Category Archives: Science News

Guest Post: How do birds see the world?

Professor Aaron Blaisdell

Professor Aaron Blaisdell

Today’s guest post is from Professor Aaron Blaisdell and graduate student Julia Schroeder in the Department of Psychology at the University of California Los Angeles. Prof. Blaisdell’s area of research is animal learning and comparative cognition. He received his Ph.D. in Experimental Psychology and Behavioral Neuroscience at Binghamton University in 1999. Julia Schroeder is a graduate student in the Psychology Department at UCLA. This project is the basis of her dissertation research which she hopes to complete by May, 2016. She received a BS in Psychology at Whitman College where she compared rational decision processes in pigeons and humans. You can support their research through their crowdfunding campaign.

How do birds see the world?

How do birds fly around objects without crashing into them? Their object perception must be similar to ours, despite having a dramatically different brain and separate evolutionary history. Birds and mammals share a last common ancestor roughly 275 million years ago! Nevertheless, most birds and mammals, especially primates, rely on sight to navigate their world, find mates, avoid foes and predators, seek food and water, and care for their young.

Vision, both sensation and perception, has been one of the top areas of research in experimental psychology and neuroscience, going back to the visual psychophysics scientists of 19th century Germany. Visual perception and cognition is currently a dominant area of study in cognitive neuroscience. Much of what we’ve learned about human vision actually comes from research in nonhuman primates, especially the macaque monkey. This makes sense, since the human visual system is like that of just monkeys and apes.

One picture that has emerged is that, when we open our eyes, we see a world populated with objects. Our object-centered view of the world is also shared with the rest of the primates.

Pigeon in a test of comparative cognition.

Pigeon in a test of comparative cognition.

What about birds? They also navigate their world using vision. Flying puts high demands on the ability to rapidly detect and process visual information. The last thing a birds wants to do is to fly into an object because it couldn’t see it in time! This suggests that bird brains also engage in visual computational processes similar to that of the primate. But we currently don’t know much about how they do so. We want to know if birds solve the incredibly complex computational process of object perception the same way that primates do.

In our next research project, we plan to test whether bird brains handle object perception the same way that the human brain does. Pigeons will play a video game where they have to rapidly peck objects as they appear on a computer touchscreen located in a Skinner box. As soon as the object is pecked, a small food reward will be delivered to the pigeon from a hopper located below the screen. The faster the pigeons peck at the object, the sooner they get fed. The speed of their responses will tell us how the birds see the objects.

Specifically, we will show the pigeons four different objects, A, B, C, and D, one at a time (actual objects are different colored geometric shapes). The objects will appear in one of four locations on the screen (see a demo here). The objects will appear in a specific order that repeats. The locations in which the objects appear will also repeat. This will allow us to test how pigeons bind features into objects. If pigeons integrate features as humans and other primates have been shown to do, then they should learn that specific objects always appear in a specific location. This is called object-place learning. The object’s identity and location become bound as shared properties of a unique, coherent object. After the pigeons learn to play the game, we can then test for object-place learning by presenting special non-reinforced probe test trials. On these test trials, we will change the order of some of the objects, locations, or both.

Stimuli in learning sequence.

Stimuli in learning sequence.

Changing only the object or location should break the object-place association. Changing both together, however, preserves the object-place association, even though the sequence order has changed. If, like humans, pigeons bind object and location information together into perceptual memory, then changing only the object or the location order should be more disruptive than changing both!

What is life like for a laboratory pigeon?

Like all other vertebrates in research, housing and laboratory conditions for pigeons are well regulated. All research protocols go through the same stringent processes of review by the University’s IACUC, and the health and welfare of each pigeon is overseen by the Division of Laboratory Animal veterinarian staff. They receive the best possible care. In a typical pigeon laboratory, the pigeons are maintained as part of a flock in a vivarium. Birds are typically individually housed in large, comfortable cages, with constant access to water and grit. Feeding times are typically restricted to the afternoon after all subjects have completed their behavioral training. This keeps them motivated to work for food reinforcement in the operant chamber, and maintains subjects at a healthy weight similar to that of pigeons in the wild. Despite being housed in individual cages, the birds can see, hear, and smell the birds in the surrounding cages, thereby simulating a flock as it would be found in the wild. Unlike most mammals, or even parrots, pigeons do not engage in much touching or grooming of each other. Rather, pigeons in a flock hang out in close proximity to one another.

While some labs acquire wild-caught pigeons from their local area, we purchase ours from a vendor that breeds pigeons and other fowl for research purposes. Pigeons are a domesticated species, having lived in human environments since the dawn of agriculture in the Mediterranean region of Europe, Asia, and North Africa. Darwin was a known pigeon fancier, and bred pigeons as part of his own experimental investigations into the process of evolution by natural selection! To this day, there are pigeon fanciers and clubs around the world that breed pigeons for show, racing, and aerial acrobatics.

Why is this research significant?

The bird brain has a very different organization than the brains of humans and other mammals. Birds don’t have a visual cortex, for example. Thus, our research can lend insight into how a brain of such different structure solves the same computational process as does the mammalian brain.

Also, the brain of a pigeon is the size of your thumb! So how can birds, like pigeons, see objects the way that we do with far fewer neurons than in the human or monkey brain? Knowing how birds see the world can tell us a lot about what is unique about human vision, and what we share with other species.

Finally, we can also use our knowledge of how small bird brains efficiently create visual objects out of messy input to find new and powerful ways to build artificial visual systems for small mobile devices, such as drones and robots.

Many neuroscientists believe object perception is one of the most important and central processes of human vision. Nevertheless, object vision has been incredibly difficult to build into robot vision using AI approaches. Perhaps we can reveal the secrets to complex object perception in the small pigeon brain that will allow for breakthroughs in computer vision. This would be a huge win for human society!

Aaron Blaisdell and Julia Schroeder

Behind the Scenes of Zebrafish Research

Today we have the 2nd in a series of articles by Jan Botthof, a PhD Student at the Cambridge University Department of Haematology and the world renowned Wellcome Trust Sanger Institute. Following his first article “Zebrafish: the rising star of animal models”, Jan discusses here how Zebrafish used in scientific research are housed, cared for and bred.

Today I am going to look at some of the things that have to happen in the background to allow scientists to carry out their research. These things include the rules and regulations covering zebrafish use in research, general care and daily work in the fish facility.

Regulations
Zebrafish research in the UK is covered by the same laws that govern research on all other vertebrates, as outlined in the Animals (Scientific Procedures) Act (ASPA), originally instated in 1986 and recently revised to implement the provisions of the new EU directive. This means that the standard of care is just as high for fish as for mammals. All institutes housing fish need a licence ensuring that standards are met, every research project is evaluated for possible harm to the animals and all of the people involved in research or care for the fish receive mandatory training in order to ensure that the fish are treated correctly. Everyone takes utmost care to ensure that the fish lead a comfortable life in the zebrafish facility.

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish housing
Now that we have covered the basic legislation, let’s talk about essential zebrafish care. Nowadays, fish are usually housed in special rooms, unlike the beginning of their use in research back in the 1970’s, when they were commonly just kept in a few tanks on a shelf in the lab. These rooms are designed to keep a constant temperature (between 24 and 28°C depending on the institution) and the lights are programmed to give a constant light-dark cycle to simulate the sun (usually around 16 hours of light and 8 hours of darkness).

Various commercial fish housing systems exist, but most of their features are very similar. The basic components of such a system are the fish tanks, racks to hold them, an integrated water supply, as well as water filtration and monitoring components.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

The tanks are designed to allow a constant inflow of fresh water, easy removal from the rack and convenient access for feeding. These tanks used to be made of glass, but currently different kinds of plastics are much more popular due to the lower weight, making it much easier to handle them. Unless a procedure requires identification or separation of a specific fish, they are always kept in groups, not only for practical reasons, but also because zebrafish are very social animals and need interaction with other fish.

The water filtration and monitoring system ensures that the water is free of contaminants, has the right pH, salinity, hardness, enough dissolved oxygen and does not contain too much nitrite and nitrate stemming from waste products (i.e. fish excretions, excess food). Apart from the constant flow of fresh water, tanks are cleaned regularly to prevent the accumulation of waste products, as well as microbial and algal growth.

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

A separate quarantine room is also very important. This is where incoming fish from outside facilities are kept on a separate water system to prevent the introduction of parasites and diseases into the main facility. These fish are preferably received as early embryos, which are disinfected before shipping to kill any germs.

Diet
Just like there are different housing systems, there are different possible food sources, ranging from commercially available dry fish flakes to adult or larval brine shrimp. This diet is often supplemented with paramecia (small single celled organisms) to achieve optimal growth and survival rates when the fish are raised to adulthood. Exactly which diet is chosen depends on the individual facility. At the Sanger Institute, fish are fed adult brine shrimp, which are very rich in protein, soft and easily digestible (especially compared to brine shrimp cysts) and they are able to survive and swim even in fresh water. This is much closer to the natural diet that the fish would obtain in the wild than most commercially available diets.

Brine Shrimp. Image: Hans Hillewaert

Brine Shrimp. Image: Hans Hillewaert

Disease prevention
During the daily cleaning and feeding tasks, all tanks are monitored for diseased or injured fish, which are then humanely euthanized to minimize suffering. Euthanasia is usually carried out using an overdose of a common fish anaesthetic followed by destruction of the brain to ensure the death of the animal. Detailed records are kept to identify recurrent problems, such as potential parasitic infections. Dead fish are also removed from the tanks and their data recorded. This monitoring is especially important for fish that have been treated with drugs or that carry mutations likely to cause disease.

Breeding
One essential component of working with fish is setting up matings between them. This is essential if you want to obtain embryos for studying them, or when crossing different genetically modified lines and many other procedures. Fish are placed in small mating tanks in the late afternoon before the actual mating, as zebrafish begin to mate right after sunrise in the wild. These mating tanks have a removable insert between the fish and the floor of the tank, so the fish cannot consume their own eggs, which they would otherwise do.

It is also possible to tilt the separation between the floor of the tank and the fish to further stimulate the fish, as they prefer shallow water for egg laying. If you need the embryos at a specific stage you can use tanks with a separator between the male and female, so you can control the time of the mating. An occasion when you would need to do this is when using the gene editing technique CRISPR to modify zebrafish genes, a process which requires injections of the Cas9 enzyme and appropriate guide RNAs during the first stage of development. Matings can be done in small groups or in pairs. It is very important to be able to correctly identify the sex of the animals – not only do you obviously need a male and a female to have a successful mating, but you also need to know this when you combine different transgenic lines. Here you would take a male from one line and a female from another, so you can put them back in the correct tank after the mating, as it is otherwise nearly impossible to identify individual fish.

Zebrafish mating tank with removable separation before and after assembly.

Zebrafish mating tank with removable separation before and after assembly.

Once the eggs have been laid and fertilized, you can collect them in a sieve, and place them in petri dishes containing water with some salts and minerals essential for development. Embryos are then raised at 28.5°C. Here at the Sanger, the zebrafish larvae are placed in nursery tanks when they are five days old and the yolk that feeds them during early development has run out. These fry reach sexual maturity within three months, from which point on they are considered adults and housed in the main facility. Zebrafish in the lab can live about two to three years, but usually we use younger fish for breeding, as they  lay more eggs.

In summary, a lot of work needs to be done before any actual research can be carried out. Moreover, a lot of effort is put into ensuring the health and welfare of all laboratory animals. The next time you read about some exciting new discovery made using animal research, try to picture how much effort was needed before any actual science was done!

Jan Botthof

Cotton Rats, Calves and Clinical Trials: New RSV vaccine shows great promise.

Respiratory syncytial virus (RSV) affects almost two-thirds of babies in their first year of life, and is a leading cause of bronchiolitis and severe respiratory disease in infants, young children, immunocompromised individuals, and the elderly throughout the world. It is a major cause of hospital admission for infants, and results in up to 200,000 deaths per year in children under the age of 5 years worldwide. Development of an effective vaccine is a public health priority, but has proven difficult, in part due to fact that RSV infection cannot be easily studied in the standard mouse and rat species that are most commonly used in laboratory research.

Oxford University researchers have announced the successful completion of the first trial of a vaccine against RSV in adult humans, which indicated that the vaccine was safe and could induce a robust immune response (though this Phase 1 study did not evaluate its ability to protect against RSV).

In winter RSV accounts for more than 10% of UK infant hospitan admissions.

In winter RSV accounts for more than 10% of UK infant hospital admissions.

In two papers published back-to-back in the journal Science Translational Medicine this week, the University of Oxford team and their colleagues at The Pirbright Institute and the Italian biotechnology firm Okairos (now Reithera Srl) report on the successful Phase 1 clinical trial in adult human volunteers, and the animal studies that led to the trial (1,2).

The basis for their vaccine was a vector derived from the chimpanzee adenovirus PanAd3, which was modified to express several highly conserved human RSV (HRSV) proteins, which they had shown could provide a good level of protection against RSV infection in cotton rats, which are one of the less commonly used laboratory animals, but a very useful model of viral infection of the respiratory system. The cotton rat immune system more closely resembles that of humans at a genetic level than that of more commonly used laboratory mouse and rat species, and because of this similarity cotton rats have been successfully used to evaluate novel therapies for RSV prior to clinical trials, including predicting the efficacy of these drugs in children. As we discussed in an earlier post on the development of gene therapy for Hemophilia B, choosing the right adenoviral vector for the task is critical, and the chimpanzee adenovirus was chosen because there is no preexisting immunity to it in the human population that would compromise its effectiveness as a vaccine vector.

Because studies with other virus-vectored vaccines had shown that heterologous prime/boost with Chimpanzee adenovirus based vector followed several weeks later by a modified vaccinia Ankara (MVA) – a vector used in several experimental vaccines, including HIV vaccines – generates a stronger  immune response than with chimpanzee adenoviral vector alone, the researchers next examined this strategy in cotton rats, demonstrating that intranasal prime/boost immunization was effective in protecting against infection, and did not lead to adverse effects.

The cotton rat - a valuable model for studying lung disease. Image: J.N. Stuart

The cotton rat – a valuable model for studying lung disease. Image: J.N. Stuart

While the cotton rat is a valuable model for the study of respiratory infections, it does not demonstrate all the clinical features of RSV infection, the infection tends to be less severe and to be cleared more quickly in cotton rats than in human infants, and  the extent to which vaccine efficacy in the cotton rat model of HRSV can predict efficacy in humans  is unclear. The scientists therefor evaluated the prime/boost vaccine in a more stringent model, calves, which are the natural hosts of the bovine form of RSV – called bovine RSV (BRSV). The disease course and epidemiology of BRSV infection in calves is very similar to that of HRSV in children, and the very high degree of similarity in sequences of the BRSV proteins to their HRSV equivalents used to develop the vaccine  suggested that the calf would be a valuable preclinical animal model to evaluate the safety and efficacy of their prime/boost vaccine strategies (2).

The results of this evaluation indicated that the different vaccination strategies they assessed were safe and did not cause any adverse immune responses, and showed that heterologous vaccination strategies that used an intranasal administration of the  PanAd3-based vector followed by intramuscular administration of the MVA-vased vector provided superior protection against BRSV infection compared to the PanAd3-based vector alone, or repeated doses of the PanAD3-based vector. As the authors described in the article reporting on the successful phase 1 trial (1) noted in their introduction, these studies paved the way for the evaluation of this vaccine strategy in 42 human volunteers:

In developing this approach toward an RSV vaccine in humans, homologous and heterologous combinations of PanAd3-RSV, including IN vaccination route, and MVA-RSV were tested in preclinical models. The genetic vaccines elicited RSV-specific neutralizing antibodies and T cell immunity in nonhuman primates and protective efficacy in challenge experiments in rodents with human RSV and in young seronegative calves with bovine RSV (32, 33). Of critical importance in both rodent and bovine challenge models was the absence of immunopathology associated with ERD after vaccination, with the calf model acting as a translational model for the development of a vaccine for the pediatric population. All regimens fully protected the lower respiratory tract from bovine RSV infection in the calf, and heterologous combinations resulted in sterilizing immunity in both upper and lower respiratory tracts (33).

The demonstration that the prime/boost vaccine strategy developed by the Oxford University team can safely induce a strong immune response in adult humans, and protect against RSV infection in both the cotton rat and calf models, is very promising, and pave the way for further clinical trials.  Professor Andrew Pollard of the Oxford Vaccine Group, who lead the clinical trial, is keen to now move the development of this much needed vaccine forward:

Both components of the vaccine were found to be safe and to create an immune response.

‘While I am delighted with these results, this was just a first trial. We need this vaccine for children and the elderly and that is where the efforts in vaccine development will now focus.’

Paul Browne

  1. Christopher A. Green et al. “Chimpanzee adenovirus– and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults” Science Translational Medicine, Vol 7, Issue 300, 300ra126, 12 August 2015 Link
  2. Taylor G. et al.”Efficacy of a virus-vectored vaccine against human and bovine respiratory syncytial virus infections” Science Translational Medicine, Vol 7, Issue 300, 300ra127, 12 August 2015 Link

Animal models are essential to biological research: issues and perspectives

The following article by Françoise Barré-Sinoussi and Xavier Montagutelli was published on 31 July 2015 in the journal Future Science OA, and is reproduced here under a Creative Commons Attribution 4.0 License

Françoise Barré-Sinoussi leads the Regulation of Retroviral Infections Division at the Institut Pasteur in Paris, and was awarded the Nobel Prize in Physiology or Medicine in 2008 for her role in the discovery of HIV, and Xavier Montagutelli is head of the Central Animal Facility of the Institut Pasteur. This article follows the recent decision by the European Commission to reject the Stop Vivisection Initiative that sought to repeal European Directive 2010/63/EU on the protection of animals used for scientific purposes and ban animal research in the EU.

Animal models are essential to biological research: issues and perspectives

Françoise Barré-Sinoussi (1) & Xavier Montagutelli*,(2)

The use of animals for scientific purposes is both a longstanding practice in biological research and medicine, and a frequent matter of debate in our societies. The remarkable anatomical and physiological similarities between humans and animals, particularly mammals, have prompted researchers to investigate a large range of mechanisms and assess novel therapies in animal models before applying their discoveries to humans. However, not all results obtained on animals can be directly translated to humans, and this observation is emphasized by those who refute any value to animal research. At the same time, the place of the animals in our modern societies is often debated, particularly the right to use animals to benefit human purposes, with the possibility that animals are harmed. These two aspects are often mixed in confusing arguments, which does not help citizens and politicians to get a clear picture of the issues. This has been the case in particular during the evaluation of the European Citizen Initiative (ECI) ‘Stop Vivisection’ recently presented to the European Commission [1].

European-Parliament

Humans and other mammals are very complex organisms in which organs achieve distinct physiological functions in a highly integrated and regulated fashion. Relationships involve a complex network of hormones, circulating factors and cells and cross-talk between cells in all the compartments. Biologists interrogate organisms at multiple levels: molecules, cells, organs and physiological functions, in healthy or diseased conditions. All levels of investigations are required to get a full description and understanding of the mechanisms. The first two, and in some instances three, levels of organization can be studied using in vitro approaches (e.g., cell culture). These techniques have become very sophisticated to mimic the 3D and complex structures of tissues. They represent major scientific advances and they have replaced the use of animals. On the other hand, the exploration of physiological functions and systemic interactions between organs requires a whole organism. It is, for example, the case for most hormonal regulations, for the dissemination of microorganisms during infectious diseases or for the influence of the intestinal microorganisms on immune defense or on the development of brain functions. In these many cases, no in vitro model is currently available to fully recapitulate these interactions, and investigations on humans and animals are still necessary. Hypotheses and models can emerge from in vitro studies but they must be tested and validated in a whole organism, otherwise they remain speculative. Scientists are very far from being able to predict the functioning of a complex organism from the study of separate cells, tissues and organs. Therefore, despite arguments put forward by the promotors of the ECI, studies on animals cannot be fully replaced by in vitro methods, and it is still a long way before they can.

Animal models have been used to address a variety of scientific questions, from basic science to the development and assessment of novel vaccines, or therapies. The use of animals is not only based on the vast commonalities in the biology of most mammals, but also on the fact that human diseases often affect other animal species. It is particularly the case for most infectious diseases but also for very common conditions such as Type I diabetes, hypertension, allergies, cancer, epilepsy, myopathies and so on. Not only are these diseases shared but the mechanisms are often also so similar that 90% of the veterinary drugs used to treat animals are identical or very similar to those used to treat humans. A number of major breakthroughs in basic science and medical research have been possible because of observations and testing on animal models. Most vaccines, which save millions of human and animal lives every year, have been successfully developed using animal models. The treatment of Type I diabetes by insulin was first established in the dog by Banting and McLeod who received the Nobel Prize in 1921 [2]. Cellular therapies for tissue regeneration using stem cells have been engineered and tested in animals [3]. Many surgical techniques have been designed and improved in various animal species before being applied to humans. The discoveries in which animal models played a critical role are indeed numerous and led to many Nobel Prizes.

It is, however, noticeable that the results obtained on animals are not necessarily confirmed in further human studies. Various reasons can be evoked. First, despite large similarities, there are differences between a given animal species and humans. For example, over 95% of the genes are homologous between mice and humans but there are also differences for example in the members of genes families, in gene redundancies and in the fine regulation of gene-expression level. These genetic differences translate into physiological differences which are increasingly better described and understood. While some people like the ECI promotors use these differences to refute the value of animal models, many including ourselves strongly advocate for further improving our knowledge and understanding of these differences and for taking them into account in experimental designs and interpretation of observations [4]. Moreover, these differences may provide opportunities to unravel novel mechanisms and imagine innovative therapies.

Research in mice has led to many medical advances - most recently the development of PD-1 inhibitors for treating cancers http://speakingofresearch.com/2015/05/30/immunotherapy-lung-cancer-pd-1-knockout-mice/

Research in mice has led to many medical advances – most recently the development of PD-1 inhibitors for treating cancers http://speakingofresearch.com/2015/05/30/immunotherapy-lung-cancer-pd-1-knockout-mice/

The second reason is due to genetic and physiological variations within each species or between closely related species. Laboratory mice have been developed as inbred strains which have highly homogeneous genetic composition to increase the reproducibility of results and the statistical power of experiments. Reports on animal models of human conditions often speak of ‘the mouse model of…’, referring in fact to observations made in a given genetic background. However, the clinical presentation often varies if another mouse strain is considered. A striking example is provided by a study published in November 2014 in Science by a team who reported that some mouse strains are fully resistant to Ebola virus, others die without specific symptoms and others develop fatal hemorrhagic fever [5]. Another example is the difference of responses to SIV, the monkey homolog to human HIV, between Rhesus macaques which develop simian AIDS and sooty mangabeys which do not develop symptoms despite high levels of circulating virus [6]. This range of responses reflects in fact the variety of clinical observations among human patients. These examples illustrate how animal models must be considered: no single animal model is able to mimic a given human disease which is itself polymorphic between patients, but the differences between strains or species provide unmatched opportunity to understand disease development and differential host response, and to eventually find new cures.

The second issue regarding the use of animals for scientific purposes is animal protection and welfare. This is the scope of the European Directive 2010/63/EU, which has set the regulatory framework for all animal research. Scientists have recognized for decades the importance of giving full consideration to three fundamental principles [7], which have become the backbone of the European Directive. First, animals must not be used whenever other, non-animal-based, experimental approaches are available, with similar relevance and reliability. Second, the number of animals used must be adjusted to the minimum needed to reach a conclusion. Third, all provisions must be taken throughout the procedures to minimize any harm inflicted to the animals. These principles, known as ‘the three Rs rules’, for replacement, reduction and refinement, have become the standard to which every project involving the use of animals is evaluated.

Animal research is conducted in compliance with regulatory provisions which cover the inspection and licensing of animal premises, the training and competence of all personal designing projects, performing animal procedures and taking care of animals and the mandatory authorization of every project by a competent authority upon ethical evaluation by an Animal Ethics Committee. The criteria for evaluation are based on the 3Rs rules and a cost–benefit analysis to evaluate if the potential harm to the animals, which must be reduced to the lowest possible level, is outweighed by significant progress in terms of knowledge on human or animal health. Regulation imposes that ethics committees include members concerned by animal protection and not involved in animal research. In response to the ECI, the European Commission has underlined, in a statement issued on 3 June 2015 [8], that animal experimentation remains important for improving human and animal health. At the same time, it is committed to promoting the development and validation of non-animal-based approaches, and to enforcing the application of the 3Rs rules by all players, including the research community. Europe has therefore implemented one of the strictest regulatory frameworks for the protection of animals used in research.

21st century medical research is highly interdisciplinary, a fact that is reflected in the design of new research institutions such as the Francis Crick Institute in London

21st century medical research is highly interdisciplinary, a fact that is reflected in the design of new research institutions such as the Francis Crick Institute in London

The greatest challenges faced by modern biomedical research concern complex, multifactorial, diseases such as cancer, cardiovascular diseases, infectious diseases, neurodegenerative disorders, pathological consequences of aging among others, for which all experimental approaches are indispensable because of their complementarity: biochemistry, genomics, cell culture, computer modeling, animal model and clinical studies. Research on relevant, carefully designed, well-characterized and controlled animal models will remain for a long time an essential step for fundamental discoveries, for testing hypotheses at the organism level and for the validation of human data. Animal models must be constantly improved to be more reliable and informative. Likewise, animal protection requires permanent consideration. These two objectives, far from being antagonistic, must be anchored in high-quality science.

References:

1. The European Citizens ‘Initiative – Stop vivisection. http://ec.europa.eu
2. Nobelprize.Org – The discovery of insulin. www.nobelprize.org
3. Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98(1), 216–224 (1996). [CrossRef] [Medline] [CAS]
4. Ergorul C, Levin LA. An example on glaucoma research: solving the lost in translation problem: improving the effectiveness of translational research. Curr. Opin. Pharmacol. 13(1), 108–114 (2013). [CrossRef] [Medline] [CAS]
5. Rasmussen AL, Okumura A, Ferris MT et al. Host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance. Science 346(6212), 987–991 (2014). [CrossRef] [Medline] [CAS]
6. Liovat AS, Jacquelin B, Ploquin MJ, Barre-Sinoussi F, Muller-Trutwin MC. African non human primates infected by SIV – why don’t they get sick? Lessons from studies on the early phase of non-pathogenic siv infection. Curr. HIV Res. 7(1), 39–50 (2009). [CrossRef] [Medline] [CAS]
7. Russell WMS, Burch RL. The Principles of Human Experimental Technique. Methuen, London, UK (1959).
8. European Commission – Annex to the communication from the commission on the European Citizen’s Initiative, ‘Stop Vivisection’. http://ec.europa.eu

Affiliations:

Françoise Barré-Sinoussi
1. INSERM & Unité de Régulation des Infections Rétrovirales, Institut Pasteur, 75724 Paris, France
Xavier Montagutelli
2. Animalerie Centrale, Institut Pasteur, 75724 Paris, France

USDA publishes 2014 Animal Research Statistics

Congratulations to the USDA/APHIS for getting ahead of the curve and making the US the first country to publish its 2014 animal research statistics. Overall, the number of animals (covered by the Animal Welfare Act) used in research fell 6.4% from 891,161 (2013) to 834,453 (2014).

These statistics do not include all animals as most mice, rats, and fish are not covered by the Animal Welfare Act – though they are still covered by other regulations that protect animal welfare. We also have not included the 166,274 animals which were kept in research facilities in 2014 but were not involved in any research studies.

Types of Animals used in research and testing 2014Statistics from previous years show that most of the “All other animals” species are rodents (but not mice or rats). 53% of research is on guinea pigs, hamsters and rabbits, while 10% is on dogs or cats and 7% on non-human primates. In the UK, where mice, rats, fish and birds are counted in the annual statistics, over 97% of research is on rodents, birds and fish. Across the EU, which measures animal use slightly differently, 93% of research is on species not counted under the Animal Welfare Act. We would expect similar patterns to be true in the US – although there are no statistics to confirm this.

Changes in number of animals used in research from 2013 to 2014 - Click to Enlarge

Changes in number of animals used in research from 2013 to 2014 – Click to Enlarge

If we look at the changes between the 2013 and 2014 statistics we can see a drop in the number of animals of most species , with only the “all other animals” category showing a rise. This is the second year in which the number of many species has fallen. For example, the number of rabbits used in 2014 fell 11.4% from 2013, following a 9.2% fall from 2012.

Most notably the number of non-human primates has fallen by 9.9%, the number of dogs fell 12.4% and the number of cats fell by 13%. This has shown these species taking up a smaller proportion of the research animals used, as can be seen below:

Trend in number of animals used in research 1973 - 2014 - Click to Enlarge

Trend in number of animals used in research 1973 – 2014 – Click to Enlarge

Clearly there has been a downward trend in the number of animals used since the early 1990s with a 61% drop in numbers between 1992 and 2014. It is also likely that, similar to the UK, a move towards using more genetically altered mice and fish has reduced the numbers of other AWA-covered animals used.

Rises and falls in the number of animals used reflects many factors including the level of biomedical activity in a country, trending areas of research, changes to legislations at home and abroad, outsourcing research to and from other countries, and new technologies (which may either replace animal studies or create reasons for new animal experiments).

It is important to note that the number of animals cannot be tallied across years to get an accurate measure of total number of animals. This is because animals in longitudinal studies are counted each year. Thus, if the same 10 animals are in a research facility for 10 years, they would appear in the stats of each year – adding these numbers would incorrectly create the illusion of 100 animals being used.

Speaking of Research welcomes the open publication of these animal research statistics as offering the public a clear idea of what animal research goes on in their country.

Clinical trial success for Cystic Fibrosis gene therapy: built on animal research

This morning the Cystic Fibrosis Gene Therapy Consortium (GTC) announced the results of clinical trial in 140 patients with cystic fibrosis, which demonstrate the potential for gene therapy to slow – and potentially halt – the decline of lung function in people with the disorder. It is a success that is built on 25 years of research, in which studies in animals have played a crucial role.

Cystic fibrosis is one of the most commonly inherited diseases, affecting about one in every four thousand children born in the USA, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene produces a channel that allows the transport of chloride ions across membranes in the body, and the many mutations identified in cystic fibrosis sufferers either reduce the activity of the channel or eliminate it entirely. This defect in chloride ion transport leads to defects in several major organs including the lungs, digestive system, pancreas, and liver. While the severity of the disease and the number of organs affected varies considerably, cystic fibrosis patients often ultimately require lung transplant s, and too many still die early in their 20’s and 30’s as the disease progresses.

In a paper published in Lancet Respiratory Medicine today (1), the GTG members led by Professor Eric Alton of Imperial College London compared monthly delivery to the airway of a non-viral plasmid vector containing the CFTR gene in the liposome complex pGM160/GL67A using a nebuliser with a placebo group who received saline solution via the nebuliser. They reported stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group after a year. This is the first time that gene therapy has been shown to safely stabilise the disease, and while the difference between the treated and control group was modest, and the therapy is not yet ready to go into clinical use, it provides a sound bases for further development and improvement.

Blausen_0286_CysticFibrosis

The Chief Executive of the Cystic Fibrosis Trust, which is one of the main funders of the GTC, has welcomed the results, saying:

Further clinical studies are needed before we can say that gene therapy is a viable clinical treatment. But this is an encouraging development which demonstrates proof of concept.

“We continue to support the GTC’s ground-breaking work as well as research in other areas of transformational activity as part of our mission to fight for a life unlimited by cystic fibrosis.”

So how did animal research pave the way for this trial?

Following the identification of the CFTR gene in 1989 scientists sought to create animal models of cystic fibrosis with which to study the disease, and since the early 1990’s more than a dozen mouse models of cystic fibrosis have been created. In some of these the CFTR gene has been “knocked out”, in other words completely removed, but in others the mutations found in human cystic fibrosis that result in a defective channel have been introduced. These mouse models show many of the defects seen in human cystic fibrosis patients and over the past few years have yielded important new information about cystic fibrosis, and in 1993 Professor Alton and colleagues demonstrated that it is possible to deliver a working copy of the CFTR gene using liposomes to the lungs of CFTR knockout mice and correct some of the deficiencies observed.

To get a working copy of the CFTR gene to the lungs of cystic fibrosis patients Professor Alton and colleagues needed three things:

• A DNA vector containing the working CFTR gene that is safe and  can express sufficient amounts of the CFTR channel protein in the lungs to correct the disease

• A lipid-like carrier that can form a fatty sphere around the DNA vector to so that it can cross the lipid membrane of cells in the lung, as “naked” DNA will not do this efficiently.

• A nebuliser device that produces an aerosol of the gene transfer agent so that it can be inhaled into the lungs of the patient.

Several early attempts to use gene therapy using viral vectors to deliver the working copy of the CFTR gene to patients failed because the immune response rapidly neutralised the adenoviral vector (see this post for more information on challenges using adenoviral vectors), and while attempts to use non-viral vectors were more promising, it was found that they caused a mild inflammation in most patients, which would make then unsuitable for long term use. As reported in a paper published in 2008 the GTC members developed and assessed in mice a series of non-viral DNA vectors, repeatedly modifying them and testing their ability to both drive CFTR gene expression in the lungs and avoid inducing inflammation. They finally hit on a vector – named pGM169 – which fulfilled both key criteria.

Earlier the consortium had undertaken a study to determine which carrier molecule to use in their non-viral gene transfer agent (GTA). To do this they assessed 3 GTA’s, each consisting of a lipid like molecule that could form a sphere around the non-viral DNA vector; either the 25 kDa-branched polyethyleneimine (PEI), the cationic liposome GL67A, or as a compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. Because there are significant differences in airway physiology between mouse and human they carried out this study in sheep, whose lung physiology more closely matches that of humans. The study identified the cationic liposome GL67A as the most promising candidate, resulting in robust expression of the CFTR transgene in the sheep lungs.

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

It now remained to bring the DNA vector and carrier together. In a 2013 publication the consortium reported that repeated aerosol doses of pGM169/GL67A to sheep over a 32 week period were safe and induced expression of the CFTR transgene in the sheep lungs, although the level of expression varied between individuals (this variation was also observed in human CF patients in the clinical trial reported today). A final study, this time in mice, assessed the suitability of the Trudell AeroEclipse II nebuliser as a device to create stable pGM169/GL67A aerosols, finding that it did so in a reproducible fashion. When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. These studies provided the evidence to support the gene delivery system and dosage strategy used in the clinical trial reported today.

The trial results announced today are an important accomplishment, but they mark a beginning rather than the end for Cystic Fibrosis gene therapy. It will be necessary to improve the efficiency of the therapy before it can enter widespread clinical use. Animal research will certainly play an important part in this work, notably the observation that the efficiency of CFTR gene delivery using this strategy was varied between individuals in both sheep and humans indicates that sheep are a good model in which to assess changes to improve the consistency and effectiveness of the gene therapy.

If you would like to know more about this cystic fibrosis gene therapy clinical trial you can watch two videos recorded at a meeting for cystic fibrosis patients at ICL on the  Cystic Fibrosis Trust website.

Paul Browne

1) Alton E.W.F.W. et al. “Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial” Lancet Respiratory Medicine Published online July 3, 2015

Lung cancer immunotherapy, from PD-1 knockout mice to clinical trials

This morning many news outlets, including the BBC, covered a very promising development in lung cancer therapy; the successful clinical trial of the cancer immunotherapy Nivolumab in 582 patients with advanced lung cancer. While the extension of survival was modest in most patients, it is to be remembered that these were patients with advanced lung cancer, which is notoriously difficult to treat, so to see the survival time doubling in some patients was quite dramatic. Future trials will examine whether greater benefits are seen when Nivolumab is given earlier in the course of the disease.

Dr Alan Worsley, Cancer Research UK’s senior science information officer, told the BBC that harnessing the immune system would be an “essential part” of cancer treatment, and adding:

This trial shows that blocking lung cancer’s ability to hide from immune cells may be better than current chemotherapy treatments. “Advances like these are giving real hope for lung cancer patients, who have until now had very few options.”

Nivolumab works by blocking the activation of the PD-1 receptor protein found on the surface of many of the immune cells that infiltrate tumours. Another protein named PD-L1 binds to PD-1 and initiates a regulatory pathway that leads to the immune response being dampened down. Usually this is a good thing as it maintains immune tolerance to self-antigens and prevents auto-immune damage to healthy tissue, but unfortunately many solid tumour cells, such as lung cancer cells, also secrete PD-L1, and by activating PD-1 can evade destruction by the immune system. By blocking PD-1 Nivolumab turns off this protective mechanism and allows the immune cells to detect and destroy the tumour cells.

X-ray of a lung cancer patient. Image credit: "LungCACXR" by James Heilman, MD - Own work.

X-ray of a lung cancer patient. Image credit: “LungCACXR” by James Heilman, MD – Own work.

So how was this discovered? This is where the knockout mice come in. Scientists had observed in the 1990’s that PD-1 was highly expressed on the surface of circulating T- and B- immune cells in mice, but didn’t know what role PD-1 played, suspecting that it may be involved in increasing the magnitude of the immune response. To examine the role of PD-1 researchers at Kyoto University in Japan creates a knock-out mouse line where the PD-1 gene was absent, and observed that this lead to some immune responses being augmented. In a paper published in 1998 they reported than rather than being an activator of the immune response PD-1 was actually involved in dampening down the immune response (1).

Subsequent studies in a range of PD-1 knockout mouse strains over the next decade explored the role of PD-1 in regulating the immune system, and also demonstrated that its ligand, PD-L1, could block immune-mediated tissue damage (2).  At the same time as these studies were taking place other research was demonstrating that PD-L1 was produced at high levels by tumour cells, first in   renal cell carcinoma in 2004 (3), but later in many other solid tumours including in lung cancer (4), and that this expression was associated with a decrease in the immune response to the tumour and a poorer prognosis.

This raised an obvious question: would blocking PD-1 improve the immune response against these tumours?

Work was already underway to find out. A paper published in 2007 by scientists from Nara Medical University in Japan demonstrated that blocking PD-L1 binding to PD-1 with monoclonal antibodies enhanced the immune response against established tumours in a mouse model of pancreatic cancer and acted synergistically with chemotherapy to clear the tumours without obvious toxicity (5). Subsequent studies with other monoclonal antibodies in a range of mouse and in vitro models of cancer showed similar results, including the humanized monoclonal antibody MDX-1106, now called Nivolumab, which was obtained by immunizing mice which had been genetically modified to produce human antibodies with human PD-1 (6).

Laboratory Mice are the most common species used in research

Cancer Immunotherapy – adding another accomplishment to an already impressive CV!

MDX-1106/Nivolumab showed promising results in a phase 1 trial against metastatic melanoma, colorectal cancer, castrate-resistant prostate cancer, non-small-cell lung cancer, and renal cell carcinoma, and following larger clinical trials (7) it was approved by the FDA for the treatment of melanoma that cannot be removed by surgery or is metastatic and no longer responding to other drugs, and more recently for metastatic squamous non-small cell lung cancer.

The story of the development of anti-PD-1 cancer immunotherapy is an illustration of how basic or fundamental biological research in animals informs medical science, and drives the discovery of new therapies. As cancer immunotherapy begins to transform the treatment of many previously untreatable cancers, it is well worth remembering that this revolution has its origin in the hard work of countless scientists working around the world, many of whom could only have guessed at the time where their efforts would eventually lead.

Breaking news, 1 June 2015: In another exciting report from the American Society of Clinical Oncology meeting in Chicago, researchers have reported that in a clinical trial of 945 patients with advanced metastatic melanoma a combination of Nivolumab with  Ipilimumab (another cancer immunotherapy that works through a different mechanism) stopped cancer advancing for nearly a year in 58% of cases, with the cancer still stopped in its tracks in many patients when the study period had ended. This is substantially greater effect than is seen with existing therapies, including Ipilimumab when administered alone, and shows how powerful cancer immunotherapies may be when two or more are combined.

Paul Browne

References:

  1. Nishimura H1, Minato N, Nakano T, Honjo T. “Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses.” Int Immunol. 1998 Oct;10(10):1563-72. PubMed: 9796923
  2. Grabie N, Gotsman I, DaCosta R, Pang H, Stavrakis G, Butte MJ, Keir ME, Freeman GJ, Sharpe AH, Lichtman AH. “Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart.” Circulation. 2007 Oct 30;116(18):2062-71. PubMed 17938288
  3. Thompson RH1, Gillett MD, Cheville JC, Lohse CM, Dong H, Webster WS, Krejci KG, Lobo JR, Sengupta S, Chen L, Zincke H, Blute ML, Strome SE, Leibovich BC, Kwon ED. “Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.” Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17174-9. PubMed:15569934
  4. Zhang Y1, Huang S, Gong D, Qin Y, Shen Q. “Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer.” Cell Mol Immunol. 2010 Sep;7(5):389-95. doi: 10.1038/cmi.2010.28. PubMed: 20514052
  5. Nomi T1, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, Nakamura S, Enomoto K, Yagita H, Azuma M, Nakajima Y. “Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer.” Clin Cancer Res. 2007 Apr 1;13(7):2151-7. PubMed:17404099
  6. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, Stankevich E, Pons A, Salay TM, McMiller TL, Gilson MM, Wang C, Selby M, Taube JM, Anders R, Chen L, Korman AJ, Pardoll DM, Lowy I, Topalian SL. “Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.” J Clin Oncol. 2010 Jul 1;28(19):3167-75. doi:10.1200/JCO.2009.26.7609. PubMed: 20516446
  7. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, Leming PD, Lipson EJ, Puzanov I, Smith DC, Taube JM, Wigginton JM, Kollia GD, Gupta A, Pardoll DM, Sosman JA, Hodi FS. “Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab.” J Clin Oncol. 2014 Apr 1;32(10):1020-30. doi: 10.1200/JCO.2013.53.0105. PubMed:24590637