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1.  Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing 
PLoS Medicine  2014;11(4):e1001628.
Gary Peltz, Jeffrey Glenn, and colleagues report that a pre-clinical mouse toxicology model can detect liver toxicity of a drug that caused liver failure in several early clinical trial participants in 1993.
Please see later in the article for the Editors' Summary
Background
Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.
Methods and Findings
Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.
Conclusions
FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Before new drugs are approved for clinical use, they undergo extensive preclinical (laboratory-based) and clinical testing. In the preclinical studies, scientists investigate the causes of diseases, identify potential new drugs, and test promising drug candidates in animals. Animal testing is performed to determine whether the new drug is likely to work, and to screen for drug-induced toxicity. In preclinical toxicology studies, new drugs are given to two or more animal species to find out whether the drug has any short- or long-term toxic effects such as damage to the liver (hepatotoxicity). Drugs that pass these animal tests enter clinical trials. Phase I clinical trials test new drugs in a handful of healthy volunteers or patients to evaluate their safety and to identify possible side effects. In phase II trials, a larger group of patients receives the new drug to evaluate its safety further and to get an initial idea of its effectiveness. Finally, in phase III trials, very large groups of patients are randomly assigned to receive the new drug or an established treatment for their disease. These randomized controlled trials provide detailed information about the effectiveness and safety of a candidate drug, and must be completed before a drug can be approved for clinical use.
Why Was This Study Done?
Since animals are not perfect models for people, candidate drugs can cause toxicities in clinical trials that were not predicted by preclinical toxicology testing performed using animal species. For example, in 1993, 15 participants in a phase II trial were given a nucleoside analogue called fialuridine to treat hepatitis B virus infection (nucleoside analogues often have antiviral activity). Seven participants developed liver failure and lactic acidosis (buildup of lactic acid in the blood). Analysis of liver tissue from the affected participants revealed steatosis (fatty degeneration), intracellular fat droplets, and swollen mitochondria (these organelles are the powerhouses of the cell). Five participants subsequently died, and two had to have a liver transplant. In preclinical toxicology testing in mice, rats, dogs, and primates, there had been no indications that fialuridine would be hepatotoxic in people. It now seems that the expression of a nucleoside transporter in the mitochondria of humans but not of other animals may underlie the human-specific mitochondrial toxicity and hepatotoxicity of fialuridine. With several other nucleoside analogues in development, a better screening tool for human-specific mitochondrial toxicity is needed. In this study, the researchers investigate whether fialuridine toxicity can be detected in TK-NOG mice with chimeric (humanized) livers. TK-NOG mice are immunodeficient mice that have been genetically engineered so that human liver cells (hepatocytes) transplanted into these animals establish a long-lived mature “human organ.”
What Did the Researchers Do and Find?
The researchers treated chimeric (with transplanted human liver cells) and control (without transplanted human liver cells) TK-NOG mice with several doses of fialuridine. After treatment with the highest dose (1,600-fold above the dose used in the phase II trial) for four days, the chimeric mice developed liver failure and lactic acidosis. Moreover, steatosis and lipid and mitochondrial abnormalities developed in the regions of their livers that contained human hepatocytes but not in regions that contained mouse hepatocytes. Notably, the control mice had not developed liver toxicity after 14 days of treatment with the highest dose of drug. Liver toxicity was also easily detectable in chimeric mice that had been treated for 14 days with a fialuridine dose only 10-fold above that used in the human trial. Treatment with another nucleoside analogue that does not cause liver toxicity in people did not cause liver toxicity in the chimeric mice.
What Do These Findings Mean?
These findings show that fialuridine-induced liver toxicity can be readily detected using TK-NOG mice that have humanized livers at drug doses only 10-fold higher than those that caused liver failure in the phase II trial. Although the liver toxicity developed much more quickly in these mice than in the human trial participants, the clinical features, laboratory abnormalities, and structural changes seen in the fialuridine-treated chimeric TK-NOG mice closely mirrored those seen in fialuridine-treated people. The use of TK-NOG mice containing humanized livers in toxicology testing will not reveal whether drugs have human-specific toxicities outside the liver. Since they are highly immunocompromised, chimeric TK-NOG mice cannot be used to detect immune-mediated drug toxicities. Nevertheless, these findings suggest that the use of chimeric mice in toxicology studies could help improve the safety of candidate drugs that are tested in humans.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001628.
The US Food and Drug Administration, the body that approves drugs for clinical use in the US, provides an overview for patients about the drug development process from the laboratory to the clinic
The UK Medicines and Healthcare Products Regulatory Agency (MHRA) provides more detailed information for patients and the public about the drug development process, including a section on preclinical research, which includes information on animal testing
The US National Institutes of Health provides information about clinical trials, including personal stories from people who have taken part in clinical trials
The UK National Health Service Choices website has information for patients about clinical trials and medical research, including personal stories about participation in clinical trials
Understanding Animal Research is a UK advocacy group that provides information about the importance of animal research to the public, teachers, scientists, journalists, and policy makers
Wikipedia has a page on animal testing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001628
PMCID: PMC3988005  PMID: 24736310
2.  The rights of man and animal experimentation. 
Journal of Medical Ethics  1990;16(3):160-161.
Since emotions give contradictory signals about animal experimentation in medical science, man's relationship to animals must be based upon reason. Thomas Aquinas argues that man is essentially different from animals because man's intellectual processes show evidence of an abstract mechanism not possessed by animals. Man's rights arise in association with this essential difference. The consequence is that only man possesses true rights by Aquinas's definition; animals have them only by analogy. However, cruelty to animals is illicit and they should be protected, principally not because they have rights, but because he who is cruel to animals is more likely to be cruel to his fellowman. If there is a need for animal experimentation in science for the good of man, this approach gives philosophical justification for experimentation, since man's well-being must come before that of animals because of his unique possession of rights. However, those experiments should be carried out in the kindest way possible, to promote kindness towards man. To see man as solely part of a biological continuum in competition for rights with those beings close to him biologically, detracts from man's dignity.
PMCID: PMC1375891  PMID: 2135948
3.  Gene Expression Signatures That Predict Radiation Exposure in Mice and Humans 
PLoS Medicine  2007;4(4):e106.
Background
The capacity to assess environmental inputs to biological phenotypes is limited by methods that can accurately and quantitatively measure these contributions. One such example can be seen in the context of exposure to ionizing radiation.
Methods and Findings
We have made use of gene expression analysis of peripheral blood (PB) mononuclear cells to develop expression profiles that accurately reflect prior radiation exposure. We demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, ranging from 50 cGy to 1,000 cGy. Likewise, a molecular signature of radiation response developed solely from irradiated human patient samples can predict and distinguish irradiated human PB samples from nonirradiated samples with an accuracy of 90%, sensitivity of 85%, and specificity of 94%. We further demonstrate that a radiation profile developed in the mouse can correctly distinguish PB samples from irradiated and nonirradiated human patients with an accuracy of 77%, sensitivity of 82%, and specificity of 75%. Taken together, these data demonstrate that molecular profiles can be generated that are highly predictive of different levels of radiation exposure in mice and humans.
Conclusions
We suggest that this approach, with additional refinement, could provide a method to assess the effects of various environmental inputs into biological phenotypes as well as providing a more practical application of a rapid molecular screening test for the diagnosis of radiation exposure.
John Chute and colleagues report that gene expression patterns in peripheral blood mononuclear cells from mice and humans reflect prior radiation exposure.
Editors' Summary
Background.
Everyone living on earth is constantly exposed to low levels of ionizing radiation—energy in the form of waves or particles that is powerful enough to strip electrons out of atoms and to break chemical bonds in important biomolecules. These low levels of ionizing radiation come from radioactive chemicals in the ground and cosmic rays, for example, and are relatively harmless. Occasionally, though, individuals are exposed to larger amounts of ionizing radiation, often as a result of medical tests and treatments but sometimes through the accidental or deliberate release of radioactive chemicals. These larger doses, which permanently damage or kill cells, can cause radiation sickness, a condition characterized by bone marrow failure, gut problems, susceptibility to bacterial infections, and other symptoms that develop days or months after exposure to ionizing radiation. Particularly large doses can be lethal but even moderate doses can increase an individual's risk of developing cancer later in life.
Why Was This Study Done?
Some of the effects of ionizing radiation can be reduced if suitable treatment is started immediately after exposure. Unfortunately, it takes several days to estimate the amount of ionizing radiation to which an individual has been exposed. It would be useful to measure personal exposures more quickly, especially in emergency situations where ideally doctors would be able to distinguish rapidly and accurately between the “worried well” and exposed individuals. As cells respond to irradiation by altering the expression of some genes, the researchers in this study investigated whether gene expression profiling (a molecular biology technique that catalogues all the genes expressed by a cell) can be used to define a set of gene expression changes—called a metagene—that differentiates between irradiated and non-irradiated cells.
What Did the Researchers Do and Find?
The researchers exposed mice to no ionizing radiation, a low dose that causes no medical problems, an intermediate dose that damages blood cells, or a lethal dose. Six hours later, they isolated blood cells from the mice, and catalogued which genes each sample expressed. Using this information, the researchers identified and validated metagenes that accurately distinguished between blood samples from non-irradiated and irradiated animals and between samples from animals exposed to different radiation doses. The researchers then developed a metagene for human radiation exposure using blood samples taken from patients before and after total body irradiation given as part of their medical treatment. This metagene correctly identified 18 of 20 pre-irradiation samples and 17 of 20 post-irradiation samples. Finally, the researchers tested whether the radiation metagenes developed in mice could distinguish between samples taken from irradiated and non-irradiated people. Although the high-dose mouse metagene correctly identified all of the samples from healthy donors as being non-irradiated, it correctly identified only two-thirds of the pre-irradiated samples from patients.
What Do These Findings Mean?
These findings indicate that metagenes can be generated that recognize different levels of radiation exposure in mice and people. In the mouse study a metagene was identified that correctly identified in all cases whether a sample came from a non-irradiated mouse or an animal exposed to the lowest dose of radiation. This result suggests that it might be possible to use a metagene to identify exposed individuals among thousands of “worried well” after a radiation emergency. First, however, the mouse and human metagenes identified here need to be refined to improve their accuracy and then validated in more people. The current high-dose mouse metagene may be bad at identifying non-irradiated patients, for example, because of gene expression changes that are a result of the patients' underlying disease or previous medical treatments. By studying additional patients, it might be possible to improve the accuracy of the metagene by taking these radiation-independent changes into account. Finally and more generally, these findings suggest that the metagene approach could be used to monitor people's exposure to other dangerous environmental agents.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040106.
US Environmental Protection Agency offers information on understanding radiation and factsheets on ionizing radiation
MedlinePlus provides links to information on radiation exposure and pages on radiation sickness
US Centers for Disease Control and Prevention has information on emergency preparedness and response to radiation emergencies
Wikipedia has pages on ionizing radiation, radiation poisoning, and expression profiling (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040106
PMCID: PMC1845155  PMID: 17407386
4.  Lithium Therapy Improves Neurological Function and Hippocampal Dendritic Arborization in a Spinocerebellar Ataxia Type 1 Mouse Model 
PLoS Medicine  2007;4(5):e182.
Background
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder characterized by progressive motor and cognitive dysfunction. Caused by an expanded polyglutamine tract in ataxin 1 (ATXN1), SCA1 pathogenesis involves a multifactorial process that likely begins with misfolding of ATXN1, which has functional consequences on its interactions, leading to transcriptional dysregulation. Because lithium has been shown to exert neuroprotective effects in a variety of conditions, possibly by affecting gene expression, we tested the efficacy of lithium treatment in a knock-in mouse model of SCA1 (Sca1154Q/2Q mice) that replicates many features of the human disease.
Methods and Findings
Sca1154Q/2Q mice and their wild-type littermates were fed either regular chow or chow that contained 0.2% lithium carbonate. Dietary lithium carbonate supplementation resulted in improvement of motor coordination, learning, and memory in Sca1154Q/2Q mice. Importantly, motor improvement was seen when treatment was initiated both presymptomatically and after symptom onset. Neuropathologically, lithium treatment attenuated the reduction of dendritic branching in mutant hippocampal pyramidal neurons. We also report that lithium treatment restored the levels of isoprenylcysteine carboxyl methyltransferase (Icmt; alternatively, Pccmt), down-regulation of which is an early marker of mutant ATXN1 toxicity.
Conclusions
The effect of lithium on a marker altered early in the course of SCA1 pathogenesis, coupled with its positive effect on multiple behavioral measures and hippocampal neuropathology in an authentic disease model, make it an excellent candidate treatment for human SCA1 patients.
Huda Zoghbi and colleagues show that lithium treatment initiated before or after disease onset improves multiple symptoms of neurodegeneration in a mouse model of spinocerebellar ataxia.
Editors' Summary
Background.
Spinocerebellar ataxia type 1 (SCA1) is an inherited, incurable neurodegenerative disease in which the neurons (cells that transmit information between the brain and body) in the cerebellum (the brain region that coordinates movement) gradually die. Symptoms of the disease, which usually begins in early adult life, include poor coordination of movement (ataxia), slurred speech, and cognitive (learning and memory) problems. As more neurons die, these symptoms get worse until breathing difficulties eventually cause death. SCA1 is a “triplet repeat disease.” Information for making proteins is stored in DNA as groups of three nucleotides (codons), each specifying a different amino acid (the building blocks of proteins). In triplet repeat diseases, patients inherit a mutant gene containing abnormally long stretches of repeated codons. In SCA1, the repeated codon is CAG, which specifies glutamine. Consequently, SCA1 is a “polyglutamine disease,” a group of neurodegenerative disorders in which an abnormal protein (a different one for each disease) containing a long stretch of glutamine forms nuclear inclusions (insoluble lumps of protein) in neurons that, possibly by trapping essential proteins, cause neuronal death. In SCA1, the abnormal protein is ataxin 1, which is made in many neurons including the cerebellar neurons (Purkinje cells) that coordinate movement.
Why Was This Study Done?
Early in SCA1, the production of several messenger RNAs (the templates for protein production) decreases, possibly because transcription factors (proteins that control gene expression) interact with the mutant protein. Could the progression of SCA1 be slowed, therefore, by using an agent that affects gene expression? In this study, the researchers have investigated whether lithium can slow disease progression in an animal model of SCA1. They chose lithium for their study because this drug (best known for stabilizing mood in people with bipolar [manic] depression) affects gene expression, is neuroprotective, and has beneficial effects in animal models of Huntington disease, another polyglutamine disease.
What Did the Researchers Do and Find?
The researchers bred mice carrying one mutant gene for ataxin 1 containing a very long CAG repeat and one normal gene (Sca1154Q/2Q mice; genes come in pairs). These mice develop symptoms similar to those seen in people with SCA1. After weaning, the mice and their normal littermates were fed normal food or food supplemented with lithium for several weeks before assessing their ability to coordinate their movements and testing their cognitive skills. Dietary lithium (given before or after symptoms appeared) improved both coordination and learning and memory in the Sca1154Q/2Q mice but had little effect in the normal mice. Lithium did not change the overall appearance of the cerebellum in the Sca1154Q/2Q mice nor reduce the occurrence of nuclear inclusions, but it did partly reverse hippocampal neuron degeneration in these animals. The researchers discovered this effect by examining the shape of the hippocampal neurons in detail. These neurons normally have extensive dendrites—branch-like projections that make contact with other cells—but these gradually disappear in Sca1154Q/2Q mice; lithium partly reversed this loss. Finally, lithium also restored the level of Icmt/Pccmt mRNA in the cerebellum to near normal in the Sca1154Q/2Q mice—this mRNA is one of the first to be reduced by ataxin 1 toxicity.
What Do These Findings Mean?
These findings show that treatment with lithium slows neurodegeneration in a mouse model of SCA1, even when it is given only after the first symptoms of the disease have appeared. Unfortunately, lithium did not improve the life span of the Sca1154Q/2Q mice (although this could be because the mutant SCA1 protein has some deleterious effects outside the brain). Thus, lithium is unlikely to cure SCA1, but it could provide some help to people with this devastating disease, even if (as is usual), their condition is not diagnosed until the disease is quite advanced. However, because drugs that work in animal models of diseases do not necessarily work in people, patients with SCA1 (or other polyglutamine diseases, which might also benefit from lithium supplementation) should not be treated with lithium until human trials of this approach have been completed.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040182.
The US National Ataxia Foundation provides information for patients
International Network of Ataxia Friends has information for patients and carergivers on ataxias, including SCA1
GeneTests provides information for health care providers and researchers about SCA1
Online Mendelian Inheritance in Man (OMIM) has detailed scientific information on SCA1
Huntington's Outreach Project for Education offers information for lay people from Stanford University on trinucleotide repeat disorders including SCA1
doi:10.1371/journal.pmed.0040182
PMCID: PMC1880853  PMID: 17535104
5.  IFN-γ Mediates the Rejection of Haematopoietic Stem Cells in IFN-γR1-Deficient Hosts 
PLoS Medicine  2008;5(1):e26.
Background
Interferon-γ receptor 1 (IFN-γR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-γR1 deficiency.
Methods and Findings
We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr1+/+ donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr1−/− recipients. However, Ifngr1−/− mice previously infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rejected HSCT. Like infected IFN-γR1-deficient humans, infected Ifngr1−/− mice displayed very high serum IFN-γ levels before HSCT. The administration of a recombinant IFN-γ-expressing AAV vector to Ifngr1−/− naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr1−/− × Ifng−/− double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-γ depletion in infected Ifngr1−/− mice in vivo allowed subsequent engraftment.
Conclusions
High serum IFN-γ concentration is both necessary and sufficient for graft rejection in IFN-γR1-deficient mice, inhibiting the development of heterologous, IFN-γR1-expressing, haematopoietic cell lineages. These results confirm that IFN-γ is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-γR1-deficient patients through IFN-γ depletion from the blood. They further raise the possibility that depleting IFN-γ may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor.
Claire Soudais and colleagues investigated the mechanism of rejection of hematopoietic stem cell transplants in patients with interferon-gamma receptor 1 (IFN-γR1) deficiency and show that IFN-γ is an anti-hematopoietic cytokine in vivo.
Editors' Summary
Background.
Normally, the body's immune system efficiently recognizes and kills bacteria and viruses, but in some rare inherited disorders (“primary immunodeficiencies”) part of the immune system works poorly or is missing. This leaves affected individuals susceptible to infections. People with one of these disorders—interferon-gamma receptor 1 (IFN- γR1) deficiency—are very susceptible to infections with mycobacteria. Except for Mycobacterium tuberculosis and M. leprae (which cause tuberculosis and leprosy, respectively), mycobacteria rarely cause human disease. However, most people with IFN-γR1 deficiency die during childhood from multiple, widespread mycobacterial infections, because IFN-γR1 deficiency disables a specific part of their immune system. When most bacteria enter the body, immune system cells called macrophages engulf and kill them, but mycobacteria actually multiply inside macrophages. This infection stimulates lymphocytes and other immune system cells to release IFN-γ, which binds to IFN-γR1 on uninfected macrophages, activates them, and recruits them to the infection site. Here, they form a “granuloma,” a mass of macrophages and activated lymphocytes that “walls off” the infection. Granuloma formation does not occur in patients with IFN-γR1 deficiency, so mycobacteria (including the usually benign tuberculosis vaccination strain M. bovis BCG) spread throughout the body with disastrous consequences.
Why Was This Study Done?
The only effective treatment for patients with IFN-γR1 deficiency is hematopoietic stem cell transplantation (HSCT). HSCs are the source of all the immune system cells, so transplantation of HSCs from a donor with a normal IFNGR1 gene can provide a patient who has IFN-γR1 deficiency with a new immune system that can combat mycobacterial infections. Unfortunately, in this particular immune deficiency, the new HSCs cannot engraft, even when the patient's own immune system is disabled before HSCT by intensive chemotherapy, and when the donor cells come from a close relative and are a good immunological match. In this study, the researchers have investigated why rejection is so common in IFN-γR1 deficiency using a mouse strain called C57BL/6 Ifngr1−/−—C57BL/6 denotes the genetic background of these mice and Ifngr1−/− indicates that, like human patients, these mice make no IFN-γR1.
What Did the Researchers Do and Find?
Ifngr1−/− mice, the researchers report, cannot control M. bovis BCG infections and do not form mature granulomas just like human patients with IFN-γR1 deficiency. Wild-type C57BL/6 (Ifngr1+/+) mice, however, rapidly control M. bovis BCG infections and form mature granulomas. Ifngr1+/+ HSC transplanted into mycobacteria-free Ifngr1−/− mice survived well and protected the mice against later mycobacterial challenge but Ifngr1−/− mice infected with M. bovis BCG before HSCT rejected the transplanted HSCs. Mycobacteria-infected Ifngr1−/− mice and human patients with IFN-γR1 deficiency have blood high levels of IFN-γ. Could this be responsible for HSCT rejection? To find out, the researchers expressed IFN-γ in uninfected Ifngr1−/− mice before HSCT. As in infected mice, these grafts failed. Conversely, transplanted HSCs survived when transplanted into Ifngr1−/− mice that had been genetically altered to express no IFN-γ, even when these mice were infected with M. bovis BCG before transplantation. Finally, when the researchers used antibodies (proteins made by the immune system that recognize specific molecules) to remove circulating IFN-γ from infected Ifngr1−/− mice, HSCT worked well in the animals with the lowest IFN-γ levels.
What Do These Findings Mean?
These findings indicate that in a mouse model of IFN-γR1 deficiency, high circulating IFN-γ concentrations drive the rejection of transplanted HSCs and prevent the development of antimycobacterial immunity, probably by directly killing the transplanted cells and/or stopping them multiplying. They also suggest how HSCT could be improved in patients with IFN-γR1 deficiency although, as with all animal studies, the situation in people might turn out to be very different. Importantly, antibodies that reduce circulating IFN-γ are already being used to treat other human immune diseases, so the clinical use of these antibodies in patients with IFN-γ deficiency before HSCT is feasible. Finally, the researchers speculate that the use of IFN-γ–depleting antibodies might be beneficial in other situations where HSCT often fails such as when a close relative is not available as a donor. However, this possibility will need to be thoroughly tested in mice before human clinical trials can be started.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050026.
General information about primary immunodeficiencies is available from the US National Institute of Child Health and Human Development
Online Mendelian Inheritance in Man (OMIM) provides information about familial predisposition to mycobacterial disease
Wikipedia has pages on hematopoietic stem cell transplantation and on interferon-γ (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Human Genetics of Infectious Diseases Lab focuses on the genetic basis of predicposition or resistance to infectious diseases in humans
doi:10.1371/journal.pmed.0050026
PMCID: PMC2214797  PMID: 18232731
6.  Wound Healing and the Dressing* 
The evolution of surgical dressings is traced from 1600 b.c. to a.d. 1944.
The availability of an increasing variety of man-made fibres and films from 1944 onwards has stimulated work on wound dressings, and some of the more important contributions, both clinical and experimental, are discussed. The functions of a wound dressing and the properties which the ideal wound dressing should possess are given. The necessity for both histological and clinical evaluation of wound dressings in animals and in man is stressed.
Wound dressings are the most commonly used therapeutic agents, but there is no means whereby their performance can be assessed. An attempt should be made either nationally or internationally to establish a standard method of assessing the performance of wound dressings. For this it is necessary to have an internationally agreed standard dressing which could be used as a reference or control dressing in all animal and human work. The only animal with skin morphologically similar to that of man is the domestic pig. Three types of wounds could be used: (1) partial-thickness wounds; (2) full-thickness excisions; and (3) third-degree burns.
The development of standard techniques for the assessment of the efficiency of wound dressings would be of considerable benefit to the research worker, the medical profession, the patient, and the surgical dressings industry.
PMCID: PMC1038310  PMID: 13976490
7.  Schofield memorial lecture. Bull semen and muscle ATP: some evidence of the dawn of medical science in ancient Egypt. 
The importance of animal experimentation to human and animal health is not well understood by an increasingly articulate segment of the public. This could have very unfortunate consequences for man and his domestic animals. Even veterinarians and physicians are not as conversant as they need be about the great extent to which advances in human health have depended upon animal observations and experiments. Some believe that resort to "animal models" of biomedical phenomena, including diseases--a comparative or analogical approach to medical studies--is a relatively recent event. Even medical historians often treat these subjects as occasionally recurring aberrations which began with the Greeks, thus largely overlooking the historical meaning and continuing importance of "one medicine" irrespective of species. In fact, comparative medicine has probably been basic to medical progress ever since the dawn of a medical science. Recent research indicates that this approach to biomedical mysteries began to evolve in the minds of Egypt's healer-priests long before Aristotle and the later Alexandrian Greeks made the whole process explicit. Here we examine the origins of what were possibly the first two biomedical theories profounded from inferences based upon dissections, confirmed in at least one instance by experiment, and then applied to medical practice.
PMCID: PMC1255182  PMID: 3530413
8.  Precision animal breeding 
We accept that we are responsible for the quality of life of animals in our care. We accept that the activities of man affect all the living things with which we share this planet. But we are slow to realize that as a result we have a duty of care for all living things. That duty extends to the breeding of animals for which we are responsible. When animals are bred by man for a purpose, the aim should be to meet certain goals: to improve the precision with which breeding outcomes can be predicted; to avoid the introduction and advance of characteristics deleterious to well-being; and to manage genetic resources and diversity between and within populations as set out in the Convention on Biological Diversity. These goals are summed up in the phrase precision animal breeding. They should apply whether animals are bred as sources of usable products or services for medical or scientific research, for aesthetic or cultural considerations, or as pets. Modern molecular and quantitative genetics and advances in reproductive physiology provide the tools with which these goals can be met.
doi:10.1098/rstb.2007.2171
PMCID: PMC2610171  PMID: 17656344
animal breeding; molecular genetics, application of; quantitative genetics, application of; reproductive technologies, application of; responsibility for breeding outcomes
9.  Medications as a source of human exposure to phthalates. 
Environmental Health Perspectives  2004;112(6):751-753.
Phthalates are a group of multifunctional chemicals used in consumer and personal care products, plastics, and medical devices. Laboratory studies show that some phthalates are reproductive and developmental toxicants. Recently, human studies have shown measurable levels of several phthalates in most of the U.S. general population. Despite their widespread use and the consistent toxicologic data on phthalates, information is limited on sources and pathways of human exposure to phthalates. One potential source of exposure is medications. The need for site-specific dosage medications has led to the use of enteric coatings that allow the release of the active ingredients into the small intestine or in the colon. The enteric coatings generally consist of various polymers that contain plasticizers, including triethyl citrate, dibutyl sebacate, and phthalates such as diethyl phthalate (DEP) and dibutyl phthalate (DBP). In this article we report on medications as a potential source of exposure to DBP in a man who took Asacol [active ingredient mesalamine (mesalazine)] for the treatment of ulcerative colitis. In a spot urine sample from this man collected 3 months after he started taking Asacol, the concentration of monobutyl phthalate, a DBP metabolite, was 16,868 ng/mL (6,180 micro g/g creatinine). This concentration was more than two orders of magnitude higher than the 95th percentile for males reported in the 1999-2000 National Health and Nutrition Examination Survey (NHANES). The patient's urinary concentrations of monoethyl phthalate (443.7 ng/mL, 162.6 micro g/g creatinine), mono-2-ethylhexyl phthalate (3.0 ng/mL, 1.1 micro g/g creatinine), and monobenzyl phthalate (9.3 ng/mL, 3.4 micro g/g creatinine) were unremarkable compared with the NHANES 1999-2000 values. Before this report, the highest estimated human exposure to DBP was more than two orders of magnitude lower than the no observable adverse effect level from animal studies. Further research is necessary to determine the proportional contribution of medications, as well as personal care and consumer products, to a person's total phthalate burden.
PMCID: PMC1241971  PMID: 15121520
10.  An Antibiotic-Responsive Mouse Model of Fulminant Ulcerative Colitis  
PLoS Medicine  2008;5(3):e41.
Background
The constellation of human inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease, which both display a wide spectrum in the severity of pathology. One theory is that multiple genetic hits to the host immune system may contribute to the susceptibility and severity of IBD. However, experimental proof of this concept is still lacking. Several genetic mouse models that each recapitulate some aspects of human IBD have utilized a single gene defect to induce colitis. However, none have produced pathology clearly distinguishable as either ulcerative colitis or Crohn's disease, in part because none of them reproduce the most severe forms of disease that are observed in human patients. This lack of severe IBD models has posed a challenge for research into pathogenic mechanisms and development of new treatments. We hypothesized that multiple genetic hits to the regulatory machinery that normally inhibits immune activation in the intestine would generate more severe, reproducible pathology that would mimic either ulcerative colitis or Crohn's disease.
Methods and Findings
We generated a novel mouse line (dnKO) that possessed defects in both TGFβRII and IL-10R2 signaling. These mice rapidly and reproducibly developed a disease resembling fulminant human ulcerative colitis that was quite distinct from the much longer and more variable course of pathology observed previously in mice possessing only single defects. Pathogenesis was driven by uncontrolled production of proinflammatory cytokines resulting in large part from T cell activation. The disease process could be significantly ameliorated by administration of antibodies against IFNγ and TNFα and was completely inhibited by a combination of broad-spectrum antibiotics.
Conclusions
Here, we develop to our knowledge the first mouse model of fulminant ulcerative colitis by combining multiple genetic hits in immune regulation and demonstrate that the resulting disease is sensitive to both anticytokine therapy and broad-spectrum antibiotics. These findings indicated the IL-10 and TGFβ pathways synergize to inhibit microbially induced production of proinflammatory cytokines, including IFNγ and TNFα, which are known to play a role in the pathogenesis of human ulcerative colitis. Our findings also provide evidence that broad-spectrum antibiotics may have an application in the treatment of patients with ulcerative colitis. This model system will be useful in the future to explore the microbial factors that induce immune activation and characterize how these interactions produce disease.
Paul Allen and colleagues describe the development of a mouse model of fulminant ulcerative colitis with multiple genetic hits in immune regulation which can be moderated by anti-cytokine therapy and broad-spectrum antibiotics.
Editors' Summary
Background.
Inflammatory bowel disease (IBD), a group of disorders characterized by inflammation (swelling) of the digestive tract (the tube that runs from the mouth to the anus), affects about 1.4 million people in the US. There are two main types of IBD. In Crohn's disease, which can affect any area of the digestive tract but most commonly involves the lower part of the small intestine (small bowel), all the layers of the intestine become inflamed. In ulcerative colitis, which primarily affects the colon (large bowel) and the rectum (the part of the bowel closest to the anus), only the lining of the bowel becomes inflamed, the cells in this lining die, and sores or ulcers form. Both types of IBD most commonly develop between the ages of 15 and 35 years, often run in families, and carry an increased risk of cancer. Symptoms—usually diarrhea and abdominal cramps—can be mild or severe and the disorder can develop slowly or suddenly. There is no medical cure for IBD, but drugs that modulate the immune system (for example, corticosteroids) can help some people. Some people benefit from treatment with drugs that specifically inhibit “proinflammatory cytokines,” proteins made by the immune system that stimulate inflammation (for example, TNFα and INFγ). When medical therapy fails, surgery to remove the affected part of the bowel may be necessary.
Why Was This Study Done?
Exactly what causes IBD is not clear, but people with IBD seem to have an overactive immune system. The immune system normally protects the body from harmful substances but in IBD it mistakenly recognizes the food substances and “good” bacteria that are normally present in the human gut as foreign and hence reacts against them. As a result, immune system cells accumulate in the lining of the bowel and cause inflammation. Several different pathways usually prevent inappropriate immune activation, so could IBD be caused by alterations in one or several of these immune regulatory pathways? In previous studies, mice with a defect in just one pathway have developed mild intestinal abnormalities but not the problems seen in the most severe forms of IBD. In this study, therefore, the researchers have generated and characterized a new mouse line with defects in two immune regulatory pathways to see whether this might be a better animal model of human IBD.
What Did the Researchers Do and Find?
To make their new mouse line, the researchers mated mice that had a defective TGFβ signaling pathway in their T lymphocytes with mice that had a defective IL-10 signaling pathway. Both these pathways are anti-inflammatory, and mice with defects in either pathway develop mild and variable inflammation of the colon (colitis) by age 3–4 months. By contrast, the doubly defective mice (dnKO mice) failed to thrive, lost weight, and died by 4–6 weeks of age. The colons of 4- to 5-week old dnKO mice were inflamed and ulcerated (some changes were visible in 3-week-old mice) and contained many immune system cells. Mice with a single defective signaling pathway had no gut abnormalities at this age. The dnKO mice, just like people with IBD, had higher than normal blood levels of IFNγ, TNFα, and other proinflammatory cytokines; these raised levels were the result of abnormal lymphocyte activation. Treatment of the dnKO mice with a combination of agents that neutralize IFNγ and TNFα (anti-cytokine therapy) greatly reduced the colitis seen in these mice; neutralization of IFNγ alone had some beneficial effects, but neutralization of TNFα alone had no effect. Finally, early treatment of the dnKO mice with broad-spectrum antibiotics completely inhibited colitis.
What Do These Findings Mean?
These findings suggest that dnKO mice are a good model for fulminant (severe and rapidly progressing) ulcerative colitis and support the idea that IBD involves multiple genetic defects in immune regulation. They also indicate that the IL-10 and the TGFβ signaling pathways normally cooperate to inhibit the inappropriate immune responses to intestinal bacteria seen in IBD. This new mouse model should help researchers unravel what goes wrong in IBD and should also help them develop new treatments for ulcerative colitis. More immediately, these findings suggest that combined anti-cytokine therapy may be a better treatment for ulcerative colitis than single therapy. In addition, they suggest that clinical studies should be started to test whether broad-spectrum antibiotics can ameliorate ulcerative colitis in people.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050041.
The Medline Plus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
Information is available from the UK National Health Service Direct Health Encyclopedia about Crohn's disease and ulcerative colitis
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
Information and support for patients with inflammatory bowel disease and their caregivers is provided by the Crohn's and Colitis Foundation of America and by the UK National Association for Colitis and Crohn's Disease
doi:10.1371/journal.pmed.0050041
PMCID: PMC2270287  PMID: 18318596
11.  Glycosylation Defects and Virulence Phenotypes of Leishmania mexicana Phosphomannomutase and Dolicholphosphate-Mannose Synthase Gene Deletion Mutants 
Molecular and Cellular Biology  2001;21(23):8168-8183.
Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (ΔPMM and ΔDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana ΔDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana ΔPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.
doi:10.1128/MCB.21.23.8168-8183.2001
PMCID: PMC99981  PMID: 11689705
12.  TUMOR INOCULATION INTO ORGANS AND THE ANALOGY BETWEEN HUMAN CANCER AND THE TUMORS OF WHITE MICE AND WHITE RATS 
The analysis of the experiments described above indicates that tumors of the white rat or white mouse inoculated into parenchymatous organs acquire a different biological character from those inoculated subcutaneously. The latter are a great deal more benign in their behavior than human cancer or spontaneous tumors in the same species of animals. Tumors inoculated into organs, on the other hand, are quite identical in their biological behavior with the malignant tumors of animal and man. A conclusion must then be drawn, even a priori, that the method of inoculation into organs is a very important aid in the experimental investigation of cancer. It is true that the method is a great deal more complicated and time-consuming than the ordinary subcutaneous inoculation. The subcutaneous method is satisfactory for a number of cancer problems. One of these is the study of general susceptibility and resistance of the organism of the host to the inoculation of the tumors, and this is a subject of paramount importance in cancer research. On the other hand, the investigations of the writer (10) have shown that an animal may be susceptible to a subcutaneous inoculation of a certain tumor and resist the inoculation of the same tumor into the testicle. Undoubtedly this method of inoculation will reveal the existence of a number of other phenomena. The discovery of specific therapeutic measures is certainly the greatest problem in cancer research. A great deal of work has been done already on the subject, and the latest investigations of Wassermann on the chemotherapy of experimental tumors seem to be of great promise. But here also the therapeutic methods must be tried on animals in which the inoculations of tumor cells have been made into parenchymatous organs before the growths thus treated will have any analogy to human cancer. In this connection one must bear in mind the fact that all the empirical so-called specific cancer remedies, which are continually being devised, are usually successful in treating localized skin cancers and fail utterly in the malignant growths of the internal organs. It is comparatively easy to produce a localized necrosis and softening in a circumscribed growth of the skin and subcutaneous tissue, but whether the same result will be produced on a diffuse and better nourished tumor growing inside of a parenchymatous organ cannot be decided a priori. To determine this it is necessary to have experimental proof on animals in which the tumor was inoculated into organs.
PMCID: PMC2125241  PMID: 19867561
13.  Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation 
PLoS Medicine  2007;4(6):e230.
Background
RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).
Methods and Findings
RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation.
Conclusions
Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.
The study by Samuel Jacobson and colleagues suggests that retinal gene therapy can improve retinal, visual pathway, and visual cortex responses to light stimulation, even after prolonged periods of blindness and in congenitally blind patients.
Editors' Summary
Background.
The eye captures light but the brain is where vision is experienced. Treatments for childhood blindness at the eye level are ready, but it is unknown whether the brain will be receptive to an improved neural message. Normal vision begins as photoreceptor cells in the retina (the light-sensitive tissue lining the inside of the eye) convert visual images into electrical impulses. These impulses are sent along the optic nerve to the visual cortex, the brain region where they are interpreted. The conversion of light into electrical impulses requires the activation of a molecule called retinal, which is subsequently recycled by retinal pigment epithelium (RPE) cells neighboring the retina. One of the key enzymes of the recycling reactions is encoded by a gene called RPE65. Genetic changes (mutations) in RPE65 cause an inherited form of blindness called Leber congenital amaurosis (LCA). In this disease, retinal is not recycled and as a result, the photoreceptor cells cannot work properly and affected individuals have poor or nonexistent vision from birth. Previous studies in dog and mouse models of the human disease have demonstrated that the introduction of a functional copy of RPE65 into the RPE cells using a harmless virus (gene therapy) dramatically restores retinal activity. Very recently, a pioneering gene therapy operation took place in London (UK) where surgeons injected a functional copy of RPE65 into the retina of a man with LCA. Whether this operation results in improved vision is not known at this time.
Why Was This Study Done?
Gene therapy corrects the retinal defects in animal models of LCA but whether the visual pathway from the retina to the visual cortex of the brain can respond normally to the signals sent by the restored retina is not known. Early visual experience is thought to be necessary for the development of a functional visual cortex, so replacing the defective RPE65 gene might not improve the vision of people with LCA. In this study, the researchers have studied the visual cortex of RPE65-deficient dogs before and after gene therapy to see whether the therapy affects the activity of the visual cortex. They have also investigated visual pathway integrity and responsiveness in adults with LCA caused by RPE65 mutations. If the visual pathway is disrupted in these patients, they reasoned, gene therapy might not restore their vision.
What Did the Researchers Do and Find?
The researchers used a technique called functional magnetic resonance imaging (fMRI) to measure light-induced brain activity in RPE65-deficient dogs before and after gene therapy. They also examined the reactions of the dogs' pupils to light (in LCA, the pupils do not contract normally in response to light because there is reduced signal transmission along the visual pathway). Finally, they measured the electrical activity of the dogs' retinas in response to light flashes—the retinas of patients with LCA do not react to light. Gene therapy corrected the defective retinal and visual pathway responses to light in the RPE65-deficient dogs and, whereas before treatment there was no response in the visual cortex to light stimulation in these dogs, after treatment, its activity approached that seen in normal dogs. The recovery of cortical responses was permanent and occurred soon after treatment, even in animals that were 4 years old when treated. Next, using structural MRI, the researchers studied human patients with LCA and found that the optic nerve diameter in young adults was within the normal range and that the structure of the visual cortex was very similar to that of normal individuals. Finally, using fMRI, they found that, although the visual cortex of patients with LCA did not respond to dim light, its reaction to bright light was comparable to that of normal individuals.
What Do These Findings Mean?
The findings from the dog study indicate that retinal gene therapy rapidly improves retinal, visual pathway, and visual cortex responses to light stimulation, even in animals that have been blind for years. In other words, in the dog model of LCA at least, all the components of the visual system remain receptive to visual inputs even after long periods of visual deprivation. The findings from the human study also indicate that the visual pathway remains anatomically intact despite years of disuse and that the visual cortex can be activated in patients with LCA even though these people have very limited visual experience. Taken together, these findings suggest that successful gene therapy of the retina might restore some functional vision to people with LCA but proof will have to await the outcomes of several clinical trials ongoing or being planned in Europe and the USA.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040230.
General information on gene therapy is available from the Oak Ridge National Laboratory
Information is provided by the BBC about gene therapy for Leber congenital amaurosis (includes an audio clip from a doctor about the operation)
The National Institutes of Health/National Eye Institute (US) provides information about an ongoing gene therapy trial of RPE65-Leber congenital amaurosis
ClinicalTrials.gov gives details on treatment trials for Leber congenital amaurosis
The Foundation Fighting Blindness has a fact sheet on Leber congenital amaurosis (site includes Microsoft Webspeak links that read some content aloud)
The Foundation for Retinal Research has a fact sheet on Leber congenital amaurosis
Find more detailed information on Leber congenital amaurosis and the gene mutations that cause it from GeneReviews
WonderBaby, information for parents of babies with Leber congenital amaurosis
doi:10.1371/journal.pmed.0040230
PMCID: PMC1896221  PMID: 17594175
14.  Staphylococcus aureus Cap5O Has UDP-ManNAc Dehydrogenase Activity and Is Essential for Capsule Expression 
Infection and Immunity  2001;69(2):917-923.
The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a repeating unit composed of (→4)-3-O-acetyl-β-d-ManNAcA-(1→4)-α-l-FucNAc (1→3)-β-d-FucNAc-(1→)n. Sixteen chromosomal genes (cap5A through cap5P) are involved in the synthesis of CP5. We recently demonstrated that Cap5P, a 2-epimerase, catalyzes the conversion of UDP–N-acetyl glucosamine (UDP-GlcNAc) to UDP–N-acetylmannosamine (UDP-ManNAc). In this study, we show that UDP-ManNAc is oxidized to UDP–N-acetylmannosaminuronic acid (UDP-ManNAcA) by a UDP-ManNAc dehydrogenase encoded by S. aureus cap5O. We expressed Cap5O in Escherichia coli and purified the recombinant protein. The UDP-ManNAc dehydrogenase activity of purified Cap5O was assessed by incubating Cap5P and UDP-GlcNAc (to produce UDP-ManNAc), together with Cap5O, NAD+, and a reducing agent. Enzymatic activity was quantitated indirectly by measuring the increase in absorbance at 340 nm resulting from NADH formation. The product of the reaction was confirmed as UDP-ManNAcA by gas chromatography-mass spectroscopy. A cap5O mutation, created by deletion of 727 bp in the 5′ end of the gene, was introduced by allelic replacement into S. aureus Reynolds, rendering it CP5 negative. Mice inoculated intravenously or subcutaneously with the wild-type strain Reynolds had greater numbers of S. aureus recovered from their kidneys (P = 0.019) or their subcutaneous abscesses (P = 0.0018), respectively, than did animals inoculated with the cap5O mutant. The results of this study indicate that S. aureus cap5O is essential for capsule production and that capsule promotes staphylococcal virulence in mouse models of abscess formation.
doi:10.1128/IAI.69.2.917-923.2001
PMCID: PMC97970  PMID: 11159986
15.  Infiltrating Blood-Derived Macrophages Are Vital Cells Playing an Anti-inflammatory Role in Recovery from Spinal Cord Injury in Mice 
PLoS Medicine  2009;6(7):e1000113.
Using a mouse model of spinal injury, Michal Schwartz and colleagues tested the effect of macrophages on the recovery process and demonstrate an important anti-inflammatory role for a subset of infiltrating monocyte-derived macrophages that is dependent upon their expression of interleukin 10.
Background
Although macrophages (MΦ) are known as essential players in wound healing, their contribution to recovery from spinal cord injury (SCI) is a subject of debate. The difficulties in distinguishing between different MΦ subpopulations at the lesion site have further contributed to the controversy and led to the common view of MΦ as functionally homogenous. Given the massive accumulation in the injured spinal cord of activated resident microglia, which are the native immune occupants of the central nervous system (CNS), the recruitment of additional infiltrating monocytes from the peripheral blood seems puzzling. A key question that remains is whether the infiltrating monocyte-derived MΦ contribute to repair, or represent an unavoidable detrimental response. The hypothesis of the current study is that a specific population of infiltrating monocyte-derived MΦ is functionally distinct from the inflammatory resident microglia and is essential for recovery from SCI.
Methods and Findings
We inflicted SCI in adult mice, and tested the effect of infiltrating monocyte-derived MΦ on the recovery process. Adoptive transfer experiments and bone marrow chimeras were used to functionally distinguish between the resident microglia and the infiltrating monocyte-derived MΦ. We followed the infiltration of the monocyte-derived MΦ to the injured site and characterized their spatial distribution and phenotype. Increasing the naïve monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. Selective ablation of infiltrating monocyte-derived MΦ following SCI while sparing the resident microglia, using either antibody-mediated depletion or conditional ablation by diphtheria toxin, impaired recovery. Reconstitution of the peripheral blood with monocytes resistant to ablation restored the lost motor functions. Importantly, the infiltrating monocyte-derived MΦ displayed a local anti-inflammatory beneficial role, which was critically dependent upon their expression of interleukin 10.
Conclusions
The results of this study attribute a novel anti-inflammatory role to a unique subset of infiltrating monocyte-derived MΦ in SCI recovery, which cannot be provided by the activated resident microglia. According to our results, limited recovery following SCI can be attributed in part to the inadequate, untimely, spontaneous recruitment of monocytes. This process is amenable to boosting either by active vaccination with a myelin-derived altered peptide ligand, which indicates involvement of adaptive immunity in monocyte recruitment, or by augmenting the naïve monocyte pool in the peripheral blood. Thus, our study sheds new light on the long-held debate regarding the contribution of MΦ to recovery from CNS injuries, and has potentially far-reaching therapeutic implications.
Please see later in the article for Editors' Summary
Editors' Summary
Background
Every year, spinal cord injuries paralyze about 11,000 people in the US. The spinal cord, which contains bundles of nervous system cells called neurons, is the communication highway between the brain and the body. Messages from the brain travel down the spinal cord to control movement, breathing and other bodily functions; messages from the skin and other sensory organs travel up the spinal cord to keep the brain informed about the body. The bones of the spine normally protect the spinal cord but, if these are broken or displaced, the spinal cord can be cut or compressed, which interrupts the information flow. Damage near the top of the spinal cord paralyzes the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause other medical problems, including the loss of bladder and bowel control. Currently, there is no effective treatment for spinal cord injuries, which usually cause permanent disability because the damaged nerve fibers rarely regrow.
Why Was This Study Done?
After a spinal cord injury, immune system cells called macrophages accumulate at the injury site. Some of these macrophages—so-called monocyte-derived macrophages—come into (infiltrate) the spinal cord from the blood in response to the injury, whereas others—microglia—are always in the nervous system. Although macrophages are essential for wound healing in other parts of the body, it is unclear whether they have good or bad effects in the spinal cord. Many experts believe that immune system cells hinder healing in the spinal cord and should be suppressed or eliminated, but other scientists claim that macrophages secrete factors that stimulate nerve regrowth. Furthermore, although some macrophages elsewhere in the body have proinflammatory (potentially deleterious) effects, others have anti-inflammatory (beneficial) effects. So do the infiltrating monocyte-derived macrophages and the resident microglia (which are proinflammatory) have different functions at spinal cord injury sites? In this study, the researchers try to answer this important question.
What Did the Researchers Do and Find?
The researchers bruised a small section of the spinal cord of adult mice and then investigated the effect of infiltrating monocyte-derived macrophages on the recovery process. Monocyte-derived macrophages and microglia cannot be distinguished using standard staining techniques so to study their behavior after spinal cord injury the researchers introduced labeled monocyte-derived macrophages into their experimental animals by using adoptive transfer (injection of genetically labeled monocytes into the animals) or by making bone marrow chimeras. In this second technique, the animals' monocyte-derived macrophages (but not their microglia) were killed by irradiating the animals before injection of genetically labeled bone marrow, the source of monocytes. Using these approaches, the researchers found that monocyte-derived macrophages collected at the margins of spinal cord injury sites whereas microglia accumulated throughout the sites. When the pool of monocyte-derived macrophages in the mice was increased by adoptive transfer or by using a technique called “CNS-specific vaccination,” more monocyte-derived macrophages infiltrated the injury site and the animals' physical recovery from injury improved. Conversely, removal of the infiltrating monocyte-derived macrophages from the injury site reduced the animals' physical recovery. Other experiments indicated that the infiltrating monocyte-derived macrophages have a beneficial, local anti-inflammatory effect that is dependent on their expression of interleukin-10 (an anti-inflammatory signaling molecule).
What Do These Findings Mean?
These findings provide new information about the contribution of monocyte-derived macrophages to spontaneous recovery from spinal cord injury, a contribution that has long been debated. In particular, the findings suggest that this subset of macrophages (but not the resident microglia) has a beneficial effect on spinal cord injuries that is mediated by their production of the anti-inflammatory molecule interleukin-10. The findings also show that the effect of these monocyte-derived macrophages can be boosted, at least in mice. Although results obtained in experiments done in animals do not always accurately reflect what happens in people, this new understanding of the different functions of microglia and infiltrating monocyte-derived macrophages after injury to the spinal cord may eventually lead to the development of better treatments for spinal cord injuries.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000113.
The MedlinePlus encyclopedia provides information about spinal cord injuries (in English and Spanish)
The US National Institute of Neurological Disorders and Stroke provides detailed information about spinal cord injury, including information on current research into the problem (in English and Spanish)
MedlinePlus provides an interactive tutorial on spinal cord injury and a list of links to additional information (in English and Spanish)
doi:10.1371/journal.pmed.1000113
PMCID: PMC2707628  PMID: 19636355
16.  Brain tumors in man and animals: report of a workshop. 
This report summarizes the results of a workshop on brain tumors in man and animals. Animals, especially rodents are often used as surrogates for man to detect chemicals that have the potential to induce brain tumors in man. Therefore, the workshop was focused mainly on brain tumors in the F344 rat and B6C3F1 mouse because of the frequent use of these strains in long-term carcinogenesis studies. Over 100 brain tumors in F344 rats and more than 50 brain tumors in B6C3F1 mice were reviewed and compared to tumors found in man and domestic or companion animals. In the F344 rat, spontaneous brain tumors are uncommon, most are of glial origin, and the highly undifferentiated glioblastoma multiforme, a frequent tumor of man was not found. In the B6C3F1 mouse, brain tumors are exceedingly rare. Lipomas of the choroid plexus and meningiomas together account for more than 50% of the tumors found. Both rodent strains examined have low background rates and very little variability between control groups.
Images
PMCID: PMC1474266  PMID: 3536473
17.  Methods for evaluating the effects of environmental chemicals on human sperm production. 
Sperm tests provide a direct and effective way of identifying chemical agents that induce spermatogenic damage in man. Four human sperm tests are available: sperm count, motility, morphology (seminal cytology) and the Y-body test. These sperm tests have numerous advantages over other approaches for assessing spermatogenic damage, and they have already been used to assess the effects of at least 85 different occupational, environmental, and drug-related chemical exposures. When carefully controlled, seminal cytology appears to be statistically more sensitive than the other human sperm tests and should be considered an integral part of semen analysis when assessing induced spermatogenic damage. Human sperm studies have complex requirements and, before sampling, careful consideration should be given to exposure details, group size and makeup, as well as animal and human data that indicate spermatogenic effects. Several study designs are possible and should include questionnaires covering medical and reproductive histories as well as known confounding factors. Animal sperm tests, such as the mouse morphology test, may be used to identify the toxic components of a complex mixture. Animal tests may also help assess the chemical effects on fertility and reproductive outcome in cases when human data are incomplete. Further efforts are needed in these areas to develop improved human sperm tests sensitive to induced spermatogenic damage, to develop improved animal models of induced spermatogenic damage, to understand the relationships among sperm changes, fertility, and reproductive outcome, and to develop sperm tests with express mutational end points.
PMCID: PMC1569065  PMID: 6825635
18.  Mutation of von Hippel–Lindau Tumour Suppressor and Human Cardiopulmonary Physiology 
PLoS Medicine  2006;3(7):e290.
Background
The von Hippel–Lindau tumour suppressor protein–hypoxia-inducible factor (VHL–HIF) pathway has attracted widespread medical interest as a transcriptional system controlling cellular responses to hypoxia, yet insights into its role in systemic human physiology remain limited. Chuvash polycythaemia has recently been defined as a new form of VHL-associated disease, distinct from the classical VHL-associated inherited cancer syndrome, in which germline homozygosity for a hypomorphic VHL allele causes a generalised abnormality in VHL–HIF signalling. Affected individuals thus provide a unique opportunity to explore the integrative physiology of this signalling pathway. This study investigated patients with Chuvash polycythaemia in order to analyse the role of the VHL–HIF pathway in systemic human cardiopulmonary physiology.
Methods and Findings
Twelve participants, three with Chuvash polycythaemia and nine controls, were studied at baseline and during hypoxia. Participants breathed through a mouthpiece, and pulmonary ventilation was measured while pulmonary vascular tone was assessed echocardiographically. Individuals with Chuvash polycythaemia were found to have striking abnormalities in respiratory and pulmonary vascular regulation. Basal ventilation and pulmonary vascular tone were elevated, and ventilatory, pulmonary vasoconstrictive, and heart rate responses to acute hypoxia were greatly increased.
Conclusions
The features observed in this small group of patients with Chuvash polycythaemia are highly characteristic of those associated with acclimatisation to the hypoxia of high altitude. More generally, the phenotype associated with Chuvash polycythaemia demonstrates that VHL plays a major role in the underlying calibration and homeostasis of the respiratory and cardiovascular systems, most likely through its central role in the regulation of HIF.
Editors' Summary
Background.
Human cells (like those of other multicellular animals) use oxygen to provide the energy needed for daily life. Having not enough oxygen is a problem, but having too much is also dangerous because it damages proteins, DNA, and other large molecules that keep cells functioning. Consequently, the physiological systems—including the heart, lungs, and circulation—work together to balance oxygen supply and demand throughout the body. When oxygen is limiting (a condition called hypoxia), as happens at high altitudes, the cellular oxygen supply is maintained by increasing the heart rate, increasing the speed and depth of breathing (hyperventilation), constricting the blood vessels in the lung (pulmonary vasoconstriction), and increasing the number of oxygen-carrying cells in the blood. All these physiological changes increase the amount of oxygen that can be absorbed from the air, but how they are regulated is poorly understood. By contrast, researchers know quite a bit about how individual cells respond to hypoxia. When oxygen is limited, a protein called hypoxia-inducible factor (or HIF) activates a number of target proteins that help the cell get enough oxygen (for example, proteins that stimulate the growth of new blood vessels). When there is plenty of oxygen, another protein, called von Hippel–Lindau tumor suppressor (abbreviated VHL), rapidly destroys HIF. Recently, researchers discovered that a genetic condition called Chuvash polycythaemia, characterised by the overproduction of red blood cells, is caused by a specific defect in VHL that reduces its ability to destroy HIF. As a result, the expression of certain HIF target proteins is increased even when oxygen levels are normal.
Why Was This Study Done?
Chuvash polycythaemia is very rare, and so far little is known about how this genetic abnormality affects the physiology and long-term health of patients. By studying heart and lung function in patients with Chuvash polycythaemia, the researchers involved in this study hoped to discover more about the health consequences of the condition and to find out whether the VHL–HIF system controls systemic responses to hypoxia as well as cellular responses.
What Did the Researchers Do and Find?
The researchers recruited and studied three patients with Chuvash polycythaemia, and, as controls for the comparison, several normal individuals and patients with an unrelated form of polycythaemia. They then measured how the lungs and hearts of these people reacted to mild hypoxia (similar to that experienced on commercial air flights) and moderate hypoxia (equiv alent to being on the top of an Alpine peak). They found that patients with Chuvash polycythaemia naturally breathe slightly quicker and deeper than normal individuals, and that their breathing rate increased dramatically and abnormally when oxygen was reduced. They also found that at normal oxygen levels the pulmonary blood vessels of these patients were more constricted than those of control individuals, and that they reacted more extremely to hypoxia. Similarly, the normal heart rate of the patients was slightly higher than that of the controls and increased much more in response to mild hypoxia.
What Do These Findings Mean?
The physiological differences measured by the researchers between Chuvash polycythaemia patients and control individuals are similar to the adaptations seen in people traveling to high altitudes where oxygen is limited. Thus, the VHL–HIF proteins may regulate the response to different oxygen concentrations both in individual cells and at the systemic level, although more physiological studies are needed to confirm this. Because the pulmonary blood vessels of patients with Chuvash polycythaemia are always abnormally constricted, and even more so when oxygen is limited, these people should avoid living at high altitude and should minimise air travel, suggest the researchers. The increased blood pressure in their lungs (pulmonary hypertension) could conceivably cause heart failure under such circumstances. Finally, this study has implications for the development of drugs directed at the VHL–HIF system. Agents are currently being designed to promote the development of new blood vessels after strokes or heart attacks by preventing the destruction of HIF, but based on the findings here such agents might have undesirable physiological affects. Conversely, HIF inhibitors (which act as anti-cancer reagents by increasing hypoxia in the centre of tumors and so inhibiting their growth) might be useful in the treatment of pulmonary hypertension.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030290.
• Online Mendelian Inheritance in Man page on Chuvash polycythaemia
• Information from the VHL Family Alliance on von Hippel–Lindau disease, including information on Chuvash polycythaemia
• Wikipedia page on polycythaemia and von Hippel–Lindau disease (note: Wikipedia is a free online encyclopaedia that anyone can edit)
Physiological study of patients with Chuvash polycythemia (caused by mutation of VHL) reveals characteristics similar to those associated with acclimatization to the hypoxia of high altitude.
doi:10.1371/journal.pmed.0030290
PMCID: PMC1479389  PMID: 16768548
19.  Immunological effects of chlorinated dibenzo-p-dioxins. 
Environmental Health Perspectives  1995;103(Suppl 9):47-53.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar halogenated aromatic hydrocarbons cause a broad range of immunologic effects in experimental animals including decreased host resistance to infectious disease and suppressed humoral and cell-mediated immune responses. In the mouse, TCDD immunotoxicity has been shown to be an aryl hydrocarbon (Ah) receptor-dependent process. However, despite considerable research, the biochemical and molecular alterations that occur subsequent to Ah receptor activation that lead to altered immune reactivity remain to be elucidated. In addition to immune suppression, TCDD promotes inflammatory responses. This effect may result from an upregulation of the production of inflammatory cytokines such as interleukin-1 and tumor necrosis factor. Nonhuman primates exposed to TCDD show suppressed antibody responses and changes in lymphocyte subsets in the peripheral blood. The immunotoxic effects of TCDD in humans are poorly characterized, and few studies have examined the immune status of individuals with known, documented exposure to TCDD. It is important for laboratory research to focus on defining TCDD-sensitive immunologic biomarkers in animal models that can also be used in human subjects. Understanding the mechanisms that underlie species differences in TCDD immunotoxicity is also of critical importance for extrapolation of effects seen in laboratory animals to man.
PMCID: PMC1518816  PMID: 8635439
20.  Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems 
British Journal of Cancer  2002;87(11):1328-1335.
The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the α-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 106 murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse−1 were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5×107 cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted.
British Journal of Cancer (2002) 87, 1328–1335. doi:10.1038/sj.bjc.6600573 www.bjcancer.com
© 2002 Cancer Research UK
doi:10.1038/sj.bjc.6600573
PMCID: PMC2408903  PMID: 12439725
TaqMan PCR; xenografts; metastasis; leukaemia; haematological cells
21.  Threats to Validity in the Design and Conduct of Preclinical Efficacy Studies: A Systematic Review of Guidelines for In Vivo Animal Experiments 
PLoS Medicine  2013;10(7):e1001489.
Background
The vast majority of medical interventions introduced into clinical development prove unsafe or ineffective. One prominent explanation for the dismal success rate is flawed preclinical research. We conducted a systematic review of preclinical research guidelines and organized recommendations according to the type of validity threat (internal, construct, or external) or programmatic research activity they primarily address.
Methods and Findings
We searched MEDLINE, Google Scholar, Google, and the EQUATOR Network website for all preclinical guideline documents published up to April 9, 2013 that addressed the design and conduct of in vivo animal experiments aimed at supporting clinical translation. To be eligible, documents had to provide guidance on the design or execution of preclinical animal experiments and represent the aggregated consensus of four or more investigators. Data from included guidelines were independently extracted by two individuals for discrete recommendations on the design and implementation of preclinical efficacy studies. These recommendations were then organized according to the type of validity threat they addressed. A total of 2,029 citations were identified through our search strategy. From these, we identified 26 guidelines that met our eligibility criteria—most of which were directed at neurological or cerebrovascular drug development. Together, these guidelines offered 55 different recommendations. Some of the most common recommendations included performance of a power calculation to determine sample size, randomized treatment allocation, and characterization of disease phenotype in the animal model prior to experimentation.
Conclusions
By identifying the most recurrent recommendations among preclinical guidelines, we provide a starting point for developing preclinical guidelines in other disease domains. We also provide a basis for the study and evaluation of preclinical research practice.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
The development process for new drugs is lengthy and complex. It begins in the laboratory, where scientists investigate the causes of diseases and identify potential new treatments. Next, promising interventions undergo preclinical research in cells and in animals (in vivo animal experiments) to test whether the intervention has the expected effect and to support the generalization (extension) of this treatment–effect relationship to patients. Drugs that pass these tests then enter clinical trials, where their safety and efficacy is tested in selected groups of patients under strictly controlled conditions. Finally, the government bodies responsible for drug approval review the results of the clinical trials, and successful drugs receive a marketing license, usually a decade or more after the initial laboratory work. Notably, only 11% of agents that enter clinical testing (investigational drugs) are ultimately licensed.
Why Was This Study Done?
The frequent failure of investigational drugs during clinical translation is potentially harmful to trial participants. Moreover, the costs of these failures are passed onto healthcare systems in the form of higher drug prices. It would be good, therefore, to reduce the attrition rate of investigational drugs. One possible explanation for the dismal success rate of clinical translation is that preclinical research, the key resource for justifying clinical development, is flawed. To address this possibility, several groups of preclinical researchers have issued guidelines intended to improve the design and execution of in vivo animal studies. In this systematic review (a study that uses predefined criteria to identify all the research on a given topic), the authors identify the experimental practices that are commonly recommended in these guidelines and organize these recommendations according to the type of threat to validity (internal, construct, or external) that they address. Internal threats to validity are factors that confound reliable inferences about treatment–effect relationships in preclinical research. For example, experimenter expectation may bias outcome assessment. Construct threats to validity arise when researchers mischaracterize the relationship between an experimental system and the clinical disease it is intended to represent. For example, researchers may use an animal model for a complex multifaceted clinical disease that only includes one characteristic of the disease. External threats to validity are unseen factors that frustrate the transfer of treatment–effect relationships from animal models to patients.
What Did the Researchers Do and Find?
The researchers identified 26 preclinical guidelines that met their predefined eligibility criteria. Twelve guidelines addressed preclinical research for neurological and cerebrovascular drug development; other disorders covered by guidelines included cardiac and circulatory disorders, sepsis, pain, and arthritis. Together, the guidelines offered 55 different recommendations for the design and execution of preclinical in vivo animal studies. Nineteen recommendations addressed threats to internal validity. The most commonly included recommendations of this type called for the use of power calculations to ensure that sample sizes are large enough to yield statistically meaningful results, random allocation of animals to treatment groups, and “blinding” of researchers who assess outcomes to treatment allocation. Among the 25 recommendations that addressed threats to construct validity, the most commonly included recommendations called for characterization of the properties of the animal model before experimentation and matching of the animal model to the human manifestation of the disease. Finally, six recommendations addressed threats to external validity. The most commonly included of these recommendations suggested that preclinical research should be replicated in different models of the same disease and in different species, and should also be replicated independently.
What Do These Findings Mean?
This systematic review identifies a range of investigational recommendations that preclinical researchers believe address threats to the validity of preclinical efficacy studies. Many of these recommendations are not widely implemented in preclinical research at present. Whether the failure to implement them explains the frequent discordance between the results on drug safety and efficacy obtained in preclinical research and in clinical trials is currently unclear. These findings provide a starting point, however, for the improvement of existing preclinical research guidelines for specific diseases, and for the development of similar guidelines for other diseases. They also provide an evidence-based platform for the analysis of preclinical evidence and for the study and evaluation of preclinical research practice. These findings should, therefore, be considered by investigators, institutional review bodies, journals, and funding agents when designing, evaluating, and sponsoring translational research.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001489.
The US Food and Drug Administration provides information about drug approval in the US for consumers and for health professionals; its Patient Network provides a step-by-step description of the drug development process that includes information on preclinical research
The UK Medicines and Healthcare Products Regulatory Agency (MHRA) provides information about all aspects of the scientific evaluation and approval of new medicines in the UK; its My Medicine: From Laboratory to Pharmacy Shelf web pages describe the drug development process from scientific discovery, through preclinical and clinical research, to licensing and ongoing monitoring
The STREAM website provides ongoing information about policy, ethics, and practices used in clinical translation of new drugs
The CAMARADES collaboration offers a “supporting framework for groups involved in the systematic review of animal studies” in stroke and other neurological diseases
doi:10.1371/journal.pmed.1001489
PMCID: PMC3720257  PMID: 23935460
22.  The hairless mouse in skin research 
Summary
The hairless (Hr) gene encodes a transcriptional co-repressor highly expressed in the mammalian skin. In the mouse, several null and hypomorphic Hr alleles have been identified resulting in hairlessness in homozygous animals, characterized by alopecia developing after a single cycle of relatively normal hair growth. Mutations in the human ortholog have also been associated with congenital alopecia. Although a variety of hairless strains have been developed, outbred SKH1 mice are the most widely used in dermatologic research. These unpigmented and immunocompetent mice allow for ready manipulation of the skin, application of topical agents, and exposure to UVR, as well as easy visualization of the cutaneous response. Wound healing, acute photobiologic responses, and skin carcinogenesis have been extensively studied in SKH1 mice and are well characterized. In addition, tumors induced in these mice resemble, both at the morphologic and molecular levels, UVR-induced skin malignancies in man. Two limitations of the SKH1 mouse in dermatologic research are the relatively uncharacterized genetic background and its outbred status, which precludes inter-individual transplantation studies.
doi:10.1016/j.jdermsci.2008.08.012
PMCID: PMC2646590  PMID: 18938063
Hairless mice; Ultraviolet rays; Skin neoplasms; Wound healing; Skin aging
23.  First case of a naturally acquired human infection with Plasmodium cynomolgi 
Malaria Journal  2014;13:68.
Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.
The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.
Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.
This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.
Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.
The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.
doi:10.1186/1475-2875-13-68
PMCID: PMC3937822  PMID: 24564912
Plasmodium vivax; Plasmodium cynomolgi; Molecular methods; Malaysia; Simian malaria
24.  Radiation-induced myeloid leukemia in murine models 
Human Genomics  2014;8(1):13.
The use of radiation therapy is a cornerstone of modern cancer treatment. The number of patients that undergo radiation as a part of their therapy regimen is only increasing every year, but this does not come without cost. As this number increases, so too does the incidence of secondary, radiation-induced neoplasias, creating a need for therapeutic agents targeted specifically towards incidence reduction and treatment of these cancers. Development and efficacy testing of these agents requires not only extensive in vitro testing but also a set of reliable animal models to accurately recreate the complex situations of radiation-induced carcinogenesis. As radiation-induced leukemic progression often involves genomic changes such as rearrangements, deletions, and changes in methylation, the laboratory mouse Mus musculus, with its fully sequenced genome, is a powerful tool in cancer research. This fact, combined with the molecular and physiological similarities it shares with man and its small size and high rate of breeding in captivity, makes it the most relevant model to use in radiation-induced leukemia research. In this work, we review relevant M. musculus inbred and F1 hybrid animal models, as well as methods of induction of radiation-induced myeloid leukemia. Associated molecular pathologies are also included.
doi:10.1186/1479-7364-8-13
PMCID: PMC4128013  PMID: 25062865
Radiation carcinogenesis; Leukemia; Animal models; Secondary cancers
25.  Mouse Models for Efficacy Testing of Agents against Radiation Carcinogenesis—A Literature Review 
As the number of cancer survivors treated with radiation as a part of their therapy regimen is constantly increasing, so is concern about radiation-induced cancers. This increases the need for therapeutic and mitigating agents against secondary neoplasias. Development and efficacy testing of these agents requires not only extensive in vitro assessment, but also a set of reliable animal models of radiation-induced carcinogenesis. The laboratory mouse (Mus musculus) remains one of the best animal model systems for cancer research due to its molecular and physiological similarities to man, small size, ease of breeding in captivity and a fully sequenced genome. This work reviews relevant M. musculus inbred and F1 hybrid animal models and methodologies of induction of radiation-induced leukemia, thymic lymphoma, breast, and lung cancer in these models. Where available, the associated molecular pathologies are also included.
doi:10.3390/ijerph10010107
PMCID: PMC3564133  PMID: 23271302
radiation carcinogenesis; animal models; radiation protectors; radiation mitigators; secondary cancers

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