Over the course of the past few decades, researchers have tested everything from seizure medications to Viagra on mice. Scientists have bred special strains or “lines” of rats specifically for experimentation, with names such as the albino Wistar rat, the Sprague Dawley rat, and the Lister black-hooded rat. Because they are quick to reach sexual maturity and are easily kept and bred in captivity, rodents have been praised as prime experimental subjects. But an increasing number of studies, including a wide body of molecular modeling research, have revealed substantial differences between the immune systems of rats and the immune systems of humans. These studies provide a novel line of reasoning on age-old questions - How many of our experiments are valid? Are men simply tall mice without tails? Can we really take the data derived from experiments on rats and apply it to human beings?

The answers to the above questions are important for anyone who hopes to fully understand chronic inflammatory disease. “Unraveling the intricacies of human [Vitamin] D metabolism is often made extremely difficult by the intermingling of murine [mouse] and human biologies in the literature,” says biomedical researcher Trevor Marshall PhD. Armed with an understanding of the differences between humans and mice, we can better determine the accuracy of the studies we are presented with, and detect the flaws in studies that do not support the correct model of chronic disease.

In an article in the Journal of Immunology, Javier Mestas and team at the University of California describe how there are significant differences between the way the rat and human immune systems develop, which affect “activation, and response to challenge, in both the innate and adaptive arms [of the immune system].” Such differences should not be surprising as rats and humans diverged somewhere between 65 and 75 million years ago, and differ hugely in both size and lifespan. According to Mestas, “They have also evolved in quite different ecological niches where widely different pathogenic challenges need to be met - after all, most of us do not live with our heads a half-inch off the ground.” Consequently, he argues that, “There has been a tendency to ignore differences and in many cases, perhaps, make the assumption that what is true in mice is necessarily true in humans. By making such assumptions we run the risk of overlooking aspects of human immunology that do not occur, or cannot be modeled, in mice.”^[1]

Multiple studies have demonstrated that the immune systems of rats and humans are inherently different. Molecular modeling research has revealed that the activity of the human innate immune system is controlled by the Vitamin D Receptor.^[2] In humans, the Vitamin D Receptor performs several fundamental roles. Not only does it control the activity of the innate immune system, but it transcribes 913 genes, and new research points to the fact that it may actually transcribe 27,091.^[3] It also controls the production of many of the antimicrobial peptides. These peptides kill bacteria, viruses, and fungi by a variety of mechanisms including disrupting membranes, interfering with metabolism, and targeting components of the machinery inside the cell.^[4][5]

In contrast, the rat innate immune system is not controlled by the Vitamin D Receptor. It is dependent on a cascade of nitric oxide (an important signaling molecule) that functions in a manner yet to be fully understood. Rats do have Vitamin D Receptors, but they transcribe different genes than the human VDR. By using molecular modeling software, researchers at McGill University in Canada found many differences in the genes targeted by the rat and human Vitamin D Receptors. For example, the gene encoding a calcium binding protein called osteocalcin is “robustly” transcribed by the VDR in humans, but not in mice. In what proves to be a fundamental difference between mice and men, Manisha Brahmachary and team recently determined that the rat Vitamin D Receptor does not express the cathelicidin antimicrobial peptides (AMPs) - marking an important difference in the way the two species kill invading pathogens.^[6] This means that rats and humans respond differently to molecules or drugs that affect the VDR and subsequently the innate immune system.

Consider for example, the medication Olmesartan (the generic name for Benicar). Biomedical researcher Trevor Marshall has used molecular modeling software to demonstrate that when Olmesartan is administered to human subjects it binds and activates the Vitamin D Receptor. Yet when Olmesartan is administered to rats it has the opposite effect - it turns the VDR off. In fact, Olmesartan does not bind into the rat VDR in the same way that it binds the human VDR. The rat VDR lacks the ability to bind a protein that would allow Olmesartan to attach to the receptor. In contrast, Olmesartan can bind the human VDR directly, allowing it to activate the receptor. These differences can be observed in a video that Marshall presented at the 2007 Days of Molecular Modeling Conference.

Many of the genes that the VDR transcribes are known to be associated with the pathogenesis of cancer, including the breast cancer gene BRAC2. If the proteins that bind the human VDR are different from those that bind the VDR in rats, and the two animals don’t end up transcribing the same genes, how effectively can the data from cancer studies done on mice be applied to human beings?

As Marshall states, “The murine environment (the use of mouse models) is inadequate to accurately model drug carcinogenic activity in humans. Because the VDR affects the genes associated with cancer pathogenesis, good homology between human VDR, and the animal model VDR, is exceedingly important.”^[7]

Even in hamsters and rats, two animals that have quite a bit in common, Olmesartan affects their respective VDRs in different ways. During clinical trials aimed at testing the safety of Olmesartan, the drug’s manufacturer, Sankyo Pharmaceuticals, discovered that Olmesartan has possible carcinogenic activity in hamsters. Yet the carcinogenic activity was unable to be duplicated in rats. What accounts for this difference? The hamster VDR binds proteins in a different manner than the rat VDR, and controls different genes in each animal. In hamsters, Olmesartan appears to turn on genes that may negatively affect the pathogenesis of cancer – genes that the rat VDR may not transcribe.

“There is a laundry list of problems with mouse models of cancer. ”Bob Weinberg, based at the Whitehead Institute of Biomedical Research in Massachusetts, a pioneer of molecular cancer research, agrees. “There is a laundry list of problems with mouse models of cancer” says Weinberg. He first became acquainted with the limited applicability of murine models after trying to develop a mouse model for retinoblastoma, a childhood cancer of the retina. Retinoblastoma results from the loss of a gene called Rb, so Weinberg and team genetically engineered mice to lack the same gene. Instead, the mice developed tumors in their pituitary glands. “This planted seeds of doubt in my mind,” said Weinberg.^[8]

Besides the fact that the mouse and human VDRs transcribe different cancer-related genes, there are also substantial differences between the ways that mice and humans develop tumors. For one thing, most mouse tumors originate in different types of a mouse’s tissue than in a human’s, and, unlike in humans, healthy mouse cells can maintain the ends of their chromosomes, a key factor influencing which mutations tumor cells develop. What’s more, the technology used to create tumors in mice causes them to develop tumors early on, but human tumors develop later in adult life, following a stepwise series of mutations that turn a normal cell into a cancerous cell.

This may explain why of the potential anticancer drugs that give promising results in tests on mice, only about 11% are ever approved for use on people. It is also possible that drugs which might have helped humans battle cancer failed in preclinical mouse trials, although there is no way of knowing. There are also differences in the way humans and mice metabolize drugs. For instance, two human enzymes, CYP2D6 and CYP3A4, which together metabolize more than 70% of drugs on the market have markedly different activities compared with their rodent equivalents. The consequence is that mouse models may be of limited usefulness in predicting the effectiveness or toxicity of drugs in humans.

An increasing body of evidence is also pointing to the fact that bacteria are responsible for at least part of the pathogenesis of cancer.^[9] Since the VDR controls the innate immune system in humans but not in rats, the gap between how rats and humans fight cancer-causing bacteria is enormous. The same can be said for other diseases now known to be bacterial in origin. This situation has much wider implications than commonly understood. Many illnesses that are thought to be genetic, “autoimmune”, or of unknown cause are now known to be caused by bacteria, meaning that the immune system plays a greater role than expected in a wider array of illnesses. To make matters worse, numerous studies point to yet other aspects of the immune system that differ between mice and men.

The immune systems of both rats and humans use white blood cells called macrophages to kill bacteria and other pathogens. But rat macrophages are activated in different ways than human macrophages. In the book Immunology, Roitt explains that a chemical called INF-y can completely activate macrophages in rats, allowing them to successfully combat bacteria such as mycobacterium tuberculosis. However, when INF-y acts on human macrophages, it causes “at best, feeble inhibition of mycobacterium tuberculosis or, at worst, significantly increased growth.”

“Of the potential anticancer drugs that give promising results in tests on mice, only about 11% are ever approved for use on people”In addition, three papers published in the August edition of the medical journal Nature describe multiple differences between the way rats and humans form cells of the immune system called Th-17 helper cells - cells play an important role in the inflammatory responses of the innate immune system.^[10][11][12]

Mestas and team at the University of California have detected multiple differences between how white blood cells called neutrophils affect the expression of defensins, proteins that assist the immune system in killing pathogens. In humans, neutrophils are a rich source of defensins, but these same defensins are not expressed by neutrophils in mice. In contrast, cells of the small intestine express over 20 defensins in mice but only two in humans. There are also differences in the processing of defensins, which Mestas feels are “likely reflecting different evolutionary pressures related to microorganism exposure through food intake.” The team goes on to describe over 20 differences in the pathways that control the innate and adaptive immune systems of rats and humans.

Clearly, rats target bacteria in a different manner than humans, and generalizing data from one species to another can result in inaccuracies. As Mestas argues, “While it is hard to draw global conclusions about the significance of differences between mouse and human immunology, it is worth considering the possibility that any given response in a mouse may not occur in precisely the same way in humans.”

Case in point: A study conducted by researchers at the Crash Trials Medical Center in London. The researchers analyzed the concordance between the effects of various medical treatments on rat and human subjects. When the researchers tested corticosteroid medications on humans with head injuries, they found that the drugs had absolutely no effect. Yet when corticosteroids were administered to rats with the same malady, the drugs demonstrated a beneficial effect. Similarly, drugs called antifibrinolytics, which are designed to reduce bleeding, worked successfully in human subjects but not in rats. A drug called Tiriiazad had a negative impact on humans with ischaemic heart disease, but when administered to rats, the medication had a beneficial effect on the activity of the heart. The researchers concluded that “Discordance between animal and human studies may be due to bias or to the failure of animal models to mimic clinical disease adequately.”^[13]

Experiments that target the rat genome can also yield inconclusive results. For example, Langui and team in France found that several genetic mutations that led to nervous system dysregulation in mice have no equivalent in man, causing them to conclude, “Introducing a human mutated gene in an animal does not necessarily trigger pathogenetic cascades identical to those seen in the human disease.”^[14]

“It is worth considering the possibility that any given response in a mouse may not occur in precisely the same way in humans.”Unfortunately, even animals which bear more resemblance to man such as apes and dogs have VDRs that differ substantially from the human VDR, meaning that many drugs cannot be successfully tested on these animals either. Several months ago, German biotech firm TeGenero conducted a trial on a drug designed to treat chronic inflammatory conditions and leukemia. When the drug, called TGN 1412, was tested on monkeys, no adverse reactions were observed. In fact, tens of thousands of initial phase 1 trials on TGN 1412 were conducted on animals without incident.

But when TGN 1412 was administered to six healthy human volunteers, it caused catastrophic systemic failure in the subjects, despite being administered at a dose of 0.1 mg per kg, some 500 times lower than the dose found safe in animals. All six volunteers were hospitalized, and at least four suffered from multiple organ dysfunction. One trial volunteer is said to be showing signs of cancer. According to representative of the pharmaceutical industry, problems arose due to “unforeseen biological action in humans.” TGN 1412 was intended to activate the immune system, but did so differently in humans than in apes.^[15]

The TGN 1412 story, albeit a medical disaster, confirms that differences in VDR homology detected by molecular modeling software do indeed generate variability among immune system responses observed in a clinical setting. In essence, we must cast a wary eye towards data derived from animal models.

“As therapies for human diseases become ever more sophisticated and specifically targeted, it becomes increasingly important to understand the potential limitations of extrapolating data from mice to humans. The literature is littered with examples of therapies that work well in mice but fail to provide similar efficacy in humans,” says Mestas. Of course these issues discussed above don’t mean that we should completely stop using rats in the laboratory. For one thing, mouse models can be very useful for determining the function of genes. Recently three researchers were awarded the Nobel Prize for developing precise methods that allow scientists to change mouse genes one by one. These discoveries led to the practice of deleting, or knocking out, specific genes in mice in order to discover their function – allowing scientists to create what are called “knock-out-mice.” “If for example, you see a little finger disappear, then you know that gene is important for making little fingers,” Mario R Capecchi, one of the researchers awarded the prize, said in a telephone interview.

Knock out mice can certainly offer valuable insights about disease. Studies that delete certain receptors from mice can give important clues about the role that the receptor plays in the body. For example, when mice are grown without beta thyroid receptors they are deaf. Glutocorticoid receptor knockout mice don’t even survive gestation.

Gene-targeting technology can knock out single genes to study development of the embryo, aging and normal physiology. So far 10,000 mouse genes, or about half of those in the mammalian genome have been knocked out. In theory, knock out mice can help researchers in the field of gene therapy identify faulty genes that could potentially be causing disease. It can also allow scientists to identify the function of a particular gene in mice. Although this research can generate hypotheses, the differences between mice and humans impose clear limitations - not to mention the fact that these studies may become less useful as medicine embraces the primary role of pathogens, rather than genes, in many unexplained chronic diseases.

Other groups of scientists are attempting to inject mice with human cells, engineering genes that would allow mice to encode human enzymes, and finding ways to alter the expression of certain cell signals. For example, Glen Merlino at the National Cancer Institute in Maryland used one of these methods to better understand melanoma, a cancer affected by UV irradiation of the skin. Melanocytes, the pigment-producing cells that become cancerous in melanoma are found in the outermost layer of the human skin, but in mice they are confined to hair follicles, making it difficult to accurately study the disease in rodents. Merlino’s team was able to engineer mice to over-express a cell signal that causes melanocytes to be found in the outer layers of rodents’ skin, as in humans.

They went on to show that young, but not adult mice, exposed to UV radiation develop malignant melanoma, giving strong evidence to back up studies that have highlighted the potential dangers of sun exposure in children.^[16] “This is an example of the value of mouse models, - they are highly manipulable, for which there is no comparison in humans, says Tyler Jacks, a researcher who develops mouse models at the Massachussetts Institute of Technology in Cambridge.

But unfortunately, since none of these manipulations affect the rodent immune system, they still of limited value. In situations where murine models cannot provide accurate data, how can we test medications that affect the immune system in a safe and effective manner?

The most logical solution rests in perfecting ways to collect data directly from human beings. Howard Finc of the National Cancer Institute (NCI) agrees, “The best study subject will always be the human” says Finc. In an effort to better understand the pathogenesis of cancer, he is working on a project that collects and catalogues data on a wide range of human tumors. His team performs genetic and molecular analysis of samples of human brain tumors sent from patients around the world. The study is a pilot for the NCI’s larger cancer Biomedical Informatics Grid, which will provide a global network for researchers to input information and access bioinformatics tools for mining cancer data. The study currently has data for 700 tumors and will eventually contain information for 2,000 tumors.

Other possible solutions involve molecular modeling software, which can be used to elucidate the effects of a particular substance on the VDR and the immune system. Programs exist that allow a researcher to take a model of the human VDR and mathematically calculate how a drug will interact with the receptor and other pathways of the immune system.

If the mathematical data is carefully collected, it should be sufficiently reliable so that human subjects can be used to confirm the drug’s actions in a clinical setting. One such example are the Phase 2 studies of the Marshall Protocol, a treatment that uses low-dose antibiotics and Olmesartan, a medication that stimulates the immune system, to treat chronic disease. Subjects take medication as directed, while reporting symptoms and drug reactions in online progress reports. But as of now, large human clinical trials such as the Marshall Protocol are few and far between. It is critical that the science community devote increasing amounts of energy towards research that examines how these types of studies can be conducted in the most effective way possible.

Another possibility is that studies could continue to be conducted in animals such as primates, whose VDRs more closely resemble that of man. If researchers work to pinpoint the differences between the primate and human immune systems, then molecular modeling software, which can correct for these differences, could be used to make up for anomalies in the data, ensuring that the information collected is undoubtedly relevant to human beings.

REFERENCES

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3. Wang, T., Tavera-Mendoza, L. E., Laperriere, D., Libby, E., Burton MacLeod, N., Nagai, Y., et al. (2005). Large-Scale in Silico and Microarray-Based Identification of Direct 1,25-Dihydroxyvitamin D3 Target Genes. Mol Endocrinol, 19(11), 2685-2695. [↩]
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