefits have been shown to outweigh the costs.

Since 1994, the University of Oxford Clinical Trial Service Unit Heart Protection Study has been following 20,000 patients. At the beginning of this decade, it showed the effectiveness of larger doses (80mg over 20mg) of simvastatin for reducing major vascular events in 12,000 heart attack survivors.

In 2012, Cholesterol Treatment Trialists showed that there were benefits to taking the drugs even in apparently healthy patients with a low risk of heart disease.

Figures from the British Heart Foundation suggest that the use of statins already saves 10,000 lives each year in England alone. The debate continues as to whether they should be prescribed more widely.

Despite the fact that coronary artery disease is the most common cause of death in the United Kingdom among people over the age of 65 years and almost half of all heart attacks occur in those over the age of 70 years, older people are often excluded from trials of treatments.

Doctors at the Newcastle Biomedical Research Centre are taking advantage of their partnership with Volcano Corporation, a specialist in coronary intravascular imaging, to look at the effectiveness of one particular treatment in older people, stents, for NHS National Institute for Health Research.

The results will be used to develop a risk score that will inform physicians of the patient’s future risk of developing adverse effects and provide a better understanding of how to manage older patients with coronary artery disease. It will also help to plan future studies evaluating treatment strategies that might be beneficial in improving outcomes in high risk older patients.

Other institutions undertaking research for the NIHR are focusing on different areas and a team at the Liverpool Heart and Chest Hospital (LHCH) led by Dr Rod Stables recently published results which could led to both increased survival rates and economies for a cash-strapped NHS. The study had the title Unfractionated heparin versus bivalirudin in primary percutaneous coronary intervention (HEAT-PPCI) and, with more than 1800 participants, was record-breaking in size.

Dr Stables, Consultant Cardiologist at LHCH, said: “As far as we are aware, our study is one of the first trials to recruit 100% of eligible patients presenting with the medical condition being examined, which means that it more closely resembles real life practice than many previous trials.”

Over a 22-month period, 1,829 patients undergoing emergency angiography (an x-ray examination of the heart’s arteries after a suspected heart attack) at LHCH were recruited to the trial.

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isease Continues page 24

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PATIENTS DIED WITHIN 28 DAYS OF SURGERY (12.9%) CAUSE OF DEATH ATTRIBUTABLE TO HEART DISEASE (26%)

DEATHS IN BIVALIRUDIN GROUP (5.1%) NOT HEART DISEASE RELATED (74%)

 Continued from page 24

OVER A 22-MONTH PERIOD, 1,829 PATIENTS UNDERGOING EMERGENCY ANGIOGRAPHY

More than four fifths of the participants went on to receive PPCI; approximately half of the participants received heparin, and half received bivalirudin.

Researchers then recorded how many patients in both groups experienced a major adverse cardiac event, such as death, stroke or another heart attack, within 28 days after surgery.

The results suggest that heparin should be used rather than bivalirudin during PPCI and after it has been completed.

Where heparin was used, there was a lower rate of major adverse cardiac events, although there was no significant difference between the groups in terms of the rate of bleeding complications, which are an acknowledged risk of drugs that prevent clotting.

Within 28 days of surgery, 46 patients (5.1%) in the bivalirudin group died, compared to 39 (4.3%) of patients in the heparin group; 24 patients (2.7%) in the bivalirudin group had another heart attack in the same period, compared to seven patients (0.8%) in the heparin group.

The results are interesting in light of the fact that bivalirudin is roughly 400 times more expensive than heparin and, at the time of the study, bivalirudin was in widespread use in high-income countries around the world.

MORE THAN FOUR FIFTHS OF THE PARTICIPANTS WENT ON TO RECEIVE PPCI

Dr Stables said: “This finding might provide an opportunity, rare in modern health care, to provide improved outcomes at much reduced cost.

“In our centre performing about 1,000 PPCI procedures a year, the cost saving would be about £500,000 a year.

Translated to the global stage, the potential savings could amount to several billion pounds.”

The results led to the European Society of Cardiology modifying its guidelines to bring them in line with the study, and a shift in clinical practice globally.

In a different approach, medics working in clinics in the

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Royal Liverpool University Hospital, Warrington Hospital and the Countess of Chester Hospital are looking at how a patient’s genes influence their response to the blood thinning drug warfarin, which is taken by about one per cent of the population to treat ventricular fibrilation.

An international trial in Liverpool, Newcastle and Sweden included genotype testing of patients into an algorithm to calculate dosages. This proved highly accurate and is now being put into practice at the clinics in north west England.

They are using equipment which can give a genotype result in 45 minutes with only a saliva sample. If the trial is successful, the method could be rolled out across the country.

The work is led by Professor Sir Munir Pirmohamed of the University of Liverpool’s Wolfson Centre for Personalised Medicine and Theme Leader of Delivering Personalised Health and Care for CLAHRC NWC. He said: “This is innovation and it is disruptive; it is a way of personalising care which can be replicated in many areas of medicine, creating a major paradigm shift in how we diagnose and treat people.

“This is how we get patients onto the right drugs at the right doses – using ‘precision dosing’ so that they are effective.

This improves the treatment of patients and improves the efficiency of existing and new drugs.”

Cardiovascular disease, although it is still a major problem and blights the life of millions, is an area where research is making inroads into better understanding and providing effective interventions.

With science producing refinements and new discoveries on so many different fronts as varied as using stem cells to regenerate dead heart muscle genetic testing for rarer conditions the future is exciting and the prospects for patients very positive.

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Researchers are making great strides We have made great strides in the fight against heart disease over the past 50 years. Since 1961, when the British Heart Foundation was established, the annual number of deaths from CVD in the UK has fallen by half. This reduction can be largely attributed to the ground breaking research that has taken place in the UK and overseas, but there is still a great deal of work to be done.

A recent example of the progress we have made is the massive improvement in the outcome of heart attacks. In the 1960s more than seven out of ten heart attacks in the UK were fatal.

Today at least seven out of ten people survive. This increase in survival is largely due to earlier, more accurate diagnosis, leading to more efficient and effective treatment, which has been made possibly by the troponin test. This blood test is used to detect a protein released by the heart during injury and is now recommended in clinical practice.

The British Heart Foundation continues to fund pioneering research that aims to improve the way that the troponin test works, and ensure that it has a high enough sensitivity to detect very low levels of troponin which can still be indicative of a heart attack.

BHF-funded researchers in Edinburgh have already shown that a higher sensitivity test could rule out a heart attack in two thirds of people arriving at A&E with chest pain, and that the test could even be used to identify people at risk of suffering a heart attack in the future.

We are also making great progress in basic science, the fundamental starting point for all breakthroughs in medicine.

Recently, BHF-funded researchers in Cambridge created blood vessel tissues in a petri dish which mimic Marfan syndrome in human arteries. It is estimated that 13,000 people in the UK have Marfan syndrome, which can cause life-threatening thoracic aortic aneurysms. This pioneering science will allow researchers to use the lab-generated vessel cells to test drugs for people with Marfan syndrome.

Our understanding of the genes responsible for heart conditions which are passed down in families is reshaping the way that we diagnose and treat inherited heart conditions. This new knowledge has enabled people at risk of sudden death to be identified and treated to prevent such tragic events. BHF Professor Hugh Watkins, a clinical cardiologist and laboratory scientist in Oxford, has led research which has given us the tools to find people with an inherited condition called hypertrophic cardiomyopathy (HCM) through genetic testing programmes.

With all the successes in recent years, we must not get complacent. Coronary heart disease is still the UK’s single biggest killer. More than seven million people in the UK are living with cardiovascular disease, which is responsible for more than a quarter of all deaths. The British Heart Foundation is currently providing £100 million in support of new cardiovascular research projects each year, which will yield results that will allow us to take further strides in the diagnosis treatment and care of heart patients, over the next 50 years.

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By Professor Metin Avkiran, Associate Medical Director at the British Heart Foundation

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Game changing cardiovascular research at the heart of the UK’s National Health Service Cardiovascular disease is a leading cause of death and disability worldwide. In an effort to buck this trend and improve outcomes for people with cardiovascular problems, the research community in the UK is highly focused on delivering innovative, world-leading cardiovascular clinical research. The National Institute for Health Research (NIHR) Clinical Research Network is at the hub of this research, ensuring it is delivered to the highest standard.

An example of such research is the International Study of Comparative Health Effectiveness with Medical and Invasive Approaches trial, known as the ISCHEMIA trial.

LAST YEAR EVERY NHS TRUST IN ENGLAND WAS RESEARCH ACTIVE, AN INCREDIBLE ACHIEVEMENT THAT WILL HELP ENSURE THE UK STAYS AT THE FOREFRONT OF CARDIOVASCULAR RESEARCH.

Every year tens of thousands of people in the UK with stable angina have one or more stents (a small mesh tube that is used to treat narrow or weak arteries) inserted in a blood vessel to aid healing or relieve an obstruction in their arteries. Except in emergency cases, there is no evidence that treating angina by inserting stents, followed by medical and lifestyle interventions, is better than medicine and lifestyle changes alone.

A research team at Northwick Park Hospital, part of the London North West Healthcare NHS Trust, has been leading the way in finding out which treatment is better and safer for patients - medical therapy alone or medical therapy plus a procedure.

The study opened at the Trust’s Cardiac Research Department three and a half years ago. When patients are referred to the hospital with stable angina - chest pain or discomfort that most often occurs with activity or emotional stress - they are given a stress echocardiogram, a procedure

that determines how well the heart’s own blood supply is working. The London based research team randomly selected their first participant just a month after opening for recruitment, since then they have randomised 150 patients.

The team attribute their success to a number of factors including the support of administration, clinical and research staff - from research registrars and cardiac physiologists, to cardiac nurses, catheterisation laboratory staff and cardiothoracic surgeons. All this is underpinned by the provision offered by the National Institute for Health Research (NIHR) Clinical Research Network - often referred to as the research delivery arm of the National Health Service (NHS).

Consultant Cardiologist and one of the study’s principal investigators Dr Ahmed Elghamaz says: “It’s such a complex study and getting patients is actually quite difficult. The reason we’ve been so successful is because we are working together very well in all aspects of the study - from the imaging of the heart, to the intervention, to the medical treatment.”

The Northwick Park Hospital team’s success has seen them take over responsibility for leading the ISCHEMIA trial in the UK, and with the help of the NIHR Clinical Research Network they have identified and opened 30 sites. Grace Young, ISCHEMIA’s UK study coordinator, explains: “The study is now in its fourth year of recruitment, and with so many patients to maintain in the study, we’ve relied on the support of staff from the NIHR Clinical Research Network to maintain our high enrolment rate.”

So, what makes the UK an attractive clinical trial destination for cardiovascular research? Dr Paul Clift is one of the NIHR Clinical Research Network national leads for cardiovascular disease. Specialising in pulmonary hypertension, congenital heart disease and arrhythmias. Dr Clift has carried out extensive research into congenital heart disease, he says: “Here in the UK we’re fortunate to have the research infrastructure provided by the NIHR Clinical Research Network. The Network provides researchers with the practical support they need to make research happen and supports the timely set-up and delivery of cardiovascular disease clinical studies across England.”

The NIHR actively supports and oversees a wide range of studies; from single-centre early stage evaluation of medical technology, through to larger multi-centre observational studies and randomised controlled trials of medical and surgical interventions. Since 2010, over 175,000 participants have taken part in cardiovascular disease studies on the Network’s portfolio. Dr Clift continues: “NIHR Clinical

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Research Network staff and our network of experts on the ground provide accurate feasibility and recruitment guidance for overcoming the potential barriers to successful study delivery, aided by our established links with patient groups and clinicians. We’ve continued year on year to increase opportunities for the over seven million people living with cardiovascular disease in the UK to take part in high-quality clinical research studies.

Last year every NHS trust in England was research active, an incredible achievement that will help ensure the UK stays at the forefront of cardiovascular research.”

As well as providing research delivery staff, the NIHR Clinical Research Network brings together communities of clinical practice, offering research teams national networks of expertise to call on. Areas of focus for cardiology research currently include cardiovascular disease prevention, atherothrombosis, arrhythmias, cardiac surgery, congenital heart disease, pulmonary arterial hypertension and heart failure.

With an extensive pool of potential investigators, amongst which basic study information can be rapidly disseminated, the Network provides accurate information about the delivery capabilities of potential study sites, and supports the selection of suitable study locations. Uniquely these early feasibility services are available free of charge to the life sciences industry.

Dr Elghamaz said the research team are already learning from the ISCHEMIA study, he also feels that the results of the study will be easy to apply in the NHS: “I am an interventional cardiologist so by nature I like to open up blocked arteries. Therefore, it was a surprise to me to leave patients with a very severe narrowing of the arteries and find that they are actually doing very well, some of them have gone up to three and a half years now without any need for intervention.

Studies don’t always follow real life practice but this study is trying really hard to mimic what we would normally do, making the results easier to apply to real life practice. By answering the question we don’t know, which is how best to

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treat patients with stable angina, this study is potentially a game changer in the future of cardiology.”

Another such study to benefit from NIHR support was the ERICCA: Effect of Remote Ischaemic preconditioning on Clinical outcomes in patients undergoing Coronary Artery Bypass Graft (CABG) surgery trial. Remote Ischaemic preconditioning is an experimental technique for producing resistance to the loss of blood supply, and thus oxygen to tissues. The aim of the ERICCA trial was to confirm if remote ischaemic preconditioning could improve health outcomes in high-risk patients undergoing CABG with or without valve surgery. The NIHR Clinical Research Network supported recruitment of patients from 30 cardiac centres across the UK. This invaluable support enabled the study to reach its recruitment target of 1610 patients well within its planned recruitment window of three years.

Elsewhere, the HEAT-PPCI study compared the use of heparin and bivalirudin during and after primary percutaneous coronary intervention, which is a procedure to unblock the arteries carrying blood to the heart.

Carried out at Liverpool Heart and Chest Hospital NHS Foundation Trust (LHCH), it ended up being the largest ever single-centre cardiology study in the world and largest interventional cardiology study in the UK. Keith Wilson, Patient Research Ambassador and HEAT-PPCI study team member, gives an insight into the patient perspective: “I got involved through an illness I had many years ago and thought that it was a way of taking control back by discussing my disease and taking part in research. I think the whole point of research is making sure that it fits the patient and that patients don’t have to fit the research.”

Clearly studies such as ERICCA, HEAT-PPCI and ISCHEMIA are driving forward our understanding of cardiovascular disease. With 37,446 participants recruited to cardiovascular disease studies in 2015/16 (7,295 of which were recruited to commercial contract studies) the NIHR Clinical Research Network is playing an increasingly important role in ensuring NHS patients in the UK remain at the centre of future research developments.

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For further information about the NIHR study support service visit www.nihr.ac.uk

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| BIOSCIENCE TODAY SPRING 2017 |

Cell line

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| BIOSCIENCE TODAY SPRING 2017 |

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technology provides optimism for researchers Frances Griss

One of the most exciting and rapidly growing fields of bioscience is the development of new cell lines with specific and controllable characteristics, either for research or industrial scale production.

Companies all over the world are vying to be the fastest, most accurate and most efficient producers of specific cultures tailored to individual clients.

Hundreds of individual lines of cells have been perpetuated for research over the past decades and some current lines can trace their origins back to the 1950s.

These cell lines originate in humans, many in various cancerous growths, animals, fish, plants and fungi.

Such samples can be used to test the effects of potential treatments without using animals for experimentation.

It is the ability of a cell line to replicate itself reliably and without introducing new material which means that results are reliable and comparable.

In this regard, experts say that it is important to maintain vigilance and authenticate a culture before work on it begins because samples can become contaminated over time, creating problems for all laboratories working on that culture.

Public Health England recently publicised results showing that a sample of glioblastoma U-87MG, a cell line isolated almost 50 years ago, was not genetically identical to the original tumour. They urged researchers to verify their samples and make the results widely available to other laboratories using the same cell line if differences were found.

There is a growing and competitive industry which produces genetically engineered cell lines which express a specific antibody or protein, which can then be produced in comparatively large quantities.

Some companies say that their process can increase bench top quantities of a culture to 1000l in less than a fortnight, yielding about 4g/l of product. Companies state that yields of up to 10g/l are possible in some circumstances.

Commercial laboratories offering this service suggest it is possible to create a cell line to specific requirements in as little as one month and promote their services saying it allows research teams to concentrate their abilities on the task in hand without forcing them to branch out into other areas of bioscience and develop their own cell lines.

A COMPETITIVE BUSINESS A recent Global Market Insight reported that cell line development was worth $2.81 billion globally in the previous year but predicted an almost three-fold increase in the size of the market by 2023 to $7.45 billion.

The increase will be driven by demand for new drugs, biosimilars and vaccines with some companies stepping up their capacity to manufacture.

French company LFB Biomanufacturing, for example, announced that it was going to triple production capacity at its Ales bioproduction facility, specialising in cell-based manufacture of recombinant proteins and monoclonal antibodies.

According to Global Market Insight, growth in the market will not only be in centres offering cell line development but in the service companies producing equipment from large scale production plants down to bench top equipment and cell growth media.

For example, Swiss company Selexis, a specialist in mammalian cell line generation, recently announced an agreement to expand a relationship which had been growing since 2011 and work strategically with Xencor, a biopharmaceutical company specialising in the production of monoclonal antibodies for the treatment of autoimmune diseases, asthma, allergic diseases and cancer.

Other strategic partnerships are for a specific purpose such as the recent agreement between Cobra Biologics and Alligator bioscience. Cobra was asked to develop a second cell line specifically for the production of a drug candidate ADC-1015, a bispecific immune activator developed to induce immune activation.

One of the most commonly used parental cell lines was isolated as far back as the 1950s from Chinese hamster ovaries (CHO) and there is much competition in the sector to provide the fastest and most efficient development process and scaling of culture production.

Lonza, based in Germany and Switzerland, recently announced a new range of media with improved cell growth.

The new PowerCHO Advance™ Media line offers easy filterability, improved cell-growth promotion and allows for easy scalability, so users do not need to change to a new medium as they progress from research projects through to large-scale production.

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A RECENT GLOBAL MARKET INSIGHT REPORTED THAT CELL LINE DEVELOPMENT WAS WORTH $2.81 BILLION GLOBALLY IN THE PREVIOUS YEAR BUT PREDICTED AN ALMOST THREEFOLD INCREASE IN THE SIZE OF THE MARKET BY 2023 TO $7.45 BILLION.

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| BIOSCIENCE TODAY SPRING 2017 |

FORMER PRIME MINISTER TAKES ON KEY

DEMENTIA RESEARCH ROLE Former Prime Minister David Cameron has become the new President of Alzheimer’s Research UK, the UK’s leading dementia research charity.

Mr Cameron made dementia a major focus of his time in office, launching the Prime Minister’s Dementia Challenge in 2012 and starting a drive to deliver improvements in care and research.

He officially took up his new role, the highest level of ambassador to the charity, at a visit to its Cambridge Drug Discovery Institute.

The former Prime Minister toured one of Alzheimer’s Research UK’s three Drug Discovery Institutes, during which he heard from scientists who are working to fast-track research breakthroughs into new treatments for people with dementia.

David Cameron said: “Tackling dementia was a major focus while I was Prime Minister, and although improvements

in attention and research innovation have been rapid, it remains one of our greatest health challenges.”

Among those welcoming the appointment was Shaheen Larrieux, a Champion of Alzheimer’s Research UK. Four years ago her mother Hosna was diagnosed with frontotemporal dementia, a rare form of dementia with distressing symptoms including personality changes and aggressive behaviour.

She said: “It was a long hard struggle to understand that Mum has a degenerative brain disease, and it’s heartbreaking to know that there are no treatments that can help her. Alzheimer’s Research UK’s work is absolutely vital: we must find a cure for dementia, and I’m thrilled to see David Cameron supporting the charity in this way.”

Hilary Evans, Chief Executive of Alzheimer’s Research UK, said: “We have a clear focus on pioneering research, working with leading scientists across the UK and the world.

We are committed to bringing together the brightest minds and most innovative ideas in powerful collaborations that reach breakthroughs faster.”

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| BIOSCIENCE TODAY SPRING 2017 |

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David Cameron – why I care Here, David Cameron explains why he is so committed to supporting research into dementia.

It was the end of the week that I resigned as Prime Minister. I was in my constituency, visiting a care home. A woman with dementia, who was surrounded by her loving family, grabbed my hand and stared into my eyes. As I looked back at her I could see she didn’t know either where she was, or who was sitting with her.

That moment brought home to me, once again, the desperate sadness of this debilitating condition. And while I didn’t know then what role I could play outside No10 to help with the fight, I knew it was something that I wanted to do.

Dementia steals people’s lives, turns their relationships upside down, destroys their hopes and dreams. We owe it to them, their families and their carers to find a solution.

I believe there are four great battles we need to win. The first is the battle to win a deeper public understanding. Over recent years, we have seen a gradual shift in society’s attitudes towards dementia: once hidden in the shadows, today diseases like Alzheimer’s are talked about in public more than ever before. But if the level of awareness is higher, a deeper understanding across society is still needed, with too many of us writing off dementia as an inevitability of later life – a natural condition of ageing – rather than being caused by diseases of the brain that we can and will overcome with the right medical research. Just as we did in the past with diseases like cancer and HIV, today we need to educate, inform and talk more – and more frankly – about dementia.

Second, we must win the battle of priorities. Cancer research and stroke research deserve all their funding – but dementia shouldn’t be so far behind. After all, dementia remains one of our greatest health challenges. Currently, 850,000 people are living with dementia, and we now know that each year in the UK, the condition is responsible for 360,000 years of life lost.

The condition leaves millions heartbroken across the country – whether they are battling it themselves or, as family and friends, caring for a loved-one.

Which leads to the third battle: winning continued support for scientific research that must be properly funded and promoted.

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Britain is in a great place to do this. Today, more scientists are working on dementia and there has been a renewed determination to catalyse world-class research as part of a truly global effort. I’m delighted that this focus on dementia continues under the present Government, with the creation of the UK Dementia Research Institute – signalling a clear statement of intent to the world to continue that effort for the long-term.

And this leads to the final battle: ensuring we work internationally to demonstrate that this is a global challenge that we will only beat by working together. Yes, our UK university and science base are world class, but if we are to confront dementia once and for all, we all need to pull together on a global basis. Alzheimer’s Research UK is leading the way, working with institutions across the world to fund vital research focused on preventing and delaying Alzheimer’s and improving the quality of life for those affected. The findings of this research will be made available to other scientists across the globe, helping everyone make progress faster.

When I launched the Prime Minister’s Dementia Challenge, I said combatting the condition was a personal priority.

I feel that more today than ever before, not least because dementia, both as a medical challenge and a societal issue, can still feel at times like it’s a generation behind cancer or heart disease. But the future is bright. We can take optimism from the ideas, drive and innovation in dementia research.

And we can be confident that we can emulate the successes of other areas of health research to avoid the heartbreak of dementia for the next generation. It is a goal I look forward to championing in the years ahead.

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| BIOSCIENCE TODAY SPRING 2017 |

Three-Pare

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| BIOSCIENCE TODAY SPRING 2017 |

nt Babies

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A step closer after regulations grant approval Occasionally in the world of science, a breakthrough comes along that is so startling that it turns conventional thinking on its head.

That is the case with the latest research into the idea of three-person IVF, which challenges the belief that a child will always have two biological parents.

The new technique has been developed by scientists seeking to prevent babies from inheriting often lethal genetic diseases.

Following protracted investigations into the scientific and ethical implications of the procedure, it has now been approved by UK fertility regulator the Human Fertilisation and Embryology Authority (HFEA).

As a result of the decision, the first ‘three-parent’ babies could be born in the UK at the end of this year, in work that builds on pioneering research by a team at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University.

It was in 2015 that the UK became the first country to legalise mitochondrial replacement therapy (MRT) and the HFEA’s follow-up decision in December 2016 cleared the way for clinics to apply for permission to carry out the treatment.

Mitochondrial diseases are caused by inherited mutations in the DNA contained in mitochondria, tiny structures present in every cell. They are genetic conditions affecting the batteries of the cell, with about one in 4,300 affected children born every year. Symptoms include muscle weakness, blindness, deafness, seizures, learning disabilities, diabetes, heart and liver failure. There is no cure for mitochondrial DNA disease and affected children often die in early infancy.

Known as ‘mitochondrial donation’ the new IVF technique involves replacing faulty mitochondria inherited from the mother in the egg with the healthy mitochondria of another woman.

Following the HFTEA decision, the Newcastle team immediately applied for a licence to help patients who risk transmitting mitochondrial disease to their children by using the procedure, which transfers a tiny amount of DNA from the second woman’s egg into the first women’s egg.

The procedure will be carried out on women receiving treatment as an NHS service at the Newcastle Fertility Centre and the NHS Highly Specialised Service for Mitochondrial Disease and the team aim to offer treatment

for up to 25 women a year affected by mitochondrial disease.

The procedure is not without its critics, who argue that it paves the way for the creation of ‘designer babies’, thereby interfering with the natural order of things.

During lengthy consultation, the HFEA considered such objections along with the safety of the procedure and concluded that the health benefits outweighed the ethical concerns.

HFEA Chair Sally Cheshire said: “This historic decision means that parents at very high risk of having a child with a life-threatening mitochondrial disease may soon have the chance of a healthy, genetically related child. This is lifechanging for those families.

“After a lot of hard work and invaluable advice from the expert panel, who reviewed the development, safety and efficacy of these techniques over five years and four reports, we feel now is the right time to carefully introduce this new treatment in the limited circumstances recommended by the panel.

“Although it is tempting to rush ahead with new treatments, the UK approach of testing public opinion, putting the issue to parliament and carefully monitoring laboratory research has proved to be the most responsible and sustainable of introducing new, cutting edge treatments into the clinic.

Such an approach has allowed us to balance innovation with safety, maintaining public trust as we go.”

Wellcome Director Dr Jeremy Farrar agreed that time needed to be taken to weigh up the issues, saying: “It’s right that there’s been such a thorough process to get to this stage. We must recognise, though, that for some affected families this decision may have come too late. The scientific community must continue to work closely with policy and the regulatory system so future innovative, cutting-edge techniques reach patients faster.”

For the Newcastle team, the priority has always been the welfare of families whose lives have been blighted by the disease.

Professor Mary Herbert from the Wellcome Centre for Mitochondrial Disease at Newcastle University and the Newcastle Fertility Centre, who led the IVF research leading to the developments, said: “It is enormously gratifying that our many years of research in this area can finally be applied to help families affected by these devastating diseases.”

Such views find an echo in comments from James Palmer, Clinical Director of Specialised Services at NHS England, who said: “Mitochondrial diseases can be devastating and

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“Although it is tempting to rush ahead with new treatments, the UK approach of testing public opinion, putting the issue to parliament and carefully monitoring laboratory research has proved to be the most responsible and sustainable of introducing new, cutting edge treatments into the clinic. Such an approach has allowed us to balance innovation with safety, maintaining public trust as we go.”

Sally Cheshire

HFEA Chair

Continued from page 35

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life limiting as well as hugely costly to the NHS to treat.

This trial will, for the first time, give women living with mitochondrial disease the option of having a baby without passing on their condition and is a shining example of how the NHS is leading the world in developing cutting-edge innovative new medical interventions.”Occasionally in the world of science, a breakthrough comes along that is so startling that it turns conventional thinking on its head.

The procedure is not without its critics, who argue that it paves the way for the creation of ‘designer babies’, thereby interfering with the natural order of things.

That is the case with the latest research into the idea of three-person IVF, which challenges the belief that a child will always have two biological parents.

HFEA Chair Sally Cheshire said: “This historic decision means that parents at very high risk of having a child with a life-threatening mitochondrial disease may soon have the chance of a healthy, genetically related child. This is lifechanging for those families.

The new technique has been developed by scientists seeking to prevent babies from inheriting often lethal genetic diseases.

Following protracted investigations into the scientific and ethical implications of the procedure, it has now been approved by UK fertility regulator the Human Fertilisation and Embryology Authority (HFEA).

IT WAS IN 2015 THAT THE UK BECAME THE FIRST COUNTRY TO LEGALISE MITOCHONDRIAL REPLACEMENT THERAPY (MRT) AND THE HFEA’S FOLLOW-UP DECISION IN DECEMBER 2016 CLEARED THE WAY FOR CLINICS TO APPLY FOR PERMISSION TO CARRY OUT THE TREATMENT.

As a result of the decision, the first ‘three-parent’ babies could be born in the UK at the end of this year, in work that builds on pioneering research by a team at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University.

It was in 2015 that the UK became the first country to legalise mitochondrial replacement therapy (MRT) and the HFEA’s follow-up decision in December 2016 cleared the way for clinics to apply for permission to carry out the treatment.

Mitochondrial diseases are caused by inherited mutations in the DNA contained in mitochondria, tiny structures present in every cell. They are genetic conditions affecting the batteries of the cell, with about one in 4,300 affected children born every year. Symptoms include muscle weakness, blindness, deafness, seizures, learning disabilities, diabetes, heart and liver failure. There is no cure for mitochondrial DNA disease and affected children often die in early infancy.

Known as ‘mitochondrial donation’ the new IVF technique involves replacing faulty mitochondria inherited from the mother in the egg with the healthy mitochondria of another woman.

Following the HFTEA decision, the Newcastle team immediately applied for a licence to help patients who risk transmitting mitochondrial disease to their children by using the procedure, which transfers a tiny amount of DNA from the second woman’s egg into the first women’s egg.

The procedure will be carried out on women receiving treatment as an NHS service at the Newcastle Fertility Centre and the NHS Highly Specialised Service for Mitochondrial Disease and the team aim to offer treatment for up to 25 women a year affected by mitochondrial disease.

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During lengthy consultation, the HFEA considered such objections along with the safety of the procedure and concluded that the health benefits outweighed the ethical concerns.

“After a lot of hard work and invaluable advice from the expert panel, who reviewed the development, safety and efficacy of these techniques over five years and four reports, we feel now is the right time to carefully introduce this new treatment in the limited circumstances recommended by the panel.

“Although it is tempting to rush ahead with new treatments, the UK approach of testing public opinion, putting the issue to parliament and carefully monitoring laboratory research has proved to be the most responsible and sustainable of introducing new, cutting edge treatments into the clinic.

Such an approach has allowed us to balance innovation with safety, maintaining public trust as we go.”

Wellcome Director Dr Jeremy Farrar agreed that time needed to be taken to weigh up the issues, saying: “It’s right that there’s been such a thorough process to get to this stage. We must recognise, though, that for some affected families this decision may have come too late. The scientific community must continue to work closely with policy and the regulatory system so future innovative, cutting-edge techniques reach patients faster.”

For the Newcastle team, the priority has always been the welfare of families whose lives have been blighted by the disease.

Professor Mary Herbert from the Wellcome Centre for Mitochondrial Disease at Newcastle University and the Newcastle Fertility Centre, who led the IVF research leading to the developments, said: “It is enormously gratifying that our many years of research in this area can finally be applied to help families affected by these devastating diseases.”

Such views find an echo in comments from James Palmer, Clinical Director of Specialised Services at NHS England, who said: “Mitochondrial diseases can be devastating and life limiting as well as hugely costly to the NHS to treat.

This trial will, for the first time, give women living with mitochondrial disease the option of having a baby without passing on their condition and is a shining example of how the NHS is leading the world in developing cutting-edge innovative new medical interventions.”

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The science behind the breakthrough The new technique, called “early pronuclear transfer”, involves transplanting the nuclear DNA from a fertilised egg into a donated egg, which contains healthy mitochondria, on the day of fertilisation.

Extensive studies conducted in a collaboration between the Newcastle team and researchers from University of Oxford and the Francis Crick Institute indicated that embryos created using the new technique are indistinguishable from those created by conventional IVF. Analysis of thousands of genes in single cells detected no difference between the two types of embryos. There was also no increase in chromosomal abnormalities, which can cause miscarriage and birth defects. These findings provide reassurance that the new procedure does not have a harmful effect on early embryonic development.

causing mitochondrial DNA to the embryos. The importance of keeping carryover to a minimum is highlighted by studies on embryonic stem cell lines.

The team found that one of five stem cell lines derived from embryos created using the new technique showed an increase in the percentage of mitochondrial DNA carryover.

While stem cells are very different from embryos, the observation raises the possibility that faulty mitochondrial DNA may persist in some cases. However, the research team is optimistic that the new technique will be effective in reducing the risk of disease in children of affected women.

A further important finding of the study is that the technique will work best if patient, rather than donor, eggs are frozen. It will therefore be possible for affected women to have their eggs frozen for future use. This is likely to increase the success of the treatment by helping to avoid the decline in egg quality as women get older.

The research findings also indicate that the new technique will result in a minimal amount (less than 2%) carryover of disease-

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THE NEW TECHNIQUE WILL RESULT IN A MINIMAL AMOUNT (LESS THAN 2%) CARRYOVER OF DISEASE-CAUSING MITOCHONDRIAL DNA TO THE EMBRYOS.

DRUG DE | feature |

www.thebiosciencejournal.co.uk

market witnesses significant growth

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| BIOSCIENCE TODAY SPRING 2017 |

LIVERY www.thebiosciencejournal.co.uk

| BIOSCIENCE TODAY SPRING 2017 |

Developing new ways of delivering drugs has become big business with companies now investing many millions of pounds in seeking the next big breakthrough.

Indeed, market analysts Mordor Intelligence estimates in a new report that the European New Drug Delivery Systems (NDDS) market alone is estimated to have reached USD 46.78 billion in 2016 and will grow at 8.15% a year until 2021. That makes the European market for NDDS the second largest in the world, accounting for almost 30 % of the global market share.

According to Mordor, the drug delivery system market in the United States represents the highest share globally and is also expected to grow at a good pace until 2021.

Mordor Intelligence says that the growth is being driven by unprecedented developments in genomics and molecular biology, supported by a growing acknowledgement that the method by which a drug is delivered can have a significant impact on its efficacy.

According to the report: “The industries have diverted their research focus from the conventional dosage forms to novel drug delivery technologies that have significantly improved market requirements.

“In recent years, the pharmaceutical companies have

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been struggling to maintain a balance between the pressure to drop prices and the high innovation costs. It is always advantageous for companies to develop their own innovative delivery devices to maintain a lead in the market.

“The high cost associated with … reducing healthcare budgets is leaving no alternative, but to use pharmaceutical drugs effectively in order to cut costs.

“Novel drug delivery systems can help in using drugs more effectively. Targeted delivery systems, which can specifically target the diseased part of body, can be very helpful in treating some disease with low amounts of drug being consumed.

“In addition, it can also eliminate side-effects associated with excessive drug use or drugs accumulated at the wrong site, thus decreasing the overall cost associated with the procedure.” among areas witnessing rapid developments is cardiovascular disease, which accounts for more than 17 million deaths worldwide and is the primary cause of death globally.

Cancer is another area seeing advances in the delivery of drugs. According to the American Cancer Society, there were 1.6 million new cases and approximately 600,000 deaths due to cancer in the United States in 2015 alone.

Mordor says: “1.6 million new cases is a really high number; each of these is expected to have the potential of developing some form of side-effects during cancer treatment. This is expected to again lead to the rise in demand for novel drug delivery systems, which, in turn, are expected to drive the growth of the market.”

Among the technologies driving forward the advances is bioengineering, as illustrated by five studies presented to the recently-held 58th American Society of Hematology (ASH) Annual Meeting and Exposition in San Diego.

DELIVERY SYSTEMS (NDDS) MARKET ALONE IS ESTIMATED TO HAVE REACHED USD 46.78 BILLION IN 2016

They showed how researchers are applying advanced biomedical engineering methods to improve the delivery of life-saving treatments to sites in the body where they are needed most.

Armand Keating, professor of medicine and biomedical engineering and director of the Cell Therapy Program at the University Health Network in Toronto, Canada, said: “I believe each of these has the potential to change practice.

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All of these studies represent substantial advances resulting from biomedical engineering. They build upon established science with bioengineering strategies that could make therapies significantly more effective if they are pursued and refined.”

Among them, researchers have developed the first artificial red blood cells designed to emulate vital functions of natural red blood cells. If confirmed safe for use in humans, the nanotechnology-based product could represent an innovative alternative to blood transfusions that would be especially valuable on the battlefield and in other situations where donated blood is difficult to obtain or store.

DELIVERY SYSTEMS (NDDS) MARKET ALONE IS ESTIMATED TO HAVE REACHED USD 46.78 BILLION IN 2016

The artificial cells, called ErythroMer, are designed to be freeze-dried, stored at ambient temperatures, and simply reconstituted with water when needed.

Lead study author Allan Doctor, MD, of Washington University in Saint Louis, said: “One key goal is to advance field resuscitation of civilian trauma victims in remote settings and soldiers who are wounded in austere environments without access to timely evacuation.

“ErythroMer would be a blood substitute that a medic can carry in his or her pack and literally take it out, add water, and inject it. There are currently no simple, practical means to bring transfusion to most trauma victims outside of hospitals. Delays in resuscitation significantly impact outcomes; it is our goal to push timely, effective care to field settings.”

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Proof-of-concept studies in mice, conducted in partnership with Greg Hare MD, PhD, at the University of Toronto, demonstrated that the artificial cells capture oxygen in the lungs and release it to tissues — the main functions of red blood cells — in a pattern that is indistinguishable from that seen in a control group of mice injected with their own blood. In rats, ErythroMer effectively resuscitated animals in shock following acute loss of 40 percent of their blood volume.

The donut-shaped artificial cells are formulated with nanotechnology in partnership with Dipanjan Pan, PhD, at the University of Illinois at Urbana-Champaign, and are about one-fiftieth the size of human red blood cells. A special lining encodes a control system that links ErythroMer oxygen binding to changes in blood pH, thus enhancing oxygen acquisition in the lungs and then dispensing oxygen in tissues with the greatest need.

Tests show ErythroMer matches this vital oxygen binding feature of human red blood cells within 10%, a level the researchers say should be sufficient to stabilise a bleeding patient until a blood transfusion can be obtained.

If further testing goes well, the researchers estimate that ErythroMer could be ready for use by field medics and emergency responders within 10-12 years.

Development work has been supported by the Children’s Discovery Institute at Washington University and St. Louis Children’s Hospital, the Skandalaris Center at Washington University and the BioSTL Fundamentals Program.

“ErythroMer would be a blood substitute that a medic can carry in his or her pack and literally take it out, add water, and inject it.”

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APPLYING TO USE TISSUE FROM THE MCRC BIOBANK

If you wish to use the MCRC Biobank resource please make a formal expression of interest to the MCRC Biobank Business Manager (see details below) via phone or email, detailing a summary of the research idea, and types and numbers of samples required. From here, the MCRC Biobank will support you in making a formal application to the Biobank for samples.

Although the MCRC Biobank does not benefit financially from supplying samples to researchers, it does make a charge per sample to cover its costs. The cost of access to samples will be discussed when a formal expression of interest is made.

Please contact Jane Rogan, MCRC Biobank Business Manager on 0161 446 3659, or email jane.rogan@christie.nhs.uk for all enquires.

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Anti Inflammatory

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Property of PDRN An in Vitro Study on Cultured Macrophages Chiara Castellini, Silvana Belletti, Paolo Govoni, Stefano Guizzardi Department of Biomedical, Biotechnological and Translational Sciences— Section of Histology, University of Parma, Parma, Italy.

ABSTRACT Skin aging and most age-related diseases are associated with a low-grade chronic inflammation. The nucleoside adenosine, a potent endogenous anti-inflammatory agent, is deeply involved in inflammatory diseases and, by interaction with the adenosine A2 receptor (A2AR) it immediately promotes a mechanism of defence against the inflammatory damage.

The aim of our study was to investigate whether polydeoxyribonucleotide (PDRN), a mixture of deoxyribonucleotides polymers of different lengths that like adenosine, binds the A2A receptor, can reduce the inflammatory state in the macrophage cell line. RAW264.7, murine macrophage cells, were incubated with PDRN in the presence and in the absence of lipopolysaccaride (LPS), which was the major component of the outer membrane of gram-negative bacteria and which acted as a strong macrophage activator. We assessed the production of nitric oxide and the secretion of inflammatory mediators (i.e., TNF-α, IL-10, IL12 and VEGF-A). Our data showed that PDRN produced a significant decrease of inflammation in macrophages pre-stimulated with LPS, assessed in terms of the nitric oxide content (p < 0.001) and cytokines secretion (p < 0.001). Moreover, PDRN stimulated the release of the vascular endothelial growth factor (VEGF-A), which promoted wound healing. Our study suggested that PDRN, by binding the A2A receptor, contributed to a great extent towards reducing inflammation.

Keywords Cell Culture, Macrophages, RAW264.7, Inflammation, Skin Aging Castellini, C., Belletti, S., Govoni, P. and Guizzardi, S. (2017) Anti Inflammatory Property of PDRN—An in Vitro Study on Cultured Macrophages. Advances in Bioscience and Biotechnology, 8, 13-26. doi: 10.4236/ abb.2017.81002 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

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1. INTRODUCTION Aging is a multifactorial and progressive degenerative process defined as an accumulation of damage and is closely associated with inflammation. While the etiology of the aging process is not fully understood [1], chronic inflammation clearly plays a major role, inextricably linking inflammation and aging. Indeed, the levels of inflammatory mediators increase typically with age even in the ab- sence of acute infection or other types of physiologic stress [2].

Continuous up-regulation of pro-inflammatory mediators is induced during the aging process due to an age-related redox imbalance. It is likely that it is caused by the decisive effect of weakened anti-oxidative defence systems and the increased production of reactive oxygen species, such as hydrogen peroxide (H2O2) and reactive nitric oxide (NO) [2].

This imbalance that results in the low grade chronic proinflammatory state, is referred as inflammaging [3].

The process of skin aging is complex and results from a combination of intrinsic (or chronological) and extrinsic factors, i.e. UV radiation (or photoaging), cigarette smoking, air pollution and incorrect nutrition. Intrinsic aging is ac- companied by cell loss, thinning of the dermis and fine lines. Inflammaged skin is wrinkled, is associated with dyspigmentation and shows proteolytic activation and degradation of collagen and elastic fibres in the dermis.

Inflammation and the accumulation of Reactive Oxygen Species (ROS) are believed to be the main causes of skin aging [4] [5].

It has been suggested that monocytes and macrophages contribute towards in- flammaging more than any other cell type. Macrophages are heterogeneous and are present in most tissues where they are responsible for numerous inflammatory, immunological and metabolic processes [6]. Monocytes change with age and contribute to inflammaging by causing a functional shift towards a pro- inflammatory phenotype and reduced function [7]; indeed these cells increase the production of inflammatory cytokines and the lengthen of the immune response [8].

One of the major pathways by which the oxidative damage produced by free radicals promotes inflammatory responses, involves the Toll-like-receptors (TLRs). TLRs are a family of ubiquitously expressed receptors that play an important role in the innate immune response, particularly in the initial interaction between the infecting microorganism and phagocytic cells, such as macrophages [9].

TLRs recognize molecules that are crucial for the integrity of the microorgan- ism, but that are non-self molecules either due to their chemical composition or due to their cellular localisations [10]. Microbial cell wall components such as lipopolysaccharides (LPSs), endotoxins from the outer membranes of gram- negative bacteria, lipopeptides and flagellar proteins are recognized at the plasma membrane of immune and epithelial cells by TLR4, TLR2 and TLR5, respectively [11].

In particular, TLR4 activation by LPSs initiates an inflammatory response, whose key mediators are tumour necrosis factor-α (TNF-α) and interleukin-12 (IL-12), and secrete nitric oxide (NO), a short-lived free radical, which mediates many biological functions such as host defence, neurotransmission, neurotoxic- ity and vasodilation. In elderly persons, an increased level of these pro-inflammatory cytokines is often observed both in the general circulation and in tis-sue-resident macrophages [12].

In addition to the resolution of inflammation in response to pathogens or tis- sue damage, macrophages are also involved in the promotion of proper wound healing, by inducing the production of VEGF and angiogenesis.

These specific physiological functions derive from the plasticity of macrophages that allow them to change their form and function in response to environmental signals.

Current macrophage classification recognizes polarization according to two distinct phenotypes: classically (M1) or alternatively (M2) activated macrophages [13].

M1 macrophages are activated by pathogen-associated molecular patterns, such as LPS or by the T cell-secreted cytokine interferon gamma (IFNγ). They are of the proinflammatory phenotype, increasing the production of proinflammatory cytokines (e.g., IL-1, IL-6, IL-12 and TNF-α) and oxidative me- tabolites (e.g., NO and superoxide) to promote host defence.

M2 macrophages, on the other hand, are induced by a variety of stimuli (e.g., IL-4/IL-13 and glucocorticoids) and are of a phenotype involved in the promo- tion of wound healing, tissue remodelling and the resolution of inflammation [13].

Recently, Grinberg and colleagues have defined a new sub-type of M2-like macrophages, induced by the costimulation of macrophages with TLRs and adenosine A2A receptor (A2AR) agonists; this stimulation switches macrophages from the M1 phenotype to a M2-like phenotype, known as M2d [14] [15].

The nucleoside adenosine is an endogenous antiinflammatory agent and is a potent physiologic and pharmacologic regulator that is produced by cells in response to stress due to the breakdown of ATP. In particular, activation of the A2A receptor is one of the most fundamental and immediate mechanisms for pro- tecting tissues against inflammatory damage, altering the cytokines network by decreasing the secretion of inflammatory cytokines by macrophages in vitro [16].

Several studies have indicated that adenosine is involved in the modulation of the inflammatory process [17], e.g.

A2AR agonists inhibit cartilage damage, by diminishing IL-8 expression [18] or reduce the inflammatory response in articular chondrocytes [19].

Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides poly- mers of different lengths and is an A2A receptor agonist. PDRN is commonly used in clinical settings for pre and post-surgical cutaneous treatments, diabetic foot ulcers and venous ulcers because it stimulates fibroblast metabolism and promotes an increase in dermal matrix component production [20]. Other clinical studies have also pointed out that PDRN promotes faster healing of autologous skin grafts at donor sites [21] [22].

The aim of this work was to investigate whether polydeoxyribonucleotide (PDRN), an A2A receptor agonist, can induce an anti-inflammatory phenotype in murine macrophage cells RAW264.7, endowed with high endogenous TLR4 expression, focusing on nitric oxide production and secretion of inflammatory mediators.

2. MATERIALS AND METHODS 2.1 PDRN Polydeoxyribonucleotide (PDRN, Mastelli srl, Sanremo, Italy) is a DNA fraction containing a mixture of deoxyribonucleotide polymers of different lengths, comprised between 50 and 2000 base pairs. It was obtained from fish for human consumption by using Mastelli’s peculiar methods, by means of purification and hightemperature sterilization procedures that ensure a very high percentage of purified DNA and the absence of active proteins and peptides [23].

2.2 CELL CULTURE AND EXPERIMENTAL TREATMENTS Murine peritoneal monocyte-macrophage cells (RAW264.7 line) were obtained from the Cell Bank of the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna (Brescia, Italy). The cells were routinely grown in Dulbecco’s

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IT HAS BEEN SUGGESTED THAT MONOCYTES AND MACROPHAGES CONTRIBUTE TOWARDS INFLAMMAGING MORE THAN ANY OTHER CELL TYPE.

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For nitric oxide determination, cells were seeded in 96-well plates, at a density of 30 × 103 cells per well. For cytokine and VEGF secretion, cells were seeded in 24-well plates at a density of 15 × 104 cells per well. 24 h after seeding, the cell growth medium was replaced with fresh medium supplemented with PDRN (100 µg∙ml−1) in the presence and in the absence of LPS (from E. coli, O55:B5 serotype) 1 ng∙ml−1 or 10 ng∙ml−1 (from 100× stock solutions in DMEM).

The dose of PDRN was selected as suggested by previous research, reporting that, at the dose of 100 µg∙ml−1, A2A receptor agonists significantly suppressed LPS-in- duced inflammation [16]. Depending on the experimental setting, PDRN was added immediately (0 h) or after 1 h, 2 h or 3 h after the exposure to LPS and kept throughout the experiments.

2.3 CELL VIABILITY FOR NITRIC OXIDE DETERMINATION, CELLS WERE SEEDED IN 96WELL PLATES, AT A DENSITY OF 30 × 103 CELLS PER WELL.

Cell viability was assessed with the resazurin method [24].

Resazurin is a non- fluorescent molecule which is converted by intracellular enzymes into the fluo- rescent compound resorufin (λ em = 572 nm). After 24 h of incubation, cell via- bility was tested replacing the medium with a solution of resazurin (44 μM, Sig- ma Aldrich) in serum-free DMEM. After 20 min, fluorescence was measured at 572 nm with a multi-plate reader.

2.4 DETERMINATION OF NO PRODUCTION Nitrite concentration in the culture media, as an indicator of NO production, was determined 24 h after treatments with PDRN by means of a fluorimetric approach, based on the production of the fluorescent molecule 1H-naphthotriazole from 2,3-diaminonaphthalene (DAN) in an acid environment [25]. 100 μl of medium were transferred to black 96-well plates with a clear bottom (Corning, Cambridge, MA, USA). DAN (20 μl of a solution of 0.025 mg/ml in 0.31 M HCl) was then added. After 10 min at room temperature the reaction was stopped with 20 μl of 0.7 M NaOH. Standards were tested in the same medium from a stock solution of 1 mM sodium nitrite.

Fluorescence (λex 360 nm; λem 430 nm) was determined with a Perkin Elmer Enspire multi-plate reader (Waltham, Massachussets, USA).

KEY

3. MATERIALS Unless otherwise stated, all the reagents were provided by Sigma Aldrich, Milan, Italy.

4. STATISTICAL ANALYSIS The results are expressed as means ± standard deviation (mean ± SD). Statistical analyses were performed using GraphPad Prism® software version 5.00 (Graph- Pad Software Inc., San Diego, CA). The data were compared using one-way ANOVA tests and checked with Bonferroni’s post hoc tests. Results were consi- dered significant at p < 0.05 compared to cells treated with LPS (positive con- trol).

5. RESULTS 5.1 CELL VIABILITY The effects of PDRN, alone or in combination with LPS (1 - 10 ng∙ml−1), on the viability of RAW264.7 cells were tested using the resazurin assay after exposure for 24 h.

PDRN, both alone and with LPS (1 - 10 ng∙ml−1), did not significantly reduce cell viability, as shown in Figure 1.

5.2 NITRIC OXIDE PRODUCTION Figure 2 shows the data on the production of nitric oxide (NO), a major in- flammatory mediator produced by activated macrophages, assessed on the basis of the nitrite concentration in the medium after 24 h of treatment of RAW264.7 cells with LPS (1 - 10 ng∙ml−1) or with PDRN (100 µg∙ml−1). The effects of PDRN were more evident in the presence of LPS 10 ng∙ml−1 than that of 1 ng∙ml−1 and 150 CELL VIABILITY (% OF CONTROL)

modified Eagle’s medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), 4 mM glutamine and antibiotics (streptomycin, 100 µg∙ml−1; penicillin, 100 U∙ml−1) and kept in a humidified atmosphere of 5% CO2 at 37˚C.

Figure 1 (a)

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50

0 LPS 1 NG.ML-1

2.5 CYTOKINE SECRETION PDRN 2H PDRN 1H PDRN 0H LPS 1 NG.ML-1 PDRN CTRL LPS 10 NG.ML-1

The presence of Tumour Necrosis Factor-alpha (TN F-α), Interleukin-12 (IL-12) and IL-10 in the culture media of RAW264.7 cells was determined with ELISA RayBio® kits (Ray Biotech, Norcross, GA, USA) following the manufacturer’s in- structions. After the selected incubation times with PDRN (6 h for TNF-α and IL-10, 24 h for IL-12), 100 μl of medium were transferred into 96-well plates functionalized with anti-cytokine antibodies and incubated overnight at 4˚C. 100μl of biotinylated antibody were then added to each well and incubated for 1 h at room temperature, after which 100 μl of streptavidin solution were added. After 45 min, the samples were incubated with 100 μl of the TMB One Step Reagent for 30 min. Absorbance was then immediately read at 450 nm with a plate reader.

Standards were tested in the assay buffer from a stock solution (50 ng∙ml−1) of the recombinant cytokine.

2.6 VEGF-A SECRETION After 24 h of incubation with PDRN, the presence of Vascular Endothelia Growth Factor-A (VEGF-A) in the culture media of RAW264.7 cells was deter- mined with ELISA RayBio® kits (Ray Biotech, Norcross, GA, USA) and per- formed as above described. Standards were tested in the assay buffer from a stock solution (25 ng∙ml−1) of the recombinant protein.

150 CELL VIABILITY (% OF CONTROL)

PDRN 3H

Figure 1 (b)

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0 LPS 10 NG.ML-1

Figure 1. Effects of PDRN on cell viability in RAW264.7 cells.

Cells, grown for 24 h in complete culture medium, were treated with 1 ng∙ml−1 (a) or with 10 ng∙ml−1 (b) of LPS plus PDRN (100 µg∙ml−1), added either simultaneously or 1 - 2 or 3 h after stimulation with LPS. 24 h after the treatment, cell viability was determined using the resazurin assay. The results are expressed as the means of three independent determinations ± S.D.

Continued on page 46

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Figure 2 (a)

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Figure 3 (b)

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IL-12 (PG.ML-1 )

0 LPS 1 NG.ML-1

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CELL VIABILITY (% OF CONTROL)

***

40 10

Figure 2 (b)

10

0 10

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Figure 3 (c)

***

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20 0 LPS 10 NG.ML-1

Figure 2. Effects of PDRN on NO production in RAW264.7 cells. Cells grown for 24 h in complete culture medium, were treated with 1 ng∙ml−1 (a) or with 10 ng∙ml−1 (b) of LPS plus PDRN (100 µg∙ml−1), added either simultaneously or 1 - 2 or 3 h after stimulation with LPS. 24 h after the treatment, the nitrite concentration was determined in the culture medium of the cells. The experiment was performed three times with comparable results. The results are expressed as the means of three independent determinations ± S.D. ***p <0.001 vs. positive control, cells treated with LPS. PDRN, added 2 and 3 h after stimulation with LPS, induce in a decrease in NO production of 30% compared with cells treated only with LPS (p < 0.001).

5.3 SECRETION OF PRO- AND ANTI-INFLAMMATORY CYTOKINES The secretion of the pro-inflammatory cytokines TNF-α, IL-12 and anti-in- flammatory cytokine IL-10 was quantified after the treatment of RAW264.7 cells with LPS (10 ng∙ml−1) plus PDRN (100 µg∙ml−1), added either simultaneously or 2 h after stimulation with LPS, depending on the NO production results.

TNF-α and IL-10 were determined 6 h after treatments while the production of IL-12 was assessed after 24 h after treatments with LPS and PDRN.

2000

Figure 3 (a)

IL-10 (PG.ML-1 )

CELL VIABILITY (% OF CONTROL)

15

10

0 As shown in Figure 3, the exposure to PDRN added after 2 h of stimulation with LPS produced a significant and marked decrease both of TNF-α and of Figure 3. TNF-α, IL-12 and IL-10 secretion in RAW264.7 cells. Cells grown for 24 h in complete culture medium, were treated with 10 ng∙ml−1 of LPS plus PDRN (100 µg∙ml−1), added either simultaneously or 2 h after stimulation with LPS. The cytokines indicated above were measured after 6 h for TNF-α (a) and IL-10 (c) and after 24 h of treatment for IL-12 (b). They were measured in the extracellular medium, as described in Materials and Methods. The results are expressed as the means of three independent determinations ±S.D. ***p < 0.001 vs. positive control, cells treated with LPS. IL-12, by 47% and 16% respectively (p < 0.001). Our results showing that PDRN inhibit TNF-α and IL-12 production suggest that the beneficial effects of PDRN in inflammatory processes or injury may be attributed partly to this inhibition.

Moreover, PDRN produced a pronounced increase of IL-10 compared with stimulation with LPS (2-fold induction).

Anti-inflammatory properties of IL-10 are well documented and include the control of TNF-α release in vitro and in vivo [26]. The present study showed that PDRN is able to increase IL-10 production after stimulation with LPS and this phenomenon contributed to TNF-α inhibition.

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5.4 SECRETION OF VEGF-A

TNF-α (PG.ML-1 )

1000

*** 500

The effects of PDRN on VEGF-A production by RAW264.7 cells was evaluated 24 h after treatment with LPS (10 ng∙ml−1) plus PDRN (100 µg∙ml−1), added ei- ther simultaneously or 2 h after stimulation with LPS. As shown in Figure 4, PDRN markedly increases the secretion of VEGF-A by 42%.

0

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KEY CTRL PDRN LPS 10 NG.ML-1 LPS 10 NG.ML-1 + PDRN 0H LPS 10 NG.ML-1 + PDRN H

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6. DISCUSSION Age-associated low grade inflammation (inflammaging) is now recognized to be the driving force of many ageassociated diseases [27].

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Figure 4

***

CTRL PDRN

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VEGF (PG.ML-1 )

LPS 10 NG.ML-1 + PDRN H

In addition to their anti-inflammatory effects, A2A receptor agonists promote faster wound closure in murine models [36]. During wound healing, macro- phages play a key role in inducing angiogenesis, by producing the vascular endothelial growth factor-A (VEGF-A).

Regulation of the expression of VEGF-A via adenosine receptors has been demonstrated in several cell types, including endothelial cells and smooth mus- cle cells [37] [38].

LPS 10 NG.ML-1 LPS 10 NG.ML-1 + PDRN 0H

decrease of TNF-α and IL-12 secretion in LPS-stimulated macro- phages.

Leibovich and colleagues have shown that VEGF-A expression by murine macrophages was synergistically upregulated by LPS together with adenosine or A2AR agonists [39]. Moreover, the synergistic up-regulation of VEGF-A expres- sion did not occur in macrophages from mice that lacked the adenosine A2A but not the A3 receptor and the response did not occur in macrophages from mice that lack functional TLR4 receptors, indicating a critical role of the TLR4 receptor in the signalling pathway.

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0 Figure 4. VEGF-A secretion in RAW264.7 cells. Cells grown for 24 h in complete culture medium were treated with 10 ng∙ml−1 of LPS plus PDRN (100 µg∙ml−1). After 24 h of treatment, VEGF-A was measured in the extracellular medium, as described under Materials and Methods. The results are expressed as the means of three independent determinations ± S.D. ***p < 0.001 vs. positive control, cells treated with LPS. As we age, the innate immune system becomes dysregulated and is characterized by persistent inflammatory responses that involve multiple immune and non-immune cell types. This dysregulation involves both elevated levels of basal inflammation and an associated impaired ability to mount efficient innate and adaptive immune responses [28].

It has recently been established that inflammation and innate immunity play roles in skin aging process. The immune system protects the skin from infection, removes damaged cells and prevents undesirable autoimmune reactions; it may, however, promote the aging process, due to intrinsic (or chronological) and extrinsic factors, such as exposure to the sun, air pollution, cigarette smoking, bad nutrition and through the generation of Reactive Oxygen Species (ROS) generation released by macrophages [29].

Not only inflammatory states, but also ischaemia and tissue injury are pathological conditions in which intracellular ATP metabolism is accelerated, leading to the accumulation of intracellular adenosine.

Adenosine is a purine nucleoside that is released in response to metabolic stress and is a potent endogenous regulator, acting via four cell surface receptor subtypes (A1, A2A, A2B and A3).

In particular, adenosine A2A receptors are present in the monocyte-macro-phage cell line [30] and, as a result of interaction with adenosine or agonists, these agents have been shown to be able to modulate macrophage functions, such as nitric oxide (NO) production, and to regulate cytokine production both in vitro and in vivo [16] [31].

In this paper, we study the effects of polydeoxyribonucleotide (PDRN), an adenosine A2 receptor (A2AR) agonist, on RAW264.7, a murine macrophage cell line.

Our results showed that PDRN did not affect cell viability or cause a decrease in nitric oxide production when added to a macrophages pre-stimulated with relatively high doses of LPS (10 ng∙ml−1). Our data suggest that in an on-going state of inflammation PDRN has as anti-inflammatory effect.

Our results were consistent with other studies [26] [32] [33] [34] [35], since it was observed that PDRN markedly potentiated IL-10 production and induced a significant

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In view of the promising role of adenosine and adenosine receptors in im- proving blood supply and wound healing [40] [41], we assumed that PDRN might exert an important role in angiogenesis.

In an experimental model of ischaemic skin flaps, Polito and colleagues dem- onstrated that PDRN improved blood flow by stimulating VEGF expression and restored tissue architecture: flaps treated with PDRN showed a complete re- epithelialization and well-formed granulation tissue rich in fibroblasts [42].

Moreover, PDRN significantly reduced the wound surface area and improved the condition of the pressure ulcers, healed graft donor site and diabetic foot ul- cers by stimulating neoangiogenesis [21] [43] [44].

Our observations indicated that treatment of LPSstimulated macrophages with PDRN strongly increase VEGF-A secretion to well above the level induced by PDRN or LPS alone.

In view of this, binding of the A2A receptor is one of the most important mechanisms for the protecting tissue from inflammatory damage. An important finding of our study was, therefore, that triggering of the adenosine A2A receptor by PDRN may constitute a therapeutic strategy for counteracting and reducing inflammation and reactive species.

7. CONCLUSION To conclude, PDRN may constitute a new, active and safe anti-inflammaging treatment, acting as an antiinflammatory agent that can avoid or heal damage to the cellular and extracellular components.

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EVALUATION OF PA cell migration with multiple parameters in vitro by using an optical real-time cell mobility assay device

Akira Yamauchi1* , Masahiro Yamamura2, Naoki Katase3, Masumi Itadani1, Naoko Okada2, Kayoko Kobiki1, Masafumi Nakamura4, Yoshiyuki Yamaguchi2 and Futoshi Kuribayashi1

ABSTRACT Background: Migration of cancer cell correlates with distant metastasis and local invasion, which are good targets for cancer treatment. An optically accessible device “TAXIScan” was developed, which provides considerably more information regarding the cellular dynamics and less quantity of samples than do the existing methods. Here, we report the establishment of a system to analyze the nature of pancreatic cancer cells using TAXIScan and we evaluated lysophosphatidic acid (LPA)-elicited pancreatic cell migration.

Methods: Pancreatic cancer cell lines, BxPC3, PANC-1, AsPC1, and MIAPaCa-2, were analyzed for adhesion as well as migration towards LPA by TAXIScan using parameters such as velocity and directionality or for the number of migrated cells by the Boyden chamber methods. To confirm that the migration was initiated by LPA, the expression of LPA receptors and activation of intracellular signal transductions were examined by quantitative reverse transcriptase polymerase reaction and western blotting.

Results: Scaffold coating was necessary for the adhesion of pancreatic cancer cells, and collagen I and Matrigel were found to be good scaffolds. BxPC3 and PANC-1 cells clearly migrated towards the concentration gradient formed by injecting 1 μL LPA, which was abrogated by pre-treatment with LPA inhibitor, Ki16425 (IC50 for the directionality ≈ 1.86 μM). The LPA dependent migration was further confirmed by mRNA and protein expression of LPA receptors as well as phosphorylation of signaling molecules. LPA1 mRNA was highest among the 6 receptors, and LPA1, LPA2 and LPA3 proteins were detected in BxPC3 and PANC-1 cells. Phosphorylation of Akt (Thr308 and Ser473) and p42/44MAPK in BxPC3 and PANC-1 cells was observed after LPA stimulation, which was clearly inhibited by pre-treatment with a compound Ki16425.

Conclusions: We established a novel pancreatic cancer cell migration assay system using TAXIScan. This assay device provides multiple information on migrating cells simultaneously, such as their morphology, directionality, and velocity, with a small volume of sample and can be a powerful tool for analyzing the nature of cancer cells and for identifying new factors that affect cell functions.

Keywords: Migration, Chemotaxis, Lipid mediator, Inhibitor, TAXIScan, Metastasis © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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ANCREATIC CANCER BACKGROUND

METHODS

Migration of cancer cells correlates with distant metastasis and local invasion. This phenomenon involves various molecules including chemoattractants, trophic growth factors and their receptors, adhesion molecules, intracellular signaling molecules, motor proteins, and the cytoskeleton [1]. These molecules are orchestrated to help cells migrate to specific parts of the body or even spontaneously without an apparent destination. As cancer metastasis is directly associated with prognosis, controlling cancer cell migration is an effective strategy for treating the disease. Pancreatic cancer is among those with the poorest prognosis [2]. The treatment for this type of cancer is currently restricted as there are few effective drugs and knowledge regarding the nature of this cancer type is insufficient. New insights regarding this cancer and novel approaches for its treatment have long been awaited.

Lysophosphatidic acid (LPA) is a highly bioactive lipid mediator and is known to be involved in cancer cell migration, proliferation, and production of angiogenic factors [3]. In the process of cell migration, LPA works as a potent chemoattractant for various kinds of cells. Six receptors of LPA (LPA1, LPA2, LPA3, LPA4, LPA5, and LPA6) are known and all of them are G-protein coupled [4–9].

Some cells express one of these receptors, while others express multiple receptors for LPA [10]. Several articles have re- ported that pancreatic cancer cell lines express LPA recep- tors and the cells migrate towards LPA, using Boyden chamber and/or Transwell culture methods, which involve counting the number of migrated cells [11–13].

TAXIScan is an assay device for studying cell dynamics in vitro and has been used in the analysis of both suspension (mostly hematopoietic) and adherent cells [14–22]. The device functions as an optically accessible system and provides two-dimensional images of cell migration. TAXIScan provides markedly more information including morphology as well as quantitative analysis compared to existing methods such as Boyden chamber method. This device consists of an etched silicon substrate and a flat glass plate, both of which form horizontal channels each with a micrometer-order depth and forms 2 compart- ments on either side of a channel. Cells are placed and aligned on one side, while a stimulating factor is injected to the other side (typically 1 μL each of the cells and the stimulant).

The cells react to the stable concentration gra- dient of the stimulant inside the horizontal channel [14]. The cell images are observed thereafter and filmed with a charge-coupled device camera located beneath the glass. By analyzing the cell images, many parameters can be determined including velocity, directionality, etc. [23–26].

The objective of this study is to establish TAXIScan as a system for pancreatic cancer research by using pancreatic cancer cell lines and to evaluate cancer cell migration in vitro for understanding the characteristics of this cancer cell type and for identifying new drugs to regulate cancer cell migration. Here, we show the adherence of cells to the scaffolds as well as LPA-elicited migration by TAXIScan, and by an existing method, the modified Boyden chamber method (Transwell). The LPA-elicited migration was confirmed by checking the expression of LPA receptors and the effect of an LPA inhibitor Ki16425.

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REAGENTS Fetal bovine serum (FBS) was obtained from Nichirei Biosciences Inc. (Tokyo, Japan); RPMI1640 and D-MEM were from Sigma-Aldrich (St. Louis, MO, USA); Collagen I, Matrigel (growth factor reduced), fibronectin, laminin, and collagen I pre-coated coverslips were obtained from Becton Dickinson (San Jose, CA, USA); fatty-acid-free bo- vine serum albumin (BSA) from Nacalai Tesque (Kyoto, Japan); LPA from Enzo Life Sciences Inc. (Farmingdale, NY, USA); Opti-MEM from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Anti- LPA1, LPA3, LPA5, and LPA6 rabbit polyclonal antibodies from GeneTex Inc. (Irvine, CA, USA); anti-LPA2 rabbit polyclonal antibody from Abgent (San Diego, CA, USA); and anti-LPA4 rabbit poly- clonal antibody from Acris Antibodies Inc. (San Diego, CA, USA); Ki16425 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

MAINTENANCE OF CELLS Human pancreatic cancer cell lines BxPC3 (ATCC CRL1687), PANC-1 (ATCC CRL-1469), and AsPC1 (ATCC CRL1682) were obtained from the American Type Cul- ture Collection (ATCC), and MIAPaCa-2 (RCB2094) and KATOIII (RCB2088) from Riken Cell Bank. PC3 and 211H were kindly provided by Dr. Masakiyo Sakaguchi. Cells were cultured and maintained in RPMI1640 with 10% FBS or in D-MEM with 10% FBS on 10-cm diameter dishes as the standard procedure. Passaging of the cells was performed using PBS and Trypsin/EDTA solution when they were 80-90% confluent. All samples were handled according to the Declaration of Helsinki.

MIGRATION ASSAY The Real-time cell mobility assay was performed by optical real-time cell mobility assay device “EZ-TAXIScan” (ECI, Inc., Kawasaki, Japan) as described previously [20], except for assembling the TAXIScan holder together with a cover- slip pre-coated with the extracellular matrix.

Briefly, cover- slips were coated with collagen I (100 μg/ mL), Matrigel (1/30 diluted solution with culture medium), fibronectin (100 μg/mL), laminin (100 μg/mL), or the culture medium, by incubating 100 μL of each solution on a coverslip at room temperature for 1 h before assembling the TAXIScan holder. After collagen I was selected as the scaffold, collagen I pre-coated coverslips were used for the TAXIScan. The TAXIScan holder was assembled according to the manufacturer’s instructions. Cells were harvested by detaching from culture flasks using the same conditions as passaging. One μL of suspension prepared in the culture medium containing 2 × 106 cells/mL was applied to the cellinjection side of TAXIScan holder and the cells (100 or less in most of the cases) were aligned at the edge of the microchannel. After obtaining the first round of images, 1 μL of the chemoattractant solution prepared in the chemotaxis buffer was added to the ligand-injection side of the device to initiate migration. The assay conditions were as follows: duration, 4 h; inter- val, 5 min; micro-channel depth, 10 μm; and temperature, 37 °C. Time-lapse images of cell migration were stored as electronic files on a computer hard disk and analyzed when needed. The morphologies of migrating cells were depicted by tracing the edge of cells and then superimpos- ing the resulting outlines onto the initial

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ABBREVIATIONS BSA: Bovine serum albumin; EDTA: Ethylenediamine tetraacetic acid; EGF: Epidermal growth factor; Eph: Ephrin; FBS: Fetal bovine serum; IC50: The half maximal inhibitory concentration; InsR: Insulin receptor; IRS-1:Insulin receptor substrate 1; image. Movies of the images were made and quantification of velocity and directionality was carried out through the “TAXIScan analyzer 2” software. The trajectory of each cell on the image was traced by clicking the center portion of each cell on the computer display. The velocity (V) and the directionality (D) of each cell were calculated using the traced data as described previously [20, 23]. The statistical analysis for the velocity and the directionality was done by the Kruskal-Wallis Test (Non-parametric ANOVA) followed by the Dunn’s Multiple Comparisons Test, as the data did not show normal distribution in most cases [20].

The modified Boyden chamber method was performed using collagen I-coated polycarbonate membrane inserts (8 μm pore size) in a 24-well plate (CytoSelect 24-Well Cell Haptotaxis Assay kit, Cell Biolabs, Inc. San Diego, CA, USA) or Transwell Plate with non-coated polycar- bonate membrane (Corning Incorporated, Corning, NY, USA), per the manufacturer’s protocols. Briefly, the cells grown on a culture dish were detached with Trypsin/ EDTA solution, washed with PBS, and re-suspended in RPMI1640/HEPES buffer with 0.1% fatty-acid-free BSA (the chemotaxis buffer) to attain a density of 0.5 × 106 cells/mL. A total of 1.5 × 105 cells per well were placed in the upper chamber; the chemotaxis buffer with or without LPA was injected to the lower chamber, and then the plate was incubated at 37 °C for 2 h. The migrated cells were stained with the staining solution (supplied with the kit), observed under the microscope, and then lysed with the lysis solution (supplied with the kit) to quantify the number of migrated cells by measuring the absorbance at 560 nm. The absorbance was calibrated with the numbers of cells by using the standard curve with a series of different cell numbers (0, 10, 32, 100, 320, 1000, 3200, and 10,000 cells).

QUANTITATIVE REVERSE TRANSCRIPTASE POLYMERASE REACTION (QRT-PCR) Total RNA was extracted from the cells using the RNeasy kit (QIAGEN, Hilden, Germany). Cells were seeded on 10 cmdiameter dishes until 80-90% con- fluency was attained. On the day of the experiment, the medium was removed, and the cells were washed with 5 mL PBS, followed by addition of lysis solution, per the manufacture’s recommended procedure. Template DNA was prepared with extracted total RNA of each sample using Ready-To-Go You-Prime

First-Strand Beads kit (GE Healthcare, Little Chalfont, UK) and 0.5 μL each of 1st strand DNA per sample was used for quantitative polymerase reaction (qPCR) with Fast SYBR Green Master Mix reagent (Life Technologies, Carlsbad, CA, USA). Ana- lysis was done after preparing samples in a 96-well plate; signal during PCR was detected by Step One Plus Real- time PCR system (Life Technologies). The primers used are given in Additional file 1: Table S1. β-actin was used as an internal control for normalization of data. Data were analyzed by the software accompanied with the PCR system.

PROTEIN EXPRESSION AND PHOSPHORYLATION DETECTION Cells were seeded on 10-cm-diameter dishes until 80-90% confluency was attained. On the day of the experiment, cells were rinsed once with 5 mL of serum free Opti- MEM and then stimulated with 1 μM LPA prepared in the chemotaxis assay buffer (0.1%BSA in RPMI1640) pre- warmed at 37 °C for 30 s, 2 min, or 5 min. Immediately after stimulation, the medium was replaced with ice-cold chemotaxis assay buffer and cells were kept on ice until lysis was done. Cells were lysed with ice-cold lysis buffer from the PathScan RTK Signaling Antibody Array kit (Cell Signaling Technology, Danvers, MA, USA) per the manu- facture’s procedure. Cell lysate was kept at −70 °C until the PathScan phosphorylation array or SDS-PAGE/ western blotting was performed. For western blotting, each cell lysate was subjected to SDS-PAGE, blotting, and antibody reaction.

The pre-stained protein marker (Bio-Rad, Hercules, CA, USA) or the CruzMarker protein marker (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to estimate the molecular weight of probed bands. Protein bands were visualized with ECL prime (GE Healthcare) and detected

by LAS-4000 mini device (GE Healthcare). The list of the phosphorylated proteins for the array is shown in Additional file 2: Table S2.

RESULTS ESTABLISHING THE OPTICAL REAL-TIME MIGRATION ASSAY SYSTEM FOR PANCREATIC CANCER CELLS We established the assay system for pancreatic cells using optically accessible horizontal cell mobility assay device,

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LPA: Lysophosphatidic acid; MAPK: Mitogenactivated protein kinase; RTK: Receptor tyrosine kinase; SDS-PAGE: Sodium dodecyl sulfatepolyacrylamide gel electrophoresis; Tie2: Tyrosine kinase with Ig-like loops and epidermal growth factor homology domains-2

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IN THIS STUDY, WE ESTABLISHED A PANCREATIC CANCER CELL MIGRATION ASSAY SYSTEM

BY USING THE TAXISCAN DEVICE.

Fig. 1 Adhesion and migration of pancreatic cancer cells monitored

by TAXIScan. a Morphology of BxPC3 pancreatic cancer cells after adherence to each scaffold material coated on the coverslip without chemoattractant.

Images were taken 240 min after starting the assay. Scale bar: 10 μm. b Chemotaxis of BxPC3 pancreatic cancer cells towards 100 nM LPA with or without various kinds of scaffold-coating. Images taken at time 0, 120 and 240 min are shown. The morphologies of 4 or 5 representative migrating cells throughout the assays are shown on the “Trace” column. The outlines of the migrating cells were traced every 10 min in this column.

Cells migrating at more than 1 μm/min are shown in red. All data are representative of 3 independent experiments.

Scale bar: 100 μm

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EZ-TAXIScan. This device has been used for monitoring chemotaxis assays mostly for hematopoietic cells such as neutrophils, monocytes/macrophages, den- dritic cells, eosinophils, and lymphocytes [14–25]. In the case of adherent cells, like the cancer cells, additional procedures may be required for retrieving the optimal re- sponse from cells, such as scaffold coating [26]. Therefore, we compared different coatings on glass for facilitating pancreatic cell migration. Human collagen I, fibronectin, laminin, and Matrigel (growth factor reduced) were ex- amined as scaffold substances coated on the glass plate inside the TAXIScan chamber. Among these materials, collagen I and Matrigel showed good performances (Fig. 1) (An additional movie file shows this in more detail [see Additional file 3]).

Without coating, the cells did not attach well onto the glass plate (Fig. 1a) and did not show good migration (Fig. 1b).

On the glass coated with collagen I or Matrigel, most cells attached and spread well even without a stimulant such as the chemoattractant (Fig. 1a). On the glass coated with collagen I or Matrigel, BxPC3 cells migrated towards LPA (Fig. 1b).

LPA is known as a chemoattractant for cancer cells. To observe chemotactic migration of the pancreatic cancer cells towards LPA using the TAXIScan system, we used different concentrations of LPA to seek an optimal concentration for migration and observed that 1 μM of LPA was optimal for BxPC3 and PANC-1 cells (Fig. 2a) (An additional movie file shows this in more detail [see Additional file 4]). In the case of AsPC1 and MIAPaCa-2 cells, very few cells migrated towards LPA at the concen- tration ranging from 0.1 nM to 10 μM (only the 1 μM data is shown in Fig. 2a, an additional movie file shows this in more detail [see Additional file 5]).

receptors and the migration images of the cells reflects the LPA-elicited migration.

SIGNAL TRANSDUCTION DURING MIGRATION OF PANCREATIC CANCER CELLS TOWARDS LPA To further confirm that the migration was LPA-dependent, we determined phosphorylation of various molecules in BxPC3 and PANC-1 cells using the PathScan array, which enabled us to simultaneously evaluate the phosphorylation of 39 different molecules (Additional file 2: Table S2). We carried out phosphorylation assays at the time points 0.5, 2, and 5 min following LPA stimulation, due to uniform stimulation of cells by LPA on culture dishes, which precludes the use of an LPA concentration gradient similar to that of the TAXI Scan device. Using this array system, we observed that Akt (Thr308 and Ser473), p44/42MAPK, IRS-1, InsR, c-kit, EphA2, and Tie2 were phosphorylated after LPA stimulation in both BxPC3 (Fig. 4a, b) and PANC1 cells (Fig. 4c, d). Of these phosphorylated pro- teins, Akt and MAPK are known to be key molecules in- volved in migration and proliferation. The phosphorylation of these signaling molecules after uniform stimulation was further observed by western blotting. The results obtained showed that Akt (Thr308 and Ser473), p44/42MAPK were phosphorylated after LPA stimulation, as expected, in both BxPC3 and PANC-1 cell lines within 5 min (Figs. 4e and 5c). For the record, we also checked longer time points, such as 15, 30, 60, 120, and 240 min which were similar to

BxPC3 cells were the most responsive to LPA of all the cell lines studied. Therefore, we quantitated the directionality and velocity of migration of BxPC3 cells in response to different concentrations of LPA. The directionality in response to LPA increased in a dose-dependent manner (Fig. 2b left panel). The velocity also increased in a dosedependent manner in the dose range of 1 to 10 μM LPA (Fig. 2b right panel). These results were in agreement the TAXIScan images (Fig. 2a). We confirmed the same phenomenon by an existing assay method, the Boyden chamber method. In the Boyden chamber method, BxPC3 cells showed good response to LPA in a dose-dependent manner (Fig. 2c, left). The concentrations of LPA that elicited the migration of BxPC3 cells were observed to be similar in both methods.

EXPRESSION OF RECEPTORS FOR LPA ON PANCREATIC CANCER CELLS To confirm if the migration of cells was due to the LPAdependent phenomenon, we evaluated the expression of LPA receptors. Because most published reports showed either only mRNA expression or only protein expression [12, 13, 27], we attempted to show both mRNA and protein expression systematically by using qRT-PCR and western blotting. As LPA1, LPA2, LPA3, LPA4, LPA5, and LPA6 are the known receptors for LPA; we used primers for these receptor isoforms (Additional file 1: Table S1) [27] to compare their mRNA expressions. In BxPC3 cells, based on the results of qRT-PCR, LPA1 was the most highly expressed receptor among all the 6 receptors (Fig. 3a), whereas LPA2, LPA3, and LPA6 were moderately expressed and LPA5 showed the lowest expression. In PANC-1 cells, LPA1 and LPA3 were the major receptors expressed. In AsPC1 cells, the mRNA expression of LPA1, LPA2, and LPA6 were detected, and in MIAPaCa-2 cells, the mRNA expression of most LPA receptors was ex- tremely low. LPA3 expression was highest among the receptors for the MIAPaCa-2 cells (Fig. 3a).

We also evaluated the expression of these receptors at the protein level in the 4 pancreatic cell lines by western blotting using anti-LPA antibodies. All cell lines express a certain amount of LPA1, LPA2 and LPA3 receptors, however, very low expression of LPA4, LPA5, and LPA6 receptors was observed in lysates of all cell lines compared to 211H, KATOIII or PC3 which were used as positive controls (Fig. 3b). The data from the migration assay and western blotting indicated that BxPC3 and PANC-1 cells express the LPA

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the time points used in the TAXIScan experiments, but no additional increase in phosphorylation of these molecules was observed (Fig. 4e). These data further support the establishment of the assay system of cancer cell migrtion towards LPA.

EFFECT OF INHIBITOR ON MIGRATION TOWARDS LPA We also tested the effect of an LPA inhibitor, Ki16425 [28], on LPA-elicited migration of BxPC3 cells. When the cells were treated with Ki16425, the migration of the cells towards LPA was abrogated in a dose-dependent manner (Fig. 5a, b, an additional movie file shows this in more

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clarity in the TAXIScan chamber, and this phenomenon was vigorously supported by the inhibition of the intra- cellular signaling with Ki16425.

DISCUSSION

detail [see Additional file 6]). The half maximal inhibitory concentration (IC50) value for directionality was ≈ 1.86 μM (Fig. 5b, left graph). Owing to weak in- hibition of velocity

by Ki16425, the IC50 value for velocity was >100 μM (Fig. 5b, right graph). When the cells were treated 50 μM Ki16425, the phosphorylation of Akt and MAPK was reduced, as observed during western blot analysis (Fig. 5c). The pancreatic cancer cells showed LPA-elicited chemotactic migration with

In this study, we established a pancreatic cancer cell migration assay system by using the TAXIScan device.

We found that coating of scaffolds such as collagen and Matrigel on glass, similar to that in some published studies using other methods, was necessary for successful adhesion and migration. BxPC3 and PANC-1 cells migrated towards LPA in a dose-dependent manner, which was clearly inhibited by an LPA inhibitor, Ki16425.

This is the first report of pancreatic cancer cell migration monitored by the TAXIScan system that enables analysis of multiple parameters, including directionality, velocity, and cell morphology. Additionally, this is the first report simultaneously comparing the TAXIScan and Boyden chamber methods. The Boyden chamber method has been used for over 50 years [29], the limitations of this method have been pointed out by several researchers.

In this method, a membrane of 10 μm thickness, having holes of 8 μm diameter (in this study) with random density, separates the upper and lower wells (see Additional file 7). It is thought that cells are able to sense differences in the chemoattractant concentration between these two wells. Although this method appears simple, it has certain limitations. (I) The density of holes may not be uniform. (II) The micro- structure inside the hole, e.g., a micro-channel of 10 μm length × 8 μm diameter, is unknown, and the chemo- attractant gradient is not measurable. (III) A large number of cells is necessary for this assay (1.5 × 105 cells per well in this study). (IV) A considerable amount of chemoattractant is necessary (500 μL per well in this study), which is expensive. (V) The process of cell migration is not visible.

(VI) The device only displays the numbers of migrated cells. (VII) The obtained data may have high background noise. (VIII) The density of cells migrating to the lower side of the membrane is not uniform. A few advantage of this method are as follows: (I) It has a simple structure; (II) the apparatus itself (without coating materials) is inexpensive; and (III) it is well known and widely used. On the other hands, the advantages of TAXI- Scan are as follows [14] (see also Additional file 8): (I) it has an uniform micro-channel (260 μm length × 1000 μm width × 8 μm height); (II) the chemoattractant, which is placed on one end of the microchannel, defuses uniformly through the channel, resulting in a stable concentration gradient [14]; (III) a small number of cells is required for analysis (100 or less cells per channel); (IV) a small and inexpensive amount of chemoattractant is necessary (1 μL per channel); (V) migrating cells are observable; (VI) images obtained during migration are recorded automatically; (VII) data obtained from this assay including that on morphology, behavior, directionality, and velocity, are more informative. However, some demerits of TAXIScan are as follows: (I) although the running cost is low, the initial cost is high, and (II) it is not well-known yet. In fact, it may not be appropriate to position TAXIScan as an alternate to the Boyden method, because both methods utilize completely different equipment and data collection methods, and the quality of data obtained using these methods is entirely different (Additional files 7 and 8).

However, because of lower requirement of samples and the

Fig. 2 Chemotaxis of pancreatic cancer cells towards LPA detected by TAXIScan (a and b) or Boyden chamber (c). a Four pancreatic cancer cell lines were used for the TAXIScan method. Dose-dependency of BxPC3 chemotaxis towards LPA is observed. The migration images of PANC-1, AsPC1, and MIAPaCa-2 cells in the optimal conditions are also shown. Images taken at time 0, 120 and 240 min are shown. The morphologies of 4 or 5 representative migrating cells throughout the assays are shown on the “Trace” column. The outlines of the migrating cells were recorded every 10 min in this column. Cells migrating at more than 1 μm/min are shown in red. Data are representative of 3 independent experiments. Scale bar: 100 μm. b Quantitation of the directionality and velocity of migration of BxPC3 cells towards various concentrations of LPA. The graph on the left indicates the directionality and the graph on the right indicates velocity. White circles are outliers. Statistical analysis was done by the Kruskal-Wallis Test (Nonparametric ANOVA) followed by the Dunn’s Multiple Comparisons Test. Data are representative of 3 independent experiments. c Migration of BxPC3 cells towards LPA using Boyden chamber assay kit. The migrated cells were stained with the staining solution and the numbers of the migrated cells were estimated by measuring OD 560 nm based on the standard curve (the graph on the right). The assay results with the collagen I coated membrane (black bar) or the plain membrane (white bar) are shown in the graph on the left. Mean values of data are shown and the error bars represents the standard error (n = 6). Statistical analysis was conducted using the Student’s t-test. *p < 0.05 (vs. data without LPA)

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WE ESTABLISHED A NOVEL PANCREATIC CANCER CELL MIGRATION ASSAY SYSTEM THAT PROVIDES OPTICAL AND QUANTITATIVE INFOR- MATION SIMULTANEOUSLY.

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LPA RECEPTOR EXPRESSION IN PANCREATIC CANCER CELL LINES.

Fig. 3 LPA receptor expression in pancreatic cancer cell lines. a mRNA expression in 4 pancreatic cancer cell lines determined by quantitative RT-PCR. The relative expression of each receptor was calculated based on the LPA1 expression in BxPC3. Data represent mean values of 3 independent experiments.

The error bars represent the standard error. Statistical analysis was conducted using the Studentâ&#x20AC;&#x2122;s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 (vs. BxPC3). b Protein expression in 4 pancreatic cancer cell lines detected by SDS-PAGE and western blotting. A prostate cancer cell line, PC3, a gastric cancer cell line, KATOIII, and a pleural mesothelioma cell line, 211H, were used as positive controls. β-actin was used as a loading control and its expression is also shown.

The arrow-head indicates the specific bands of each LPA receptor. M, protein marker; Mia, MIAPaCa-2; Photographs are representative of 3 independent experiments. The intensity of each band was measured and the relative expression of each receptor protein was calculated based on the receptor in BxPC3 cells. Quantitative data represent mean values of 3 independent experiments except the positive controls PC3 and 211H. The error bars represent the standard error

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Fig. 4 Phosphorylation of receptors or signaling molecules. a and c Images of phosphorylation of receptors in BxPC3 (a) or in PANC-1 (c) cell lines detected

by Antibody Array. Data are representative of 3 independent experiments. b and d The quantitation of phosphorylation by measuring density of Antibody Array with BxPC3 data (b) or with PANC-1 data (d). e Phosphorylation of Akt or p44/42MAPK in BxPC3 and PANC-1 cell lines, as indicated. Cell lysates taken after LPA stimulation at each time point were analyzed by SDS-PAGE and western blotting. Anti-β-actin antibody was used as the internal control.

Arrows indicate the specific band for each antibody. Data are representative of 2 independent experiments. collection of more informative data, the approach to cancer cell migration using TAXIScan is more useful than analysis using existing techniques such as the Boyden chamber method. With the TAXIScan system, the charac- teristics of pancreatic cancer cells can be analyzed in de- tail.

Moreover, our system can be adopted for migration studies in other types of cancer cells.

In the Boyden chamber method, a certain number of cells without LPA was observed to migrate, indicating a high background (Fig. 2c), similar to that reported previously [30–33]. This high background with the Boyden chamber method is considered to be due to the thickness of the membrane (10 μm in this study). In TAXIScan method, cells without LPA were observed to migrate for more than 10 μm (up to 100 μm) (Fig. 2a), explaining this phenomenon. From this point of view, we could argue that TAXIScan has a wider dynamic range to detect cell migration.

Herein, 4 pancreatic cancer cell lines were analyzed and only 2 of these cell-lines, BxPC3 and PANC-1, showed good migration towards LPA with reasonable co-evidence on the expression of LPA receptors. The reason why AsPC1 and MIAPaCa-2 cells do not migrate towards LPA is still unknown. BxPC3 and PANC-1 do express LPA1, LPA2, and LPA3; however, these cell lines do not express LPA4, LPA5, and LPA6 as observed during western blotting (Fig. 3b). The latter 3 receptors are likely not involved in cell migration but might be involved in other cellular functions.

LPA inhibitor, Ki16425, shown in this study is believed to block human LPA1 and LPA3 receptors [28]; 10 μM of Ki16425 significantly blocked the migration of cancer cells [13]. In our system, Ki16425 clearly inhibited BxPC3 cell migration towards LPA at 5-50 μM concen- trations, indicating that TAXIScan and BxPC3 cells are the best tools for screening inhibitors of pancreatic cell migration.

Utilizing such a new method, new molecules for regulating pancreatic cancer metastasis can be identi- fied, and the limited treatment options and the poor prognosis of this disease can be overcome. Studies on neutrophils have tested various kinds of compounds and found that some compounds inhibit neutrophil function, leading to the successful selection of several effective molecules [34].

Collectively, it can be concluded that the system established in our study can be a powerful tool for cancer research and drug discovery in seeking effectors and inhibitors for analyzing cancer cell function. We are currently looking for and screening such mole- cules that can regulate pancreatic cancer cell migra- tion; some promising molecules will be reported in the near future.

Fig. 5 Inhibition of BxPC3 chemotaxis towards LPA by Ki16425. a BxPC3 chemotaxis towards 1 μM LPA with various concentrations of Ki16425.

Cells were pre-incubated with Ki16425 for 24 h and the chemotaxis assay was performed using TAXIScan. Data are representative of 3 independent experiments. b Box-plots of the directionality and the velocity in BxPC3 migration towards LPA with Ki16425. The graph on the left indicates directionality and that on the right indicates velocity. The half maximal inhibitory concentration (IC50) values are also shown. Statistical analysis was done by the Kruskal-Wallis Test (Non-parametric ANOVA) followed by the Dunn’s Multiple Comparisons Test. ***p < 0.0001 (vs. data with 1 μM LPA and without Ki16425). Data are representative of 3 independent experiments. c Inhibition of phosphorylation of Akt or p44/42MAPK by Ki16425 in BxPC3 and PANC-1 cell lines, as indicated. Cell lysates taken after LPA stimulation at each time point were analyzed by SDS-PAGE and western blotting. Anti-β-actin antibody was used as the internal control.

Arrows indicate the specific band for each antibody. Data are representative of 3 independent experiments

CONCLUSIONS We established a novel pancreatic cancer cell migration assay system that provides optical and quantitative infor- mation simultaneously. Using this system, we demon- strated that BxPC3 and PANC-1 cells showed good migration towards LPA. The effect of an LPA inhibitor, Ki16425, was detected clearly in this system, which was confirmed by the reduction in the phosphorylation of signal transduction molecules, Akt and MAPK. As this method provides a large amount of information on mi- grating cells simultaneously, such as their morphology, directionality, and velocity, with a small volume of sample, it can be a powerful tool for analyzing the characteristics of cancer cells and for evaluating factors affecting cellular functions.

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INHIBITION OF BXPC3 CHEMOTAXIS TOWARDS LPA BY KI16425.

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