Archive for the 'Animals in Research' category

Perspective on the use of animals in research

May 09 2013 Published by under Animals in Research

We were just discussing the closure of the New England National Primate Research Center. One of the uncertainties about that decision of Harvard Medical School was where the approximately 2,000 animals of various species were to be placed. 2,000. Remember that now. Some of the news reporting also referred to the deaths of some of the NENPRC nonhuman primate subjects as a triggering cause for a two year attempt to improve their procedures. These deaths comprised a series of ones and twos going by the available news reporting...perhaps amounting to a dozen or score of animals[ETA 4, see first comment]? We have no information over what timeframe those deaths occurred.

According to the LA Times, Malaysia has culled 97,000 cynomolgous macaques. Last year. The article claims they culled 88,000 of them in the previous year.

The cynomolgous macaque, btw, is a very commonly used species in research laboratories in the US. From Speaking of Research we learn that in 2010 there were about 73,317 nonhuman primates used in 2010. Of all species. This is out of an estimated 25 million vertebrate animals used in research for that year. And remember, for the longer-lived species such as dogs or nonhuman primates, the vast majority of studies use them across multi-year and even multi-decade intervals. So across time the comparison of the yearly use of, say dogs versus mice, tends to overestimate the dogs on a per-individual basis.

We are incredibly parsimonious with the approximately 1% of animals used in research that are cats, dogs or monkeys, the ones usually of greatest concern to the average person. Parsimonious with all of them, in reality. As the Speaking of Research page puts it.

Let us put the number of animals used in perspective. Scientists in the US use approximately 26 million animals in research, of which only around 1 million are not rats/mice/birds/fish. We use fewer animals in research than the number of ducks eaten per year in this country. We consume over 1800 times the number of pigs than the number used in research. We eat over 340 chickens for each animal used in a research facility, and almost 9,000 chickens for every animal used in research covered by the Animal Welfare Act. For every animal used in research, it is estimated that 14 more are killed on our roads.

Malaysia just euthanized (how we don't know but I'm pretty sure IACUC oversight wasn't involved) 185,000 monkeys in the past two years. Why?

Tourists and many Malaysians gather near jungle edges to watch the monkeys, snap photos of them and feed them peanuts and bananas. But the wildlife department, also known as Perhilitan, says the extensive culling was necessary to curb a “pest species” that breeds prolifically, adapts with ease, and ransacks homes for food.

“It is a hard decision, but in order to safeguard the well-being of people and to maintain a stable macaques population … it might be the best option in a short run,” the department said in an email to The Times.

185,000 culled as annoying pests over two years. A problem that may or may not have been increased by tourists. Even if the above stats reflected different individuals in each year, this is about equivalent to the total number of nonhuman primates used in US research laboratories. In fact, the number is likely to be much closer to the annual 73,317 count, given the longevity of the species. And we have no idea when Malaysia will stop culling.

In a related vein the ASPCA says that about 3-4 million companion animals (dogs and cats) are euthanized in shelters in the US annually. Annually. Why? Because nobody wants them. 64,930 dogs and 21,578 cats used in research in 2010. Versus 3-4 million. That's 3,000,000 vs 86,508. Mere inconvenience and irresponsibility versus the advance of knowledge and creation of new medical treatment for humans and animals as well.

The inadvertent deaths of a handful to perhaps a score of monkeys[ETA 4, see first comment] at the NENPRC led to massive shut down of research, a two year reorganization process and ultimately the demise of the Center. The center which made demonstrable advances in AIDS, drug abuse and Parkinson's disease amongst other accomplishments.

At the very least this should give some perspective on how seriously the research enterprise and oversight system takes the humane treatment of research animals. It compares very favorably indeed with how the rest of the world (including the US) treats animals (yes, including companion species).

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Harvard to close their New England National Primate Research Center

Apr 25 2013 Published by under Animals in Research, NIH, NIH Careerism, NIH funding

Harvard has decided not to seek to renew NIH support for their New England National Primate Research Center, established by Congress in 1962. The Center has operated with a so-called "base grant" from the National Institutes of Health underpinning the not-inconsiderable costs of housing thousands of nonhuman primates and the usual grab bag of investigators' independent sources of funding. The NENPRC site lists an impressive series of accomplishments.

First unambiguous evidence that AIDS is caused by a virus.
Discovery of Simian Immunodeficiency Virus (SIV) and development of first animal model of AIDS.
Original demonstration that vaccine protection against AIDS is theoretically possible.
Discovery that a gene product of the AIDS virus activates lymphocytes necessary for disease progression.
Identification of therapeutic genes that can prevent infection of cells by the AIDS virus.
First demonstration that protective genes introduced into blood stem cells can block HIV or SIV infection.
Discovery of primitive blood stem cells lacking CD34 and their implications for bone marrow transplantation
Isolation of type-D retroviruses as major causes of illness and death in macaques.
Discovery of the oncogenic herpesvirus, Herpesvirus saimiri.
Discovery of a nonhuman primate virus closely related to the human Kaposi's sarcoma-associated herpesvirus.
First nonhuman primate models of colon cancer and inflammatory bowel disease.
Evidence leading to the use of hydroxyurea to treat sickle cell anemia.
Discovery of stunned myocardium and its role in myocardial ischemia.
Discovery of cellular organization and critical period for development of the visual cortex.
First unambiguous evidence for the addictive properties of nicotine.
Identification of major risk factors in self-injurious behavior.
First animal model for progressive neurodegeneration in Parkinson's disease.
Development of improved brain imaging techniques for early diagnosis of Parkinson's disease.
Development of novel cellular and pharmacological strategies for treatment of Parkinson's disease.
First survey of distribution of cocaine binding sites in primate brain.
Identification of the dopamine transporter as a principal target for cocaine in the brain.
First nonhuman primate model of drug relapse.
Development of novel drug classes to treat cocaine addiction and other brain dopamine disorders.

Most of the news reporting has focused on a series of lapses in the care of nonhuman primate subjects, leading to several deaths. I cannot comment on the degree to which this situation reflected lapses in the system, but clearly Harvard was undergoing major corrective measures. The news accounts describe situations which seem to me to be procedural lapses that have relatively straightforward fixes. Nothing appears to be systematically unfixable...again, going by the news accounts.

The Harvard Medical School press release is slightly more instructive, however.

The decision to conclude NEPRC operations follows a two-year period during which the Center leadership successfully addressed operating issues with input from the NIH and other governing agencies. The process resulted in new procedures that have significantly strengthened the Center’s day-to-day activities and that can serve as a model for other institutions throughout the country. Many of those changes carried additional costs, and HMS will continue to make investments in the Center to ensure ongoing compliance with all federal regulations.

Right? So the problems were fixable and they'd been investing in fixing them for two years. "Additional costs", eh? Well, no biggie if the investment is good.

But what has happened in the past several months, hmm? The sequester. The Continuing Resolution for FY2013. Obama's budget request for FY2014. None of this is good news. If you look at the NENPRC as effectively a small, soft-money research institute funded in large extent by federal grants (and let's face it, partnering with for-profits isn't going that well for academia right now either) then its prospects are pretty dim. Look at the situation through the lens of Return on Investment and everything becomes clear.

As they weighed whether to renew the base grant from the NIH, HMS leaders made a strategic decision based on a review of the long-term academic benefits and the financial cost of continuing to operate the NEPRC.

“Deciding how to best assign our limited resources is not unique to HMS,” said Jeffrey S. Flier, Dean of the Faculty of Medicine of Harvard University,

also...


Driving the decision was the fact that the external funding environment for scientific research has become increasingly challenging over the past decade. Recent funding pressures have added uncertainty to this already-challenging fiscal context. As Harvard Medical School leadership evaluated the long-term need to use its resources in the most effective manner across all of its missions, they came to the conclusion that winding down the operations of the NEPRC was more beneficial to the School than investing further resources in maintaining and renewing the NEPRC grant.

So yeah, this looks from the outside like a small, specialized research institute closing down due to the NIH funding situation to me.

Maybe I have NIH grant myopia but this is the way it looks.

I am reviewing some of the claims made about their listed accomplishments and going back to the original papers, where I can deduce them. In a few areas that I am familiar with....man. Straight up. These are valid claims, even if we recognize that no science breakthrough arrives entirely by itself. And more importantly, particularly when it came to the early days of AIDS, I am having trouble imagining how progress could have been made so rapidly without one of the National Primate Research Centers. They really do seem to serve a unique function in the NIH / US Federal extramural research enterprise and it would be a shame if this was merely the lead indicator in shuttering the whole program.
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Disclaimer: I have professional acquaintances that work at NENPRC. I am disturbed that they are losing their jobs and I do hope that they get snapped up by some other University.

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Blogrolling: unlikely activist

I invite you to put the new blog of Professor J. David Jentsch on your list. At the unlikely activist you will find fare such as:

The mystery of addictions, Part 1: Why spend money on addiction research at all?

If they are remarkably lucky and have proper medical and psychological support, they may return to a healthy life and never use again. But for most, their freedom is only temporary, and they will relapse again days, weeks, months or even years later, returning them to their suffering and to their fateful spiral. You see, drugs kill. They are powerful toxins that can stop breathing or a heart. If they are injected, they can bring infectious diseases like hepatitis and HIV along with them. And because they intoxicate the mind, they lead to reckless driving and other behaviors that risk the lives of the addict and those around them.

Ignoring science, from the bench

Put differently, juveniles and teens have a brain fully capable of feeling powerful emotions (like anger), but their ability to resist those emotions and to behave in a socially appropriate manner (like to inhibit aggressive reactions) is not at adult levels. The 5 justices who struck down harsh penalties for child offenders recognized this; it was a crucial part of their logic in this, and the earlier death penalty, case.

But like a frightening number of people in our society, the other 4 justices viewed the science as either being wrong or irrelevant. Their own ethical or philosophical views about crime and punishment appeared to trump their interest in scientific principles and facts. In this regard, they are not unlike strident animal rights activists opposed to biomedical and behavioral research involving animals.

A solemn voice in support of medical researchers

In the fall of 2010, an animal rights extremist sent me razor blades and heinous threats to cut my throat in the mail. It became a national news story, again highlighting the abject cruelty of some in the anti-vivisection movement. During this time, I turned on my phone one evening to see that I had received a voice mail. Anticipating the worst – yet another cruel, rabid and profane threat from my opponents – I found something quite different. I have kept this communication private for long enough. Now, at the wishes of the caller, I am sharing it with the broader community to demonstrate that support for humane animal research is everywhere…. It comes not from greed or ignorance, but from love and a hope that no one should ever suffer the same loss as the caller.

VoiceofSupport (click on this link to listen to this .wav file)

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On discovering new medicines

ResearchBlogging.orgA link from writedit pointed me to a review of drugs that were approved in the US with an eye to how they were identified. Swinney and Anthony (2011) identified 259 agents that were approved by the US FDA between 1999 and 2008. They then identified 75 which were "first in class", i.e., not just me-too drugs or new formulations of existing drugs or whatnot. There were 20 imaging agents, not further discussed, and 164 "follower" drugs.

The review also focused mostly on small molecule drugs instead of "biologics" because of an assumption that the latter are all exclusively "target based" discoveries. The main interest was in determining if the remaining small molecule drugs were discovered the smart way or the dumb way. That's my formulation of what the authors term "target based screening" (which may include "molecular mechanism of action") discovery and "phenotypic screening" type of discovery. As they put it:

The strengths of the target-based approach include the ability to apply molecular and chemical knowledge to investigate specific molecular hypotheses, and the ability to apply both small-molecule screening strategies (which can often be achieved using high-throughput formats) and biologic-based approaches, such as identifying monoclonal antibodies. A disadvantage of the target-based approach is that the solution to the specific molecular hypotheses may not be relevant to the disease pathogenesis or provide a sufficient therapeutic index.

A strength of the phenotypic approach is that the assays do not require prior understanding of the molecular mechanism of action (MMOA), and activity in such assays might be translated into therapeutic impact in a given disease state more effectively than in target-based assays, which are often more artificial. A disadvantage of phenotypic screening approaches is the challenge of optimizing the molecular properties of candidate drugs without the design parameters provided by prior knowledge of the MMOA.

You will note that this is related to some comments I made previously about mouse models of depression.

The authors found that 28 of the first-in-class new molecular entities (NMEs) were discovered via phenotypic screening, 17 via target based approaches and 5 via making synthetic mimics of existing natural compounds. To give you a flavor of what phenotypic screening means:

For example, the oxazolidinone antibiotics (such as linezolid) were initially discovered as inhibitors of Gram-positive bacteria but were subsequently shown to be protein synthesis inhibitors that target an early step in the binding of N-formylmethionyl-tRNA to the ribosome

and for target based approaches:

A computer-assisted drug design strategy that was based on the crystal structure of the influenza viral neuraminidase led to the identification of zanamivir

The authors even ventured to distinguish discovery approaches by disease area:

Evaluation of the discovery strategy by disease area showed that a phenotypic approach was the most successful for central nervous system disorders and infectious diseases, whereas target-based approaches were most successful in cancer, infectious diseases and metabolic diseases

Unsurprising of course, given that our state of understanding of nervous system disorders is, to most viewers, considerably less complete in comparison with some other health conditions. You would expect that if there are multiple targets or targets are essentially unknown, all you are left with are the predictive phenotypic models.

Of the follower drugs 51% were identified by target based discovery and 18% by phenotypic screening. This is perhaps slightly surprising given that in the cases of the me-too drugs, you would think target-based would be more heavily dominant. Perhaps we can think of a drug which initially looked to have property X that dominated but then in the phenotypic screening, it looked more like a property Y type of drug.

The authors take on this is that it is slightly surprising how poorly target-based discovery performed within a context of what they describe as a period in which there was a lot of effort and faith placed behind the target-based approaches. I suspect this is going to be in the eye of the beholder but I certainly agree. I can't really go into the details but there are areas where my professional career is...affected, let us say...by the smart/dumb axis of drug discovery. It should be obvious to my longer term readers that I align most closely with animal models of various things related to health and neurobiology and so therefore you may safely conclude that I have a bias for phenotypic screening. And even in the case of the target-based discovery:

at least three hypotheses that must be correct to result in a new drug. The first hypothesis, which also applies to other discovery approaches, is that activity in the preclinical screens that are used to select a drug candidate will translate effectively into clinically meaningful activity in patients. The other two hypotheses are that the target that is selected is important in human disease and that the MMOA of drug candidates at the target in question is one that is capable of achieving the desired biological response.

Right. You still need good phenotypic models and ultimately you are going to have to pass human clinical trials. The authors further worry that this higher burden, especially knowing the MMoA is going to lead to some misses.

in the case of phenotypic-based screening approaches, assuming that a screening assay that translates effectively to human disease is available or can be identified, a potential key advantage of this approach over target-based approaches is that there is no preconceived idea of the MMOA and target hypothesis.

Ultimately I think this review argues quite effectively for an "all hands on deck" approach to drug discovery but it can't help but come off as a strong caution to the folks that think that "smarter" (aka, "rational drug design") is the only solution. Yes, this points the finger at Francis Collins' big thrust for a new translational IC at the NIH but also at the BigPharma companies that seem to be shedding their traditional models-based, phenotypic discover research units as fast as they can. No matter which side you come down on, this is a great read with lots to think about for those of us who are interested in the discovery of new medicines.
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Swinney, D., & Anthony, J. (2011). How were new medicines discovered? Nature Reviews Drug Discovery, 10 (7), 507-519 DOI: 10.1038/nrd3480

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Consider the source, Ed Silverman

Oct 07 2010 Published by under Animals in Research

I admit I am not a highly regular reader of Ed Silverman's Pharmalot blog, although I do keep up with his Twitts and read the occasional post. So I have little knowledge of where he's coming from on this issue. But he had a post that basically parroted the animal rights extremist's party line without a smidgen of critical thought.

The background is that pictures of severely wounded monkeys got out from a scientific supply company. The animal rights extremist organizations are all over this. Dog bites man story. They are, of course, certain that these pictures provide smoking gun evidence indicting all of animal research and nonhuman primate research in particular, demonstrating the general incompetence and uncaring nature of the industry.

This is their a priori belief. The extremist organizations that want to halt all use of animals in research by any means necessary do not have their opinion changed by facts either supporting or undermining their arguments. They feel free to lie, misrepresent or otherwise play fast and loose with any situation. This is what they do.

Ed Silverman should know this.

In his blog post, Silverman makes at least two glaring mistakes. The most troubling one is this one because it is deployed by the author in a way that makes it sound as if he agrees with the charge.

Primate Products ceo Don Bradford recently told NBC that the conditions depicted in the photos were not caused by medical testing, but due to injuries caused by other animals, and the monkeys have since healed. But the Animal Rights Foundation of Florida does not seem to believe him

the second item is a direct quote from an extremist group- still troubling because it contains an unexamined accusation:

These serious injuries may have resulted from self-mutilation, experimental procedures, or fights between animals who had been improperly housed.

As if the beliefs of anti-research extremist groups have any bearing on anything of evidentiary or probative value. They are against research. They are against researchers. They are against medical advances that are made possible only through the use of animals in research. Period. There is nothing that can be said or proven with facts that will make them "believe" anything anyone who is in support of well regulated humane use of animals in research has to say. Facts are irrelevant.

Which is why, Ed Silverman, it is essential for those who are presumably interested in the facts of a matter to account properly for the opinions offered by the person who comes from an unfalsifiable, unassailable fundamentalist belief structure that is impervious to fact. In this case properly means "deeply suspicious".

Fortunately, the Speaking of Research organization has an excellent bit up on their blog which underlines something anyone might have come up with on only a moment's thought. Anyone who has a National Geographic level understanding of the natural world and the behavior of species, that is.

There are two observations relevant to my points about Ed Silverman's dismal coverage. First, that macaque monkeys are, at times, socially aggressive organisms in their natural social and environmental niche- this frequently results in major wounding and even death. Second, that this means that the only "proper" housing that can guarantee zero wounding is single housing. And we all know how the animal rights groups feel about the propriety of that choice.

In other words, the animal rights extremist reaction to this situation betrays their usual profound misunderstanding of the natural world. It also illustrates their theologically driven desire to make the world we actually live in conform to some Utopian ideal in which all species are somehow equivalently enlightened and interacting as truly sentient (in the real sense of the word) organisms.

Science fiction is a nice read, but it is just a fantasy.

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Additional reading is a mere PubMed click away.

Or you can visit the American Journal of Primatology and use the search box for macaque

It does not require much effort to turn half-baked opinion into even minimally-informed opinion...assuming one is unafraid to have one's uninformed views modified by facts, that is.

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Levi Leipheimer: Cyclist, Philanthropist....Idiot

Sep 27 2010 Published by under Animals in Research, Public Health

Of course there is no particular reason to think that bike racing celebrity types should be any smarter than your average Hollywood actor or even one of those ruthless self-promoting celebrities who you can't quite figure out why they are famous.

Levi Leipheimer is a US professional cyclist who has become, over the course of a long career, a top talent with a long list of accomplishments. Recently his accomplishments have been in association with Lance Armstrong who is an absolute pitbull when it comes to battling cancer. I've mentioned before that following his Twitter gives you a whole new appreciation for how much this guy works at the whole LiveStrong charity.

And it isn't like Levi is just a passive participant. He puts the name of some kid with "pineoblastomas, a rare and aggressive brain cancer that afflicts less than 2% of all juvenile brain cancer patients" on his bike. Or remembers the name of a junior high school counselor who fell to colon cancer in another race.

He sponsors and promotes "Levi's Gran Fondo", a charity ride to raise money for various causes including "The Lance Armstrong Foundation received a $10,000 donation from the GranFondo for their ongoing funding of cancer research".

Research.
So what in the hell is he doing
tweeting this?

Odesssa and I hanging w/ @richroll and @jaiseed at the 30th anniversary [famous ARA wackanut organization-DM] Gala. Great night http://yfrog.com/3upsxnj

I've said it before...we really need to get Lance Armstrong focused on including animal research as part of his message.

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Why Supposed Ethics Case Studies / Training Scenarios Are Idiotic

Aug 16 2010 Published by under Animals in Research, Ethics, Science Ethics

Dr. Isis has a post up responding to a Protocol Review question "Noncompliance in survival surgery technique" published in Lab Animal [2010; 39(8)] by Jerald Silverman, DVM. His column is supposed to be in the vein of practicum case studies that are a traditional part of the discussion of ethical issues. Given X scenario, how should person A act? What is the ethical course of action? Was there a violation? Should it be reported/evaluated/punished.

We see these sorts of examples all the time in the ethics training courses to which we subject our academic trainees, particularly graduate students and postdocs.

These exercises frequently annoy me and this IACUC / Animals-in-Research question is of the classic type. Continue Reading »

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Don't t-a-a-a-a-a-a-aze me bro!

BlackSheepDM_234.jpg
source
A report in Popular Science (authored by Jeremy Hsu) points to a recent paper published in Academic Emergency Medicine. In this, Dawes and colleagues report on an investigation on the effects of TASER on sheep intoxicated with methamphetamine (MA). I was alerted to this by Damn Good Technician who wanted a little bit of context for what would seem to be a WTF? kind of study.
ResearchBlogging.orgThe study was conducted in Dorset sheep who were anesthetized, and administered 0, 0.5, 1.0 or 1.5 mg/kg of methamphetamine HCl (curiously from dissolved Desoxyn, the approved pharmaceutical product) in an IV infusion. The drug treatment was a between subjects factor (N=4 per group) and animals were monitored for "continuous blood pressure, heart rhythm (one-lead), pulse oximetry, and capnography... Arterial blood sampling was performed at baseline, 30 minutes after the administration of the methamphetamine, and after each exposure from a TASER X26".
To answer the question of why?, and for appropriate background on the science try a PubMed search for "cardiac TASER". I note a study in which 5 sec of TASER didn't cause cardiac damage or symptoms in law enforcement trainees and another showing minimal cardiac effects on law enforcement volunteers after vigorous exercise. Also of interest are the case studies of atrial fibrillation in a previously healthy adolescent and recovery of a teen in TASER induced asystole. These, a mini-review by the Dawes group and other searched papers should give you some context and support from the feeling you might have from half-remembered MSM reports over the years that TASER is suspected of being somewhat less than "safe".
What I'm not finding right away is very much about the drug intoxicated suspect who might be TASER'd by law enforcement. Remember this guy? My best estimate was that he was acutely intoxicated with 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") although that might be one of my blog interests talking. You might also wish to consider some papers found by searching PubMed for "methamphetamine cardiac toxicity", "methamphetamine vetricular fibrillation" and "methamphetamine heart attack".
Together this background would seem to identify a situation crying out for additional study.

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Second annual rally in support of biomedical science at UCLA

Apr 08 2010 Published by under Animals in Research

Wow, has it really been a year? Time for the second annual rally in support of biomedical research. I'll quote liberally from the UCLA Pro-Test for Science bit hosted at Speaking of Research:

In 2009, Pro-Test for Science held an historic rally on the UCLA campus; bringing over 700 people onto the streets in support of the scientists and researchers who carry out lifesaving medical research using laboratory animals. Such research continues to advance scientific knowledge and plays a vital role in the development of innovative treatments for human disease. However, animal rights extremists have continued to escalate their threats against researchers and their families.
On Thursday April 8th Pro-Test for Science will respond by rallying students, scientists and members of the public to support the cause of medical science. We call on the community to stand together against the recent tide of animal rights activism which has worked to misrepresent research and coerce those that carry it out.

A video from the rally held last year.

If you cannot attend perhaps you might want to take a look around Janet Stemwedel's blog. She has a number of thoughtful entries on topic of Research with Animals that are fantastic starting points for your own discussions that you will be having with your friends, family and colleagues. My own posts on the topic are perhaps less fulfilling but you may find a nugget or two. I would point you specifically to the Lie of the Truncated Distribution, an introduction to the heavily regulated activity of animal research, a description of additional guidelines that carry the weight of law and regulation and why the use of mice and rats is well regulated despite the Helms amendment.
You might also read a computer guy demolishing the myth that animal research can be replaced with computer simulations and an extensive and link-heavy discussion of typical animal rights' extremist tropes from Orac.
Happy reading.

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CongressCritters, can you please get the left hand talking to the right hand?

Mar 25 2010 Published by under Animals in Research, Call yer CongressCritter

From this Op-Ed.

The Institute of Medicine has recently released a report outlining the ominous public-health threat of chronic hepatitis C, much of which is the result of unwitting infection through medically-necessary blood transfusions, leading to 350,000 deaths worldwide each year and infecting more than three to five times as many people in the United States as HIV.

Narsty isn't it? We should get right on that, don't you think? Any decent models for research?

Currently, chimpanzees are the only experimental animal, except for humans themselves, susceptible to infection with hepatitis C. The Great Ape Protection Act would end the use of chimpanzees in biomedical research, grinding promising studies to a halt and unconscionably delaying the release of anti-viral therapies and a vaccine for chronic hepatitis C.

Whoops.

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