Monthly Archives: November 2013

Animating Animal Research

The British Pharmacological Society and Understanding Animal Research have produced an animation explaining why animals are used in pharmacology. This under-three minute video is a great way of getting across the importance of animal research to those who are less aware of the science behind biomedicine.

It’s not the first animated video that we have mentioned. Check out a previous post for a great video on why researchers use genetically modified animals. Hopefully we will see more videos explaining everything from the use of animals in basic research to the animal testing used to ensure safety.

Happy Thanksgiving

Jerry the Beagle and the “Liberation” that Wasn’t

On Monday, Nov. 4, Jerry, a six-month old beagle allegedly “rescued from a laboratory” at UC Davis, gamboled on the grass outside California’s state capitol as news cameras looked on.

But campus veterinarian, Vic Lukas, was puzzled. He wasn’t aware of an animal being “rescued.” More concerning, one of the people in the photos with Jerry was Shannon Keith, an animal rights lawyer connected with the notorious activist group Stop Huntingdon Animal Cruelty (SHAC).

Screenshot from the Beagle Freedom Project website

Screenshot from the Beagle Freedom Project website

The mystery was solved when staffers were able to compare Jerry’s ear tattoo, shown in some photos, with university records. Jerry had indeed been at UC Davis, for a couple of weeks, but for teaching purposes, not research.

Twice a year, residents in training at the UC Davis School of Veterinary Medicine see a demonstration of the sort of electrophysiological exam that they might perform on a dog patient. This involves giving the animal an anesthetic and placing electrodes on its skin. No surgery, or invasive procedures are involved, other than that the dog is spayed or neutered at the same time.

This teaching class involves exactly two dogs a year, and the dogs are normally adopted afterwards.

UC Davis does allow animals that have been involved in research or teaching procedures to be adopted in some circumstances. So far this year, 90 animals, mostly dogs and cats, have been adopted, according to Lukas.

Paperwork for Jerry’s adoption was filed on Oct. 23 — the day the dog arrived at UC Davis. The adoption was by a “Katie Johnson” of Livermore — but the address given for a veterinary clinic was in West Hollywood, hundreds of miles away. Ms. Johnson took possession of Jerry about 5 p.m. on Sunday, Nov. 3 — and the next afternoon, he was being photographed at a media event organized by animal rights activists.

Apparently, the Beagle Freedom Project went shopping for a poster dog for their campaign, and were able to find one at UC Davis.

Who is behind the Beagle Freedom Project? Apart from SHAC lawyer Shannon Keith, one of the leaders is Kevin Kjonaas, also known as Kevin Chase, recently released from federal prison for his role in the campaign of threats, violence and harassment by SHAC-USA. As a mouthpiece for extremist groups, Shannon has made many shady friendships.

Activists pictures include Jerry Vlasak (ALPO), Peter Young, Nicoal Sheen (ALPO), Pamelyn Ferdin (SHAC), Shannon Keith (SHAC), Greg Kelly (Band of Mercy) and Steve Best.

Shannon Keith posing with animal right extremists at a Chinese restaurant.  Activists include Jerry Vlasak (ALPO), Peter Young (Voice of the Voiceless), Nicoal Sheen (ALPO), Pamelyn Ferdin (SHAC), Shannon Keith (Beagle Freedom Project), Carol Glasser (Progress for Science), Greg Kelly (Band of Mercy) and Steve Best (University of Texas at El Paso).

Speaking of Research

To learn more about the role of animal research in advancing human and veterinary medicine, and the threat posed to this progress by the animal rights lobby, follow us on Facebook or Twitter.

Successful gene therapy for hemophilia A in dogs – humans next!

On Wednesday we were saddened learned that double Nobel laureate Fred Sanger had died,  so it was fitting that yesterday also saw the announcement of an important scientific advance that owes everything to the molecular biology revolution he helped to launch – one that may improve the lives of many thousands of people with Hemophilia A.

Hemophilia can affect dogs, and research on dogs with haemophilia has helped develop therapies for the disease. Image: Understanding Animal Research.

Hemophilia can affect dogs, and research on dogs with haemophilia has helped develop therapies for the disease. Image: Understanding Animal Research.

Hemophilia A is caused by a deficiency in the production of coagulation factor VIII, which leads to an increased risk of bleeding, and is due to defects in the gene located on the X-chromosome that lead to either insufficient production of factor VIII or production of defective factor VIII. Patients with severe Hemophilia A require frequent intravenous injections of recombinant factor VIII to prevent serious bleeding.  The BBC reported on Wednesday that a team of scientists based in the US and France have developed a gene therapy that successfully treated hemophilia in 2 dogs, and continued to prevent serious bleeds more than 2 years following treatment. Their therapeutic strategy involved isolating bone marrow hematopoietic stem cells (stem cells that give rise to all types of blood cell – including red blood cells, white blood cells and the platelets that are crucial to clotting) and transforming them with a lentiviral vector containing a gene encoding factor VIII under the control of a promoter that had previously been shown to drive expression of the drives the expression the target gene in the platelets of mice and dogs.  The transformed hematopoietic stem cells were then infused back into the same dog from which they had been isolated. Writing in the Nature Communications paper (1) reporting this study lead author Dr David Wilcox of the Medical College of Wisconsin and his colleagues discuss why dogs were the ideal subjects for preclinical evaluation of this therapy (for a great article on the crucial role of dogs in haemophilia research see this article from the magazine HEMAWARE).

 A canine model for haemophilia A exists, which results from a genetic mutation causing a large inversion of the FVIII gene (that resembles a molecular genetic defect found in about 40% of humans with the severe haemophilia A)4. Likewise, canine haemophilia A is essentially identical to the human disease in its clinical presentation characterized by severe-intermittent episodes of joint bleeding and haemorrhage. Protein replacement therapy is the most common treatment of severe bleeding episodes for haemophilia A but it has been confounded by the formation of inhibitory antibodies to transfused human FVIII in 30% of patients5,6. Similarly, 100% of dogs utilized from the Chapel Hill colony for this study develop inhibitory antibodies after being transfused with human FVIII (ref. 7), albeit severe bleeding is successfully treated with canine FVIII supplements. Thus, canine haemophilia A appears to be an ideal system to determine whether platelets can be used successfully to deliver human FVIII to the site of a vascular injury as a feasible approach to improve haemostasis within a ‘large-animal’ model of haemophilia A with the ability to form inhibitory antibodies to human FVIII.”

The above quotation also refers to an advantage of the technique they used over previous gene therapy methods developed to treat hemophilia A, one that the BBC article surprisingly didn’t pick up on. The BBC article mentions that an advantage that targeting expression of factor VIII to the platelets over previous studies where factor VIII was expressed in the liver of dogs with hemophilia A is that it would be suitable for patients who have damaged livers, but this is not the main advantage. The production of inhibitory antibodies against recombinant factor VIII by the patient is a problem that reduces the effectiveness of current therapies in about a third of people with hemophilia A, and this was also a problem in gene therapy techniques previously tested in clinical trials where factor VIII is secreted into the bloodstream from tissues such as the liver. Dr Wilcox and colleagues had the idea that by targeting expressing factor VIII specifically to platelets it would not be exposed to and blocked by inhibitory antibodies.

Laboratory Mice are the most common species used in research

GM mice are aiding the development of innovative therapies for many diseases, and haemophilia A is no exception.

The test this theory they turned to a genetically modified mouse model of hemophilia A, which had already proven very useful in earlier stages of the development of gene therapy to treating hemophilia A. In a study undertaken in GM mice (2) which had been immunized so that they produced inhibitory antibodies against human factor VIII, Dr Wilcox and colleagues at the Medical college of Wisconsin demonstrated that the their lentiviral vector that directed factor VIII expression specifically to the platelets resulted in the expression of therapeutic levels of factor VIII associated with platelets in the blood, even 6 months after treatment.

What’s more, they showed that this was possible using a nonmyeloablative conditioning regime before infusing the transformed hematopoietic stem cells. Conditioning regimes reduce the immune response to a transplant (and in cancers such as leukemia also eradicate the cancerous cells, using drugs such as the nitrogen mustards that we discussed earlier this week) but the myeloablative conditioning regimes that are very effective in treating leukemia carry significant risks, for example from infections following the procedure. Nonmyeloablative conditioning that does not completely destroy the patient’s reduces the risk of infection and transplant related death, and is thus more appropriate for conditions that are not immediately life threatening. This study paved the way for the evaluation of platelet specific factor VIII therapy in dogs that was reported on Wednesday. It is noteworthy that in the dogs, which were also treated using a nonmyeloablative pre-transplant conditioning regimen,  no inhibitory antibodies were detected against human factor VIII (unlike with previous gene therapy techniques), indicating that when associated with platelets it is sequestered from the immune system.

Almost 2 years ago we reported on the success of a small clinical trial of gene therapy in the treatment of hemophilia B following studies in mice and monkeys. We hope that with the development of a gene therapy technique that requires a milder conditioning regime and can avoid inhibitory antibodies this success will soon be repeated in hemophilia A.

Paul Browne

1)      Du LM, Nurden P, Nurden AT, Nichols TC, Bellinger DA, Jensen ES, Haberichter SL, Merricks E, Raymer RA, Fang J, Koukouritaki SB, Jacobi PM, Hawkins TB, Cornetta K, Shi Q, Wilcox DA. “Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.” Nat Commun. 2013 Nov 19;4:2773. doi: 10.1038/ncomms3773. Pubmed 24253479

2)      Kuether EL, Schroeder JA, Fahs SA, Cooley BC, Chen Y, Montgomery RR, Wilcox DA, Shi Q. “Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.” J Thromb Haemost. 2012 Aug;10(8):1570-80. doi: 10.1111/j.1538-7836.2012.04791.x.PubMed 22632092. PMC3419807

Mice and Mustard Gas: A History of Chemotherapy

We write a lot of articles about the role animal research is playing in promising upcoming research. Sometimes it results in a breakthrough, sometimes it doesn’t – but it often takes years to find out. Chemotherapy is an older example of how decades of research and testing – including with animals – can build into a lifesaving treatment that improves the lives of millions.

The origins of chemotherapy are routed in the mustard gas attacks of World War I [i]. Adair and Bagg noted that mustard gas applied to the skin of mice with an chemically-induced tumour resulted in a regression in the cutaneous tumour [ii]. Further research in rabbits allowed researchers to find the correct dosage for local use. In 1931, clinical experiments on twelve patients resulted in significant therapeutic reactions and “the virtual disappearance of the tumour after the intratumoral injection” [iii].

During World War II, studies on nitrogen mustards (originally developed as a chemical weapon) in mice showed regression of advanced tumours. Although the tumours returned, these too were treated in the same way and regressed (albeit not to the same extent as the initial treatments). The treated mice survived over three times longer than those left untreated (84 days compared with 21 days) [iii]. Similar results were then shown in a human trial in 1942. It is worth noting that the bone marrow depression, a toxic side effects of the treatment, was predicted by the animal results in mice, however these symptoms were reversible. This early research led to the development of other alkylating agents, many of which are used today, such as chlorambucil, melphalan, busufan and cyclophosphamide [iv].

It had been shown in animals that a single implanted leukemic cell was sufficient to kill a mouse, thus researchers realised to need to eradicate every last leukemia cell [v]. This resulted in a more aggressive approach to using chemotherapy to treat leukaemia through the 1960s, pushing up remission rates from 25% at the start of the decade to 60% by the end of it [vi]. Mice models in the 1960s also paved the way for combination chemotherapy. Scientists showed that drug resistance to one drug could be overcome by the use of another. In the present day, the majority of children with acute lymphocytic leukaemia are cured through the use of aggressive combination chemotherapy programs.

Today studies in mice continue to be at the forefront of cancer research, for example playing a key role in the development of new immunotherapy techniques that have shown great promise in early clinical trials against chronic and acute lymphocytic leukaemia that resisted existing treatments. The development of techniques to modify the genes of mice has led to a great deal of interest in their in cancer research, and recently studies of GM mice have yielded important insights into particular types of cancer such as acute myeloid leukemia and pancreatic cancer, as well as increasing understanding of processes involved in many cancers, such as the role of cancer stem cells.

Research in mice has also helped develop non-animal methods for screening potential chemotherapeutic compounds. Fluids from the peritoneal cavity of mice have allowed scientists to grow lymphoblastic cells in vitro. These cell lines, called L1210 cells are used today for screening compounds before they are administered to animals – reducing the number of animals needed [vii].

In the past century, the contribution of mice to the development of chemotherapy has been tremendous. Without the use of mice we would not have the high remission rates for all patients that currently exist, with remission for almost 85% of all sufferers.

Chemotherapy and animal testing

Chemotherapy and animal testing: Click to Enlarge

Speaking of Research


[i] Krumbhaar, E. B. Role of the blood and the one marrow in yellow cross gas (mustard gas) poisoning. I. Peripheral blood changes and their significance. J. Am. Med. Assoc. 72:39-41, 1919.

[ii] Adair, F.E. and Bagg, H.J. Experimental and clinical studies on the treatment of cancer by dichloroethylsulphide (mustard gas) Am. J. Surg 93:190-199, 1931

[iii] Papac RJ. Origins of cancer therapy. Yale J Biol Med 2001; 74: 391–8

[iv] Burchenal, J.H. The historical development of cancer chemotherapy. Semin. Oncol. 4:135-146, 1977

[v] Furth J,Kahn MC. The transmission of leukemia of mice with a single cell. Am J Cancer 1937; 31: 276–82.

[vi] Frei E III. Potential for eliminating leukemic cells in childhood acute leukemia. Proc Am Assoc Cancer Res 1963; 5: 20 (abstract).

[vii] Potter, M. 2003 In Memoriam: Lloyd W. Law (1910-2002) Cancer Res 63: 7002

Global Trends in Animal Rights Activism 2013

In 2011 I wrote a post about the number of animal rights incidents posted on the Bite Back website (warning: AR extremist website) which logs many incidents of animal rights extremism around the world.

The analysis only looks at the July – September period as it was too cumbersome to get all the figures for the entire year. It was assumed that these figures should be representative of animal rights activity over a year. The analysis only looked at 8 countries which had the most activism – it is possible other countries now have higher levels of activism but were overlooked.

This post provides an update, adding in data from 2012 and 2013*.

Animal Rights Activity 2004 to 2013

The first thing that is clear is that in the last two years the number of incidents in the 3-month period has fallen from an average of 88 down to 55 (around 19 incidents  month across all 8 countries).

Graph 2004-13 animal rights activism - global trends

Most countries have seen a dip in the level of activism – but let’s look closer.

The USA fell to the lowest number of recorded incidents in 2012, but then rose to 31 in 2013. The latest rise seems to be due to a spate of attacks against fur farmers including that have seen over 7,500 mink released between July-October 2013.

The UK saw a spike in 2012 before falling back to the low level seen in 2010-11. Three quarters of 2012 incidents were related to the freeing of poultry from British farms (and almost all the rest related to farm animal “liberations” or anti-hunting activities).

Sweden saw a massive drop from 57 incidents in 2011 to just 5 in the 3 month periods in both 2012 and 2013. Spain has seen a slight rise in incidents, whereas Mexico, Germany and Ireland continue to see a decline in activism.

Overall it is reassuring to see the numbers of animal rights incidents decline in the last two years – however, it is possible that this could simply be down to lower levels of reporting of activism (on BiteBack).

*Disclaimer: I may have made some small errors while counting by hand, however these errors should not be big enough to affect the statistics overall. It is also worth noting that not all global incidents are likely logged on BiteBack. Furthermore, I did not investigate the nature of each incident – some are arson attacks and vandalism, others are empty threats and the release or imprisonment of activists – I have not differentiated between these incidents.

Animal research and diabetes: Now the truth must be told – Part 2

In yesterday’s post we described how animal research contributed to the understanding and treatment of diabetes – most importantly with the discovery of insulin. In this post we address some of the common misinformation that activists are circulating on social media about the role of animal experiments in diabetes research.

a)      One of the most valuable advances in understanding diabetes was made by Dr Moses Barron.

Prior to 1920, many researchers had worked on the relationship between the pancreas (or even more specifically, the islets on Langerhans) and diabetes. Banting and Best were by no means the pioneers in this field.  In the literature review of their February 1922 paper (1), they cited the article of Barron for having inspired their work and allowed them to formulate their hypothesis. They also cited precursor work of Mering & Minkowski, and Sscobolew, amongst many others. Banting and Best made use of groundwork already done before them and used this as support to bring their research to higher heights. This is something very usual in biomedical research, groundbreaking discoveries/advances are never achieved in a nutshell – they all rely on previous precursor work.

b)      Barron explained that his discovery ‘could be made in no other way, not even by experimental ligation of the ducts in animals’

In his November 1920 paper (2), Barron was not speaking about his ‘discovery’. In fact the purpose of the paper (page 1) was to present examples of typical changes in the Islets found in cases of true diabetes together with one histopathology case study of pancreatic lithiasis (formation of stones in the pancreas) and to correlate these findings with those recorded in the literature as obtained in experimental ligation of the ducts in animals.

On page 8 of his paper, while he is describing his 4th case study,  he describes the lesions of pancreatic lithiasis as ‘by their very nature, being of long standing, presents gradually progressive changes in the parenchyma, that could be obtained in no other way, not even by experimental ligation of the ducts in animals”.  This is indeed true as experimental ligation would bring an abrupt obstruction in the pancreatic duct while the formation of stone (lithiasis) would be something gradual and hence bring progressive changes in the functional parts of the pancreas. This has of course been taken completely out of context by activists.

Animal rights activists also conveniently occlude the statement of Barron on the next page of his paper after he finishes describing the case study, that:  “ The study of this case reveals results that are remarkably similar to those found in experimental ligation of the ducts”. Thus the principle impact of Barron’s work was not providing groundbreaking information, the role of the pancreas and the islets of Langerhans in diabetes was already known by the time he published his 1920 paper, but in providing additional evidence that what had been observed in animal studies was also true in humans, and of course in bringing diabetes to the attention of Banting, whose interest in the field was sparked by reading Barron’s paper.

c)       Diabetes was understood and insulin was applied primarily thanks to human clinical study and autopsy.

Prior to 11 January 1922, most of the work done to understand the mechanism of diabetes was on animals (dogs, cats, rabbits). Human clinical studies of pancreatic extracts in the treatment of diabetes were almost inexistent due to problems observed in the preclinical animal studies, and the few clinical studies that had been attempted in the previous decades produced severe adverse effects that prevented the therapy entering clinical use. Toxic reactions due to impurities in the pancreatic extract prevented its application by Banting, Macleod and Best to humans until January 1922, when a sufficiently pure extract was produced to allow human trials.

As for autopsy, it was apparent decades before the paper of Barron from autopsy/surgical findings that the pancreas might play an important role in diabetes, but it was through animal research that the role was confirmed and mechanism through which the pancreas regulated sugar levels was determined. It was only one chance autopsy that indicated to Barron that it was indeed the islets of Langerhans that played an important role in the disease in humans.  The disease that allowed this observation was very rare, in fact, Barron said that this was the first case of pancreatic lithiarsis that he encountered in a series of several thousand autopsies.

The understanding of diabetes and the role of insulin that allowed the development of insulin therapy was obtained from the interplay between crucial discoveries made through both clinical observation and animal research, so assigning primacy to one or the other is nonsensical.

d)      Claude Bernard had by 1895 experimented on dogs and come to the incorrect conclusion that diabetes had nothing to do with the pancreas.

Claude Bernard (who died in 1878 – more than a decade before the discoveries of Von Merin and Minkowski and the discovery of the first hormones) lived in a time where it was generally accepted that diabetes was a disease of the kidney due to the excessive levels of sugar in the urine. In the mid to late 19th century, various scientists had been exploring other possibilities to the cause of diabetes, including Bernard who believed that the sugar present in diabetic urine was stored in the liver as glycogen.  In a series of animal studies in the late 1840’s and early 1850’s Bernard made crucial discoveries about the role of the liver in storing and producing glucose.  He also found – correctly – that the central nervous system was involved in controlling blood glucose concentration by working with rabbits, though he believed that this was through a direct nerve communication between the CNS and liver, whereas in fact the conversion of glucose to glycogen in the liver was controlled indirectly via the hormone epinephrine – produced by the adrenal gland – and then insulin (and of course insulin also affects other tissues as well as the liver). So the situation is not that Bernard concluded that diabetes had nothing to do with pancreas, it is merely that he did not explore this avenue, principally because he did not think that it was possible to surgically remove the pancreas without killing the animal being studied. Bernard’s animal research did not provide a complete explanation of how glucose levels are regulated, but it was the starting point for the key discoveries made over the next 70 years, as Professor J. Sjöquist of the Nobel Committee for Physiology or Medicine of the Karolinska Institute noted in his presentation speech for the Nobel Prize in Physiology or Medicine on December 10, 1923.

It is true that the observation by Tidemann and Gmelin in 1827, that starchy foods are under normal conditions transformed into sugar in the intestinal canal and that this is absorbed by the blood, marks an important advance; but really epoch-making was the discovery of the great French physiologist Claude Bernard in 1857 that the liver is an organ that contains a starch-like substance, glycogen, from which sugar is constantly being formed during life; in the words of Claude Bernard, the liver secretes sugar into the blood.

In connection with his investigations into the circumstances that affect the formation of sugar, Claude Bernard observed that in certain lesions of the nervous system the sugar content of the blood was increased and that the sugar passed into the urine of the animals in the experiments. For the first time, therefore, an appearance of sugar in the urine – a glycosuria, though of a transitory nature – was experimentally produced; and consequently this discovery by Claude Bernard may be characterized as the starting-point of a series of experimental researches into the causes and nature of diabetes.”

e)      Macleod and Banting did not discover insulin as it had been identified and named before their experiments.

It was strongly hypothesized that the pancreas and more specifically the islets of Langerhans were producing a substance responsible for controlling blood sugar levels well before 1920 on the basis of animal research and clinical observations. Various names were proposed for this substance, insulin being one of them as this particular name had its Latin root derived from ‘islets’ (Latin: insula,island), but there is a big difference between inventing a name for a hypothetical substance and actually proving it exists and purifying it.

The quest for insulin was the ‘holy grail’ amongst scientists working in this field. Macleod, Banting and their fellow co workers had devised a series of experiments whereby they proved the hypothesis that insulin indeed came from the islets of Langerhans, they managed to produce an extract containing insulin and purify it to a degree that it retained its potency and efficiency in animal testing. They then further purified it to allow clinical testing, with the same positive results. They even refined their techniques to eventually reduce and replace animals that had to be used in the research and managed to produce the insulin in a stable, pure form in commercial quantities – making it available to the general public for diabetes treatment.

Irrespective of the controversy regarding who identified/named insulin, two things must be considered:

i)                    Almost all the scientists working in this field had been using animals as research models.

ii)                   The work of Macleod, Banting and their team was done over a short time (2 years) at the end of which, this resulted in clinical treatment of diabetes .Their Nobel Prize in Medicine/Physiology was amply deserved as they had according to the will of Alfred Nobel, made a discovery during the preceding year, “that conferred the greatest benefit on mankind”.

f)       Use of dogs was not necessary as human tissue was available

The study of diabetes required whole organisms – not just tissue/organ samples. The complex interactions behind production of insulin and its action in the metabolism of carbohydrates as well as the fact that diabetes affects several organ systems in the body made it crucial that whole organisms were studied. In addition insulin could not be obtained from any source other than as an extract from the pancreas, and human pancreases were not available for this purpose.

Dogs were the preferred animal model in the initial studies on diabetes because of their availability, the fact that their anatomy was well understood, hence making surgical techniques more efficient and most importantly, because at the time they began their experiments a large volume of blood needed to be sampled, thus the research animal could not be any smaller.

While in the course of their work, with newer methods being developed to test blood sugar levels in smaller volumes of blood, the team switched to smaller animals (rabbits). In fact, they acknowledge that the development of such techniques allowed their work to be done more precisely and more quickly.

beagle_UAR

g)      Synthetic insulin was developed in 1936

Synthetic insulin could not be made until a full understanding of its molecular structure was achieved. Sequencing of insulin was achieved in 1958 ; Chemical synthesis was first achieved in the lab in 1974 and it was only in the 1980s that synthetic insulin made by recombinant DNA technology was made available to the public. Until recombinant insulin became available almost all insulin was obtained from cattle, horses, pigs or fish.

h)      Diabetics owe nothing to animal experimenters.

Diabetics owe everything to animal researchers. The basic understanding of the anatomy and physiological functions of the pancreas was made possible thanks to work on animals. Duct ligations and depancreatisation experiments which gave insights into the functioning of the islets of Langerhans and allowed the isolation of pancreatic extracts containing insulin were made on animals. Although later this stage was deemed unnecessary due to the development of alcohol extraction methods, it provided the key stepping stones to the team to better understand the mechanism and properties of insulin, allowing them to move on to better techniques of obtaining insulin from whole pancreas. The testing of the pancreatic extract containing insulin of various degrees of purity had to be tested first on animals before being considered safe enough for humans. Even today, new insulin analogues need to be tested first on animals for efficacy and toxicity before moving on to human trials.

Research in the field of diabetes is still ongoing nowadays. Scientists are working to find alternative routes of insulin administration either by the oral route or by inhaling. Hopefully, in the near future, insulin injections will be something of the past. Work is being undertaken in the field of pancreatic or islet cell transplantation to cure Type I diabetes, including innovative stem cell based therapies that are being developed through animal research. Newer classes of therapy, including insulin pumps and insulin sensitizers that allow more precise control over blood sugar levels are being developed. All this progress relies on the continuing use of animals in the research.

So tomorrow on World Diabetes Day remember the many millions of lives around the world that have  been saved through animal research conducted by scientists over many decades, and also the thousands of scientists around the world who continue to strive to develop even better therapies – and even cures – for diabetes.

Nada and Paul

1)      Banting F.G. and Best C.H. “The internal secretion of the pancreas.” The journal of Laboratory and Clinical medicine, 1922;Vol VII No 5: 251-266

2)      Barron M. “The relation of the islets of Langerhans to diabetes with special reference to cases of pancreatic lithiasis.” Surg Gynec Obstet 1920;31:437-448.

Animal research and diabetes: Now the truth must be told – Part 1

Today we will take a look at the series of discoveries and innovations that led to the development of insulin therapy for type 1 diabetes, and tomorrow we will take a closer look at some of that claims made about this by animal rights activists.

With the World Diabetes Day coming up on 14th November, it is no surprise that activists are targeting diabetics by saying that cures and supportive treatment of this disease owe nothing to research on animals. They as usual, support their claim by a series of misinformation, statements taken out of context and in some arguments, deliberate distortions.

Although the remedy for diabetes was discovered in the 20th century, it is a disease that has plagued mankind since the ancient times. It was first described some 3,500 years ago by Egyptian physicians. Since then, various physicians of different civilizations – Greek, Roman, Indian, Chinese, Japanese and Arabic – have separately recorded descriptions of this disease, with the Greeks giving it the name ‘diabetes’ in reference to the increased frequency of urination that is characteristic of it. The predominant school of thought at that time was that the disease was due to kidney malfunction.

From the 16th to 18th century, diabetes was further described by European doctors, and from the idea that the disease was caused by kidney malfunction, various other hypotheses were also put forward – liver malfunction, systemic disease or even a malfunction of the central nervous system.  Amongst the various hypotheses put forward, one from Dr Thomas Cawley in the late 1700’s, linking diabetes to a damaged pancreas, based on the autopsy of a diabetic patient. During the 19th century evidence for a role of the pancreas in diabetes increased, but it was not clear if the damage seen was related to the cause of the disease or was a consequence of the disease, and how the pancreas might be regulating blood sugar. The pancreas was known to secrete digestive enzymes into the intestines via the pancreatic duct, but it was not clear how this function was related to the proposed role in controlling blood sugar levels.

The first experimental proof that the pancreas played an important role in carbohydrate metabolism and that diabetes could be of pancreatic origin was provided in 1890 by Von Merin and Minkowski  using dogs as research models. Their work showed that if the pancreas was removed from a dog, the animal got diabetes, but if the duct through which the pancreatic juices flow to the intestine was ligated the dog developed minor digestive problems but no diabetes, indicating that the roles of the pancreas in digestion and regulating blood sugar were separate (1). Later Minkowski and French scientist Edouard Hedon showed independently that if the entire pancreas was removed but some pancreatic tissue was grafted under the dog’s skin then diabetes was prevented, thus conclusively demonstrating that the  sugar regulating function of the pancreas was independent from its role in digestion (2).

While the role of acinar cells of the pancreas in the production of digestive juices was well understood at that time,  the small clusters of cells (‘islets’) discovered by Paul Langerhans in the pancreas of rabbits in 1869 still had no function attributed to them.   Dr Edouard Laguesse in 1893 suggested that these ‘islets of Langerhans’ might constitute the endocrine tissue of the pancreas responsible for the glucoregulatory role of this organ. Further evidence for this role came in 1900 from the pathologist Eugene Lindsay Opie, who noted damage to the islets of langerhans in diabetes patients (4). A year later Leonid W. Ssobolew demonstrated that a ligature of the pancreatic ducts in rabbits, cats, and dogs leads to gradual atrophy and destruction of the enzyme-secreting acinar cells, whereas the islet cells remained intact for weeks, with no evidence of excessive sugar in the urine (5).

In 1905, William Bayliss and Ernest Henry Starling, introduced the concept of hormones to designate the chemical messengers of the body’s endocrine glands, following their identification of the first hormone “secretin” through research on the regulation of digestion in dogs (3). Extrapolating on this concept, in 1913, Sir Edward Albert Sharpey-Schafer – another pioneer in the field of endocrinology – suggested that a hormone responsible for lowering blood sugar concentration was being secreted by the islets of Langerhans and he named this hypothetical hormone ‘insulin’. Independently, 4 years earlier, Jean de Meyer, a Belgian physician had produced an extract from the pancreas that lowered blood sugar concentrations and he also, concluded that the extract contained a substance from the islets of Langerhans and he named that hypothetical substance ‘insuline’ (Latin: insula,island).

Once it was clearly established that there was a link between diabetes and the pancreas – more specifically,  a substance produced by the islets of Langerhans – researchers from various parts of the world were focusing on treating diabetes with pancreatic extracts. Several workers including Zuelzer (Germany), Paulesco (Romania), Scott and Kleiner (North America) had all been able to produce pancreatic extracts that often reduced hyperglycemia or glycosuria in animals and, in a handful of cases with mixed results, in humans. However, due to toxic reactions after the initial relief of symptoms and the outbreak of the First World War, their work was discontinued or slowed down. The problem of how to isolate the hormone insulin in a form sufficiently pure for clinical use remained for now unresolved.

On 30 October 1920, while preparing for a physiology lecture, Frederick Banting came across the article by Moses Barron, “The Relation of the Islets of Langerhans to Diabetes, with special reference to cases of pancreatic lithiasis” (6). While doing routine autopsies, Dr Barron had come across a rare case of the formation of a pancreatic stone. Rarer still, the stone had completely obstructed the main pancreatic duct. Dr Barron observed that this obstruction had caused the atrophy of all acinar cells while the islets of Langerhans had remained intact, and he observed that there was a similarity to Leonid W. Ssobolew’s earlier animal studies where the ligature of the pancreatic ducts in rabbits, cats and dogs lead to the gradual atrophy of the enzyme-secreting acinar cells.

If Frederick Banting was alive, I'm sure he'd be writing about his research for us.

The Nobel Prize in Physiology or Medicine was awarded to Banting and Macleod

Although not a researcher (he was a general surgeon and part time lecturer), Banting’s thoughts were triggered by Dr Barron’s article, as he describes in his 1923 Nobel Prize Lecture. He suspected that in the failed attempts of those scientists who were using pancreatic extract to cure diabetes, the digestive enzymes of the pancreas were destroying the active principle responsible for lowering glucose levels. He started thinking about what if the extract came from a fully degenerated pancreas (through duct ligation) – something that no one had tried before.  He brought his idea to John Macleod, department head at the University of Toronto and a leading authority on carbohydrate metabolism. After much convincing, Macleod agreed to provide research facilities including 10 dogs as research animals and overview the testing of Banting’s proposal. He employed Charles Best to be a research assistant to Banting with work starting in May 1921 (7).

The team had to first refine their surgical techniques on dogs – namely to ligate the pancreatic duct in some animals, and in other individuals to completely remove the pancreas. On 30 July, they managed to obtain an extract from a duct-tied dog which was administered to a depancreatised dog that was displaying the symptoms of diabetes. The extract caused a reduction in blood sugar. The experiments were replicated and recorded frequent decreases in blood sugar as well as sugar excreted in urine. The team had experimental evidence of having isolated an extract with antidiabetic properties – they named the extract ‘Isletin’.

They next refined their technique by using a hormone, secretin, to exhaust acinar cells and obtain extracts from the pancreas free from digestive enzyme trypsin, thus bypassing the duct ligation procedure. They also explored the avenue of obtaining pancreatic extract from fetal calves – in which there was no active acinar secretion, hence free from the digestive enzymes. This was successful but still not efficient enough to be appropriate for large scale production. The next advance was to employ an extraction method that used slightly acidified alcohol rather than saline water, as the ethanol could then be evaporated to leave behind the active hormone. With the help of James Collip, a biochemist who joined the team  in early December 1921, they managed to secure the active principle from fresh whole beef pancreas, and Collip even managed to isolate the active principle as a powder, still with impurities but far purer than any previous extracts. This was tested on rabbits as newer methods for blood testing had been developed that required a far lesser volume of blood as sample. These new methods for testing blood sugar levels had been instrumental in the further purification and testing of the extract as it was more precise, more rapid and required  a small volume of blood (hence, smaller animals could be used). After the assay of tests, it was realized that the extract was sufficiently pure for testing on humans.

On 11 January 1922, the extract was administered to Leonard Thompson, a 14 yr old boy with severe diabetes, however, although they recorded a drop in blood sugar levels, it was considered not effective enough.  A second trial on 23 January 1922 with a more highly purified extract prepared by Collip was performed, and this time a more significant blood sugar level decrease as well as glucose excretion level decrease was obtained. In February 1922, 6 more patients were treated, all with favourable results (8). In later papers the active principle was re-named ‘Insulin’.

When moved to industrial production, with the help of the chief chemist from Eli Lilly and Co, Georges Walden, insulin was being produced at greater yields, better stability and much purer than what was obtained before. Diabetic patients were being treated successfully and the team was achieving fame, honours and prizes with the culmination of the Nobel Prize being awarded to Banting and Macleod in October 1923.

Further work by other scientist on Insulin resulted in its sequencing by Frederick Sanger in 1958. Chemical synthesis of the two protein chains of insulin was achieved in 1967. For many years, beef/pork insulin was the only source of insulin until 1974 where Sieber and his colleagues managed to chemically synthesise human insulin. Subsequently human insulin was produced by recombinant DNA technology by scientists working for the biotechnology company Genentech Inc. and in 1982 the first synthetic insulin analog “Humulin” was approved by the FDA. Nowadays more than 300 insulin analogues exist – with differences in their absorption and duration of action characteristics according to the needs of the patients.

Prior to the introduction of insulin therapy, most patients with diabetes died within a year of diagnosis.  Today’s life expectancy for people with diabetes is still lower than that for the general population by about 10 years, but better control is leading to longer and healthier life. Much of this improvement is thanks to animal testing.

Tomorrow, in the second part of this article,  we will take a closer look at some of the specific claims that animal rights activists are making about the role of animal research in the development of insulin therapy for type 1 diabetes.

Nada and Paul

Related Post: Animal research and diabetes: Now the truth must be told – Part 2

1)      von Mering J, Minkowski O. “Diabetes mellitus nach Pankreasextirpation”. Centralblatt für klinische Medicin, Leipzig, 1889, 10 (23): 393-394. Archiv für experimentelle Patholgie und Pharmakologie, Leipzig, 1890, 26: 37

2)      Hedon E. “sur la consommation du sucre chez la chien apres l’extirpation du pancreas” Arch Physiol Narmal Pathol Vth series 1893; 5: 154-63.

3)      Bayliss WM, Starling EH.”The mechanism of pancreatic secretion.” J Physiol. 1902 Sep 12;28(5):325-53.

4)      Opie EL. “The relation of diabetes mellitus to lesions of the pancreas. Hyaline degeneration of the islands of Langerhans.” J Exp Med 1900;5:527-540

5)      Ssobolew LW. “Zur normalen und pathologischen Morphologie der inneren Secretion der Bauchspeicheldrüse.” Archiv für pathologische und anatomie und physiologie und für klinische medizin 1902;168:91-128.

6)      Barron M. “The relation of the islets of Langerhans to diabetes with special reference to cases of pancreatic lithiasis.” Surg Gynec Obstet 1920;31:437-448.

7)      Rosenfeld L. “Insulin:Discovery and controversy.” Clinical Chemistry 2002 48:12 2270-88

8)      Banting FG, Best CH, Collip JB, Campbell WR, Fletcher AA. “Pancreatic Extracts in the Treatment of Diabetes Mellitus.” Can Med Assoc J. 1922 Mar;12(3):141-6.