Pro-Test Blogs!

When we think of the immune system we usually think of the adaptive immune system - the B-cells and T-cells that recognize and destroy specific pathogens – which isn’t surprising since this is the arm of the immune system that vaccines are designed to stimulate. However working alongside the adaptive immune system is the innate immune system which protects us form infection in a non-specific fashion. Key to this system are phagocytes, a diverse set of cells whose primary characteristic is their ability to consume and digest invading microorganisms and secrete a range of chemical messengers known as the proinflammatory cytokines which stimulate other components of the immune system. This usually a useful part of the immune response, but sometimes there is an excessive release of cytokines which causes the patient to enter a condition known as septic shock where the immune system over-reacts and causes serious tissue damage, eventually leading multiple organ failure. As a consequence of the increase in complicated surgery, implantable medical devices, elderly patients and patients with weakened immune systems, there has been an increase in the incidence of septic shock in recent years, and with around half of septic shock cases proving fatal it is now the number one cause of death in intensive care units.

This week a multinational team of scientists based in Bern, Frankfurt, Glasgow and Singapore, and led by University of Glasgow physician Professor Alirio Melendeza, have published a paper in Science (1) announcing an important development in the struggle to reduce the death rate from septic shock.

They had previously used in vitro cell culture techniques to identify an enzyme called sphingosine kinase 1 (SphK1) in human phagocytes and demonstrated using both RNAi and a specific inhibitor of SphK1 called 5c that SphK1 was involved in stimulating the cellular signaling pathways that promote release of proinflammatory cytokines. In this study they began by examining phagocytes isolated from 30 septic shock patients, finding that SphK1 levels were higher in these patients than in a control group. They next found that treating the phagocytes from septic shock patients with the inhibitor 5c blocked the production of proinflammatory cytokines by these cells in response to exposure to bacterial lipopolysaccharide, a molecule found on the exterior of some bacteria that usually provokes a strong inflammatory response.

The ability of 5c to block SphK1 dependent inflammation in-vitro was impressive but would the same happen in a whole organism where other pathways might promote inflammation? The team led by Professor Melendez next examined if 5c or RNAi could protect mice which were injected with an otherwise lethal dose of lipopolysaccharide, and they found that both methods of blocking the action of SphK1 did indeed provide complete protection against septic shock.

This was a very exciting result but acute, one-off exposure to lipopolysaccharide in vitro or in vivo is not the same as bacterial infection, where bacteria are multiplying and constantly interacting with the immune system to induce inflammation. Of course it is also vital that when turning down the inflammatory response the treatment doesn’t also compromise the immune system’s ability to fight the infection. The team therefore assessed whether pre-treatment with 5c or RNAi could prevent systemic inflammation and mortality from septic shock in a mouse model that simulates microbial infection in humans following surgery or injury, and not only was the immune system’s ability to combat the infection not compromised but the infection was cleared more quickly.

Pre-treatment is all very well but in the clinic treatment almost always starts after sepsis develops, so it was cheering to note that the inhibitor 5c reduced mortality when given up to 12 hours after infection it reduced mortality from septic shock, though it was most effective when given within 6 hours. This was as effective as the broad-spectrum the antibiotic co-amoxiclav, a standard treatment for sepsis, and when co-amoxiclav was administered along with 5c the combination was observed to be considerably more effective than either treatment used alone.

Professor Melendeza and his colleagues have identified an exciting new approach to reducing the toll from septic shock, hopefully work is already underway to translate this promising study from the bench to the bedside.

In other news the 2010 Kavli Prize in Neuroscience has gone to three scientists, Richard H. Scheller, Thomas C. Südhof, and James E. Rothman, who have “used a creative multidisciplinary set of approaches to elucidate the key molecular events of neurotransmitter release”. Their work, which involved the study of tissues from a variety of species including rats and marine rays and studies of knockout mice, has made a huge contribution to our understanding of how the release of the molecules that carry messages between the cells of the immune system work. This research may sit squarely in the realm of basic science, but the understanding of nerve cell communication that these three scientists have provided is now informing the development of new therapies for a wide range of psychiatric and neurological disorders.

Both these news items may at first seem unrelated, but what they have in common is animal research at the heart of a multidisclipinary approach that is increasingly typical of how biomedical science is done in the 21st century.
Paul Browne

1) Puneet P. et al. "SphK1 Regulates Proinflammatory Responses Associated with Endotoxin and Polymicrobial Sepsis" Science Volume 328(5983), pages 1290 - 1294 (2010) DOI:10.1126/science.1188635

One of the things that often strikes me when reading about medical advances or clinical trials is how variable the reporting of basic and applied research, including animal research, that underpins the clinical research is. In some cases it is discussed in some depth, but far too often it is either skimmed over or not mentioned at all. This is a shame since it makes it more difficult for readers to make the connection between what is happening in the clinic and animal research that may have begun years earlier. A few recent stories in the news illustrate this variability very nicely.

I’ll start with an excellent report by Miriam Falco on CNN entitled “Stem cell treatment goes from lab to operating room” which describes a clinical trial of fetal stem cells in the treatment of Amylotrophic Lateral Sclerosis (Lou Gehrig’s disease), a progressive neurodegenerative disease affecting the motor neurons that leads to severe muscle weakness and eventually death as the muscles that control breathing fail. As the CNN report points out research on rats was vital to the identification of the correct type of cells for this transplant, and Dr. Eva Feldman demonstrated that injecting fetal stem cells into rats with ALS preserved the large motor neurons and muscle strength.

'Lead researcher Dr. Eva Feldman, a neurologist at the University of Michigan, designed the trial just four years ago. After a lot of animal testing, her team determined that using fetal nerve stems rather than human embryonic or adult stem cells (such as bone marrow stem cells) was most effective, she says.

Stem cells have the ability to turn into different cells in the body. However, human embryonic stem cells, which come from 4- or 5-day-old embryos, also been found to sometimes turn into cancer cells. Fetal stem cells, such as those used in this trial, are a few weeks older and have already taken on a specific identity — in this case nerve cells.'

'Feldman says the fetal stem cells used in this trial did not become any of the unwanted cell types. “That’s very, very important,” she says.'

Basic animal research showed the potential of this therapy, but applied research also played an important part in making this clinical trial possible. Through studies on pigs Dr. Nicholas Boulis developed an apparatus that allows the stem cells to be injected at precise locations in the spine, and then practice the technique before attempting to use it on a human patient.

'Animal testing also proved very useful when it came to figuring out how to actually inject the stem cells. Emory University’s neurosurgeon Dr. Nicholas Boulis invented the device that holds the needle that injects the stem cells. The goal is to inject the cells without injuring the spine and causing even more paralysis. He practiced on 100 pigs before attempting the procedure on a human.'

Our second report is from the LA Times, and in an article entitled “A personal fight against a lethal childhood illness” reports on the work being done at the Centre for Duchenne Muscular Dystrophy at UCLA. It’s a nice report which shows how passionate scientists like Stan Nelson and Carrie Miceli are about finding effective treatments and cures for serious diseases. While the report does refer to experimental therapies such as exon-skipping and gene therapy it unfortunately does not discuss them or the research that led to their development in any depth.

Exon skipping is a particularly innovative approach to treating some cases of Duchenne Muscular Dystrophy (DMD) where the disease is due to a mutation in the dystrophin gene that stops translation from messenger RNA prematurely and prevents the production of the protein dystrophin. In exon-skippping a molecule known as an antisense oligonucleotide or morpholino acts to remove the portion of mRNA that contains the mutation and allows the translational machinery of the cell to read through and produce a working dystrophin protein. As I discussed in a post last year research in mice and dogs has been crucial to the development and refinement of exon-skipping and early versions of this therapy have already had promising results in clinical trials undertaken at Great Ormond Street Hospital in London and Royal Victoria Infirmary in Newcastle. Gene therapy, where the faulty dystrophin gene is replaced by a working version, is also being developed, though it has not yet entered human clinical trials. A recent review (1) available to read for free at PubMed Central discusses the progress that has been made in recent years, the challenges that remain before DMD can be cured, and the vital role played by animal models in overcoming these challenges. The review also covers stem cell therapy for DMD, another exciting approach to treating the disease that we have discussed previously.

The final news item is a BBC report on a successful clinical trial of stem cells to treat Multiple Sclerosis, this time using stem cells isolated from a patient’s own bone marrow. Multiple Sclerosis (MS) is an autoimmune disorder where the patient’s immune system turns on the myelin sheath that insulates the axons of nerve cells, leading to a range of often serious neurological problems. At present few effective treatments have been approved for MS, and several are currently being evaluated in clinical trials. While the improvements seen in the clinical trial were modest they do hold promise for longer and lager trials that are now being planned, and I suspect that as with other therapies the key might be to start treatment early to prevent damage as well as allowing damage to be repaired.

The trial at Frenchay Hospital in Bristol built on years of careful animal research, including research conducted by Professor Neil Scolding who lead this clinical trial. Interestingly the research, conducted in mice with experimental allergic encephalomyelitis that reproduces many of the features seen in autoimmune diseases that attack the myelin sheath, showed that rather than replacing the damaged cells that produce the myelin sheath or nerve cells the injected stem cells protected the myelin sheath and nerve cells by turning down the pathogenic immune response responsible for damaging the myelin sheath (2,3). This was important since it meant that it was not necessary to inject the stem cells directly into the site of the MS lesion, rather the cells could be as (if not more) effective if injected into the bloodstream so that migrate to tissues such as the lymph nodes where they can interact with cells of the immune system. This discovery paved the way for the clinical trial reported by the BBC.

There’s a lot of stories in the news that are relevant to animal research, the trouble is that it’s not always easy to see the connection. At Speaking of Research we believe that the onus is on scientists to make sure that when they talk to reporters they give the full picture of what their research involves, and what earlier studies it depended on. Only then can the public really begin to appreciate just how important animal research is to continued medical progress.

Paul Browne

1) Wang Z. et al. “Gene Therapy in Large Animal Models of Muscular Dystrophy” ILAR J. Volume 50(2), Pages 187-198 (2009). PMCID: PMC2765825

2) Matysiak M. et al “Stem cells ameliorate EAE via an indoleamine 2,3-dioxygenase (IDO) mechanism” J Neuroimmunol. Volume 193(1-2), Pages 12-23 (2008) DOI:10.1016/j.jneuroim.2007.07.025

3) Gordon D . et al “Human mesenchymal stem cells abrogate experimental allergic encephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration.” Neurosci Lett. Volume 448(1), Pages 71-73 (2008) DOI:10.1016/j.neulet.2008.10.040

Read more about Pro-Test for Science

On a beautiful sunny day in Los Angeles, Pro-Test for Science (A US side project of Pro-Test) organisers arrived at the junction of Le Conte and Westwood, on the edge of the UCLA campus, with armfuls of placards in support of animal research. Within ten minutes every placard had found a new owner as hundreds of scientists, students and members of the public showed up to support the cause. Those gathering chatted together, sharing their reasons for attending the rally.

Those participating were not limited to the UCLA community. Faculty from University of Southern California, California Institute of Technology, and California State University - Los Angeles, all came out to demonstrate their support for lifesaving medical research using animals. Soon the chants began to ring out - "Penicillin? ANIMAL RESEARCH! Insulin? ANIMAL RESEARCH! Vaccines? ANIMAL RESEARCH! Anaesthetics? ANIMAL RESEARCH!" A short while later, when the crowd had swelled further, the rally set off towards the center of the UCLA campus.

The mood was one of excitement and passion. Those participating exchanged ideas for public outreach in the future - sharing the best of ways of explaining to the public the clear connection between animal research and medical benefits. The rally continued to snake along Westwood and up towards Wilson Plaza.

As the rally turned into Wilson Plaza, passing the top of Bruin Walk, hundreds of students turned their heads towards the march, many shouting words of encouragement or joining in the rally.

Eventually the tail end of the rally reached the destination (some time after the front end due to the length), and Tom Holder brought the crowd together for a picture perfect moment of solidarity before shouting "What do we need?". "Animal Research" replied the hundreds of voices in unison.

Holder then introduced the first speaker, Prof. David Jentsch - founder of Pro-Test for Science and member of the Speaking of Research committee - who took the microphone to rapturous applause. David spoke of the progress of Pro-Test for Science, and the struggle against animal rights extremists in UCLA. He took the time to thank each of the individuals who had made the 2010 rally possible eliciting a cheer from the crowd as each name was called. Jentsch then passed over to Tom Holder, founder of Speaking of Research.

Holder thanked the crowd, insisting that UCLA were winning in their battle against extremists. However he warned the crowd against complacency - saying that public outreach was the only way to win this battle in the long run. Holder also announced the success of the Pro-Test Petition, which had gained 11,621 signatures over the previous year (including Nobel Prize Laureates, and every chancellor in the UC system, including UC President Mark Yudof). He finished by announcing the presentation of the signatures to Dr. Kevin Quinn, Dr. Michael Steinmetz.

Dr. Quinn, the Chief of Behavioural Science and Intergrative Neuroscience at the National Institute of Mental Health (NIMH), accepted one copy of the petition on behalf of NIMH. Quinn spoke of the important role that animal research has in our understanding of Mental Health problems:

"Animal research conducted in a humane, ethical and responsible manner is absolutely critical … to understand, treat and cure mental disorders"

Dr. Michael Steinmetz, program director of the National Eye Institute, talked of the medical breakthroughs in vision. He spoke particularly of Leber's congenital amaurosis, a form of blindess which affects thousands of people across the United States. Through research in mice and then dogs (Briards), scientists found a way of inserting a gene into the eye through a virus, which could corect the problem.

"The National Eye Institute supports strongly the use of appropriate animal models in research, not just for the big clinical advances but for the many, many years of basic science that it takes to discover the underlying biological principles"

Jentsch then returned to the stage to introduce UC Executive Vice-Chancellor, Scott Waugh. Waugh offered his continued support to researchers at UCLA, mentioning that the Pro-Test for Science movement has played an important role in bolstering support for research. He congratulated Jentsch and Ringach for organizing the February pabel debate, explaining that "violence, threats and other criminal activity are never a viable alternative to dialogue".

Jentsch and Holder finished the rally by talking of the importance of continued education and outreach.

A message from our US sister organisations: Speaking of Research and Pro-Test for Science:

Promoting Science and Rejecting Extremism

In 2009, Pro-Test for Science held an historic rally on the UCLA campus; bringing over 700 people onto the streets in support of the scientists and researchers who carry out lifesaving medical research using laboratory animals. Such research continues to advance scientific knowledge and plays a vital role in the development of innovative treatments for human disease. However, animal rights extremists have continued to escalate their threats against researchers and their families.

On Thursday April 8th Pro-Test for Science will respond by rallying students, scientists and members of the public to support the cause of medical science. We call on the community to stand together against the recent tide of animal rights activism which has worked to misrepresent research and coerce those that carry it out.

David Jentsch, founder of Pro-Test for Science, said:
“The scientific community has joined together to push back against those who seek to stall advances in biomedicine. Never before has it been more important to continue these efforts so that humane biomedical research can continue unhindered by the misguided threats of a minority who oppose it.”

This rally, on the UCLA campus seeks to:

• Communicate a better understanding of animal research to the public, its importance to medical progress, and what we all stand to lose if such work were to stop
• Celebrate the successes of animal research in the development of treatments for disease, new diagnostic procedures/instruments, and surgical techniques.
• Defend the rights of researchers to pursue their work free from harassment and intimidation.

The rally will begin on Thursday April 8th at 11:30 AM, on the north-east corner of Westwood Blvd. and Le Conte Ave., which will be followed by a march to Wilson Plaza, where speakers include UCLA Executive Vice Chancellor Scott Waugh and Dr. Kevin Quinn from the National Institute of Mental Health.

[youtube=http://www.youtube.com/watch?v=1V2RfzEu8g8]
Please check www.speakingofresearch.com and www.pro-test-for-science.org regularly for updates and further communications. For further information contact:
David Jentsch – j.david.jentsch@gmail.com – +1 310 825 8258
Tom Holder – tom@speakingofresearch.com - +1 310 498 0881

Notes to Editors:

1. The rally will take place from 11:30am, Thurday April 8th 2010 on the north-east corner of Westwood Blvd and Le Conte Ave.


2. Further speakers are to be confirmed


3. Report of the 2009 rally: http://speakingofresearch.com/get-involved/ucla-pro-test/ http://www.nbclosangeles.com/news/local-beat/West-Side-Story-Scientists-Activists-Face-Off-at-UCLA.html


4. Pro-test for Science was formed in March 2009 by David Jentsch:
   See www.pro-test-for-science.org

The new Harrison Ford film, Extraordinary Measures, hitting UK cinemas from 26 February, is a fictionalised account of the development of a treatment for Pompe disease, a rare genetic disorder. Pompe disease (glycogen storage disease type 2, acid maltase deficiency) is an enzyme deficiency with devastating effects – progressive muscle weakness and, in the severe infantile form, gross enlargement of the heart. Until fairly recently, the infantile form of the disease was invariably fatal within the first year of life. Now, however an effective treatment is in place.

While the increased awareness that the film’s fictional account brings is very welcome, the real story of how that treatment came about is a fascinating one (1) and laboratory animals play a starring role. The long road to a treatment started in 1932 with the first observation of the disease by Dr JC Pompe, after whom it is named. Pompe described accumulation of glycogen in muscle tissue, which was a puzzle, as the enzymes involved in the usual metabolism of glucose and glycogen were all present and correct. The solution to this puzzle had to wait until Christian de Duve’s 1974 Nobel Prize-winning discovery of lysosomes in 1955. These cellular compartments or organelles are the ‘recycling units’ of animal cells. They have an acid environment and their own specific set of enzymes for breaking down cellular components.

De Duve was carrying out ‘blue skies’ research, with no thought of direct medical application. However, as so often in research, a breakthrough in our basic understanding of biology led to medical applications. In this case, de Duve’s colleague Henri Hers realised that the deficiency of a lysosomal enzyme (alpha glucosidase) for the breakdown of glycogen would explain the symptoms of Pompe disease. This proved to be the case, and Hers established the principle of lysosomal storage diseases, of which around 40 have now been described, in 1965. Before moving on, let us note the role of laboratory animals in this breakthrough. I wrote to Professor de Duve and asked what part the use of animals had played in his work and he replied that “We would not have been able to make the discoveries we made without an extensive use of laboratory animals.”(2) - a statement confirmed by his Nobel Prize lecture.

Having discovered the basis of Pompe disease, the next milestone was to develop a treatment. This proved to be very difficult, largely due to the lack of animal models. A recurring refrain from the animal rights lobby is that if the humane use of animals in medical research was banned, scientists would soon find other ways to ensure medical progress. That comforting belief is belied by the series of attempts, some of them pretty desperate, to treat terminally ill children over the next 25 years. None of them worked.

The next great leap forward came from The Netherlands in 1990 and relied on the use of laboratory mice. Enzyme replacement therapy (ERT) had long been suggested as a potential treatment for lysosomal storage diseases but had never succeeded. In the case of Pompe disease, where large amounts of enzyme were needed in the muscle, introduced enzyme was simply soaked up by the liver. Two Dutch scientists, Arnold Reuser and Ans van der Ploeg, had the idea that phosphorylated enzyme would be taken via by the mannose-6-phosphate receptors in lysosomes, allowing the enzyme to be targeted.

However the supply of phosphorylated enzyme was small – nowhere near enough to treat a sick child. How could efficacy be demonstrated, in the absence of an animal model? In an ingenious experiment (3), they used specific monoclonal antibodies to demonstrate that when bovine phosphorylated alpha glucosidase was introduced to mice, it was taken up by heart and skeletal muscle lysosomes and caused a significant increase in enzyme activity – a 43% increase in skeletal muscle and 70% in the heart. An increase that, if repeated in humans, would result in the level of enzyme found in the healthy population. With characteristic understatement, van der Ploeg et al concluded "...we think that the original idea of enzyme replacement therapy for treatment of lysosomal storage diseases deserves new attention." At last, thanks to this ground-breaking work, a treatment for Pompe disease was a real possibility.

Now that there had been ‘proof of principle’ all that was needed was for a pharmaceutical company to spend millions of dollars in developing a treatment. Understandably perhaps, given the rarity of the disease and the inability to demonstrate actual efficacy, there was no immediate rush. Fortunately, at this point two animal models became available that allowed scientists to demonstrate that not only did the phosphorylated alpha glucosidase make its way to the lysosomes, it also had a beneficial effect.

From 1998 onwards, transgenic mice with Pompe disease, developed in Rotterdam and elsewhere, were used to demonstrated the efficacy of alpha-glucosidase enzyme. At the same time the potential of ERT was also illustrated, more dramatically perhaps, by YT Chen at Duke University, using quail. The quail had the same enzyme deficiency as found in humans, resulting in muscle weakness. After injection with the enzyme, they recovered to the extent of one subject actually flying around the lab (4). The evidence was therefore now pretty convincing – it was time for human trials.

The big problem was in producing enough enzyme for humans, even for babies. This required substantial industry investment. Two rival approaches were tried. A Dutch company, Pharming, produced the enzyme in the milk of transgenic animals for use in a trial led by Ans van der Ploeg, whose PhD research had led to the original breakthrough. The transgenic animal used was the rabbit, on the grounds that a human alpha-glucosidase-producing line could be established quite quickly. This work was used in a successful clinical trial, the results of which were published in The Lancet in July 2000 (5).

Another trail was carried out by YT Chen, using enzyme produced via Chinese Hamster Ovary (CHO) cell culture, by Synpac, a Taiwan-based company. This trial was also successful.

What follows next is a slightly convoluted story. The short version is that a third company, Genzyme, with an existing enzyme replacement therapy for Gaucher disease, bought out both Pharming and Synpac. In the end, they didn’t use either of the enzymes produced by these companies but developed their own, in-house CHO product, now marketed as Myozyme. This was a difficult decision – how could they decide which of the competing products should be invested in to produce a commercial treatment? The answer was what Genzyme called “The mother of all experiments” which compared the different products in transgenic Pompe mice. The result led to the availability of the treatment we have today.

However, the eventual production system is a technicality that need not concern today’s patients. Their concern is that an untreatable, terminal illness is now treatable. If you go and see Extraordinary Measures do bear in mind the starring role that doesn’t appear in the cast list – that of the mice and quail that made this treatment possible.

Kevin O’Donnell

Edinburgh

1. http://pompestory.blogspot.com
2. Letter from Christian de Duve to Kevin O’Donnell, 4 March 1997
3. Intravenous Administration of Phosphorylated Acid Alpha-Glucosidase Leads to Uptake of Enzyme in Heart and Skeletal Muscle of Mice http://www.jci.org/articles/view/115025
4. Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail http://www.jci.org/articles/view/1722/pdf
5. Recombinant human alpha-glucosidase from rabbit milk in Pompe patients http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(00)02533-2/fulltext#article_upsell (free registration required)

Biographical note.

I should declare an interest. I am a professional scientist, however my involvement with Pompe disease dates from the diagnosis of our first child, Calum, with infantile Pompe disease in 1993. At that time the disease was still untreatable and Calum died at 8 months of age. Following that I had the great privilege of participating in an international community of patients and scientists that championed the development of a treatment for Pompe disease. They don’t appear in the cast list of the film either, or the book on which it is based The Cure by Geeta Anand. This prompted me to write the real story down – I think it’s a better story than either rthe book or the film though not, sadly, as well written. Your comments welcome at http://pompestory.blogspot.com You can find out more about Pompe disease from the following sites:

International Pompe Association www.worldpompe.org
Acid Maltase Deficiency Association www.amda-pompe.org
UK Pompe Group www.pompe.org.uk
Genzyme www.pompe.com