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New neurons take baby steps in adult brain

Johns Hopkins scientists, in experiments with mice, have discovered the steps required to integrate new neurons into the brain’s existing operations. For more than a century, scientists thought the adult brain could only lose nerve cells, not gain them. But, in fact, new neurons do form during adulthood in all mammals and then become a working part of the adult brain — at least in mice.

In the first study to show how “baby” neurons are integrated into the brain’s existing networks, the Johns Hopkins researchers show a brain chemical called GABA readies baby neurons to make connections with old ones. “GABA is important during fetal development, but most scientists thought it would have the same role it has with adult neurons, which is to inhibit the cells’ signals,” said Hongjun Song, an assistant professor in the university’s Institute for Cell Engineering. “We’ve shown GABA, instead, excites new neurons and that this is the first step toward their integration into the adult brain.” Song says the discovery might help efforts to increase neuron regeneration in the brain or to make transplanted stem cells form connections more efficiently. The discovery is described in the online edition of Nature.

Science Daily
January 3, 2006

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Interactive 3-D atlas of mouse brain now available on web

Researchers at the U.S. Department of Energy’s Brookhaven National Laboratory have just launched a web-based 3-D digital atlas browser and database of the brain of a popular laboratory mouse. “Neuroscientists around the world can now download these extremely accurate anatomical templates and use them to map other data — such as which parts of the brain are metabolically active and where particular genes are expressed — and for making quantitative anatomical comparisons with other, genetically engineered mouse strains,” said project leader Helene Benveniste, who is a researcher in Brookhaven’s medical department and a professor of anesthesiology at Stony Brook University.

The database was created using high-resolution magnetic resonance (MR) microscopy at the University of Florida in collaboration with researchers from Brookhaven Lab’s Center for Translational Neuroimaging. The work was done in parallel with an international collaboration, the Mouse Phenome Database (MPD) project, which was created to establish a collection of baseline phenotypic data from commonly used inbred mice. The new brain atlas database consists of 3-D anatomical data from 10 adult male mice of the strain C57BL/6J, and contains data on 20 segmented structures, including variability of brain structures across the strain, and downloadable visualization tools. The research that makes up this database was published as a cover article in the October 2005 issue of the journal Neuroscience.

Science Daily
January 3, 2006

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Laser activates gene therapy in rats’ eyes

Laser light has been used to remotely control gene therapy in rats. This mechanism will help make gene therapy more effective by allowing the precise time and location at which new genes are activated to be controlled, meaning specific tissues can be targeted while healthy tissues are left alone. Lasers have been used in the past to perforate cells for gene therapy in cultured cells. But the new research – activating marker genes in the eyes of rats – is more sophisticated and the first time lasers have been used for gene therapy in live animals. Kazunori Kataoka, at the University of Tokyo, Japan, and colleagues developed a photosensitive molecular complex that could be activated in rats’ eyes by irradiating them with visible light from a low power laser.

The synthetic complex is designed to deliver foreign DNA by carrying it past the cell membrane – a process known as transfection. The complex consists of three components: a photosensitive anionic dendrimer, which provides the triggering mechanism, and a cationic peptide which drives the third component, its DNA payload, towards the nucleus of a cell after it has been released. The complex enters the cell by a process known as endocytosis, where the cell’s plasma membrane envelops the complex at its surface and draws it into the cell. The membrane around the complex then detaches from the cell’s membrane to form a bubble containing the complex within the cell. Applying the pulse of the laser light causes the dendrimer to break free from its bubble and simultaneously release the DNA-carrying peptide. By only causing damage to the plasma enveloping the complex the cell membrane is left intact, reducing cell death.

Jennifer West at Rice University in Houston, Texas, US, has used light to kill cancer cells by inducing localised heating in nanoparticles. She says that the scope of such “biophotonics” does not have to be limited to exposed areas such as the eye. “Some wavelengths of light can penetrate very deeply,” she says. Near-infrared, for example, could potentially be used to treat many different tissue types and organs. And fibre optics can be run through needles, catheters and laparoscopes to deliver light of any wavelength to essentially anywhere in the body. But there are good reasons for concentrating on ocular therapies, says Christopher Norbury at Pennsylvania State University, US. There is a lot of interest in using gene therapy to treat congenital blindness because gene therapy has a greater chance of success in the eye compared with other parts of the body.

There are two reasons why gene therapy often does not work, says Norbury. “Either because the cells are dividing so the gene isn’t passed on or because you get an immune response against the gene.” But in the eye, cells tend not to divide and immune responses are less severe. Norbury also notes that previous methods of breaking into cells often kill a lot of them. But, according to the Japanese researchers, evaluations of the complex carried out on cultured cells showed that, not only did the areas targeted by laser light enhance gene expression 100-fold, but cell death in those areas was minimal.

New Scientist
December 20, 2005

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Gene controls whether fear is a factor

In the Nov. 18 issue of Cell, researchers report the discovery of a gene that controls the ability to react with appropriate fear to impending danger. As a result, mice lacking the gene stathmin become daredevils of a sort, the researchers report. The basic findings may have general implications for the study of anxiety disorders and potential anti-anxiety drugs, according to researchers. The researchers found that the gene stathmin–normally present in high levels in a part of the brain called the amygdala–controls both innate and learned fear. Mice without the gene show abnormally low levels of anxiety in situations that should instinctively inspire fear. Stathmin-deficient animals also show less reaction to conditions that have previously proven unpleasant, an indication that the mice lack a normal memory for fear.

“While one of the best understood memory-related neural circuitries within the mammalian brain is that which controls fear conditioning, little is known about the molecular mechanisms underlying fear reactions,” said lead author of the study by Gleb Shumyatsky of Rutgers University. “We have now found that stathmin plays a critical role in both learned and innate fear. Knockout mice, which lack the gene, show a decreased memory for fear and fail to recognize danger in innately aversive environments.” By contrast, he added, the mice depleted of stathmin perform normally in a test of spatial learning.

Fear reactions represent a spectrum of behaviors that vary from those that are inborn to those instilled through experience, said the researchers. Instinctive fears–such as fear of heights or predators–are often species specific toward actual or potential threats. In contrast, learned fear results from particular uncomfortable or life-threatening events in the past. Because fear plays an essential role in survival, memory for fear is easily established, very resistant to extinction, and normally lasts a lifetime, Shumyatsky said. In the laboratory, fear can be conditioned by linking a neutral stimulus, such as a light or sound, to something unpleasant or painful, such as an electric shock, he explained. That process of learned association occurs in a portion of the amygdala called the lateral nucleus. As a first step to unravel the molecular events underlying fear learning, Shumyatsky’s group recently identified several genes present at particularly high levels in the lateral nucleus and in the structures that relay information about learned and instinctive fear to the amygdala. One such gene was stathmin.

In the current study, the researchers found that the brains of mice lacking stathmin showed an unusual number of microtubules, which are structural components of the cytoskeleton. Stathmin normally controls the assembly and breakdown of the cellular scaffolds, Shumyatsky explained. “For memory, the brain needs to quickly disassemble and rebuild microtubules to form connections where they are needed,” Shumyatsky said. “It appears that loss of stathmin might interfere with this ability in the amygdala, leading to the overproduction of microtubules in certain areas. In essence, the cells lose their flexibility.” Indeed, the researchers found impairments in the ability of key inputs in the animals’ brains to form connections between neurons. Such connections form the cellular basis for learning and memory.

To relate these brain abnormalities to behavior, the team then exposed normal and stathmin-deficient mice to a neutral tone while delivering a mild electric shock. While both groups displayed some fear response by freezing immediately after a shock and later after hearing the tone, knockout mice reacted less strongly, they found, suggesting that they had an impaired ability to learn fear. In other tests, the mutant mice also showed less instinctive fear of open spaces, venturing out into environments they would usually avoid naturally, Shumyatsky said. Mice lacking stathmin continued to perform normally on a water maze test, an indication that spatial learning and memory–controlled outside of the amygdala–were unaltered.

“The findings provide genetic evidence that amygdala-enriched stathmin is required for the expression of innate fear and the formation of memory for learned fear,” Shumyatsky said. “This evidence suggests that stathmin knockout mice can be used as a model of anxiety states of mental disorders with innate and learned fear components,” he added. “As a corollary, these animal models could be used to develop new anti-anxiety agents.” Together with the team’s earlier findings that the amygdala-enriched gene gastrin-releasing peptide selectively affects learned fear, the new findings support the clinical data suggesting that anxiety is a spectrum of disorders with multiple subclasses, each of which may have a unique molecular signature requiring distinctive approaches to therapy, the researchers said.

Science Daily
December 20, 2005

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Poxvirus used to fight cancer: Malignant human brain tumors ‘cured’ in mice

A cross-Canada scientific collaboration has successfully tested a potent new cancer-fighting virus that eliminates malignant brain tumors and prolongs survival in mice with a single injection. The scientists – from Calgary and London, Ontario – have shown for the first time that myxoma virus, a poxvirus, will kill human brain tumors in mice and prolong the animals’ survival. Their findings are published this month in the journal Cancer Research. “We’re extremely encouraged by these results and the apparent cure seen in the mice treated with the active virus compared to untreated mice or those injected with inactivated virus,” says virologist Grant McFadden, a scientist at Robarts Research Institute in London and Canada Research Chair in Molecular Virology. His research on poxviruses over the past two decades has shed significant light on how viruses disarm their hosts’ immune systems – and their potency as anti-cancer and anti-inflammatory agents.

For the brain tumor experiments, McFadden teamed up with Dr. Peter Forsyth of the University of Calgary, a professor in the departments of oncology, and biochemistry & molecular biology, who has developed a mouse model with human gliomas, a malignant form of brain tumor. Over the past two years, their laboratories have tested the virus against experimental models of human malignant tumors, both in cell culture and in living animals. Injecting the tumor with the virus was not only well-tolerated – with only minimal inflammation at the site of the inoculation – but 92 per cent of the 13 mice treated were alive and apparently “cured” when the experiment was finished (after more than 130 days). “Those animals continued to show a selective and long-lived myxoma virus infection in the tumors themselves but that infection did not spread and harm the animal,” says Dr. Forsyth, director, Clark H. Smith Integrative Brain Tumour Research Centre. “This and other factors suggest that myxoma virus warrants further investigation as a potential treatment for malignant brain tumors in people.”

Science Daily
December 20, 2005

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Sibling hormone rivalry

Researchers have identified a new appetite-suppressing hormone – aptly named obestatin — and its receptor in mammals. Stanford University biologists report this week in Science that obestatin is produced by posttranslational modification of a protein precursor that also gives rise to ghrelin, a peptide dubbed ‘the hunger hormone’ for its stimulatory effects on appetite. The authors suggest this work may provide new targets for the control of obesity, as well as clear up some mysteries concerning the function of its sibling and physiological opponent, ghrelin. “It’s an enormous data set, an elegant study, and beautiful data,” Matthias Tschöp of the Obesity Research Center at the University of Cincinnati, Ohio, who co-wrote an accompanying perspective, told The Scientist. David E. Cummings of the University of Washington, who was not involved in the research, described the work as a “major new find.” The study was led by Aaron Hsueh at Stanford University’s School of Medicine, whose lab usually works on reproductive endocrinology. Hsueh’s team predicted the existence of obestatin through a bioinformatic search for hormones derived from the protein precursors of known peptide hormones. The exercise yielded a 23-amino-acid region of preproghrelin that is highly conserved across a range of mammalian species. The authors confirmed the prediction when they isolated obestatin from rat stomach extracts and blood. They showed that, in contrast to ghrelin, synthetic obestatin administered to rodents peripherally and centrally resulted in reductions in appetite, weight gain and the rate of gastric emptying.

While ghrelin requires posttranslational acylation of the amino terminus of its precursor, obestatin is the product of amidation of preproghrelin’s carboxyl terminus, the researchers note. By screening orphan mammalian receptors, Hsueh’s team found that obestatin binds to GPR39, a ghrelin receptor-family member expressed in the stomach, intestine and hypothalamus. Hsueh said he is hopeful that obestatin or other compounds that activate the same receptor could one day serve as appetite-suppressing drugs. Tschöp, however, said he is more cautious about the prospects of obestatin as an obesity drug, given that its effects on weight gain were modest and that the hormone has yet to be tested in obese animal models. For instance, leptin is an appetite suppressor, but it is nevertheless high in obese subjects. Tschöp said he considers it “not an unlikely possibility” that obestatin will show a similar relationship with body weight.

It’s also not clear whether the lower weight gain observed following obestatin administration is due to reductions in fat mass, muscle mass or water content – or even illness, Tschöp noted. Hsueh told The Scientist that preliminary data suggest illness is not a side effect of obestatin, but Tschöp pointed to PYY3-36, whose appetite-suppressing effects are, according to a recent report, at least in part due to the nausea induced by its administration. “It’s important to test that before everybody gets really excited,” Tschöp told The Scientist.Cummings agreed that obestatin sheds new light on some troublesome observations that have “taken some of the wind out of ghrelin’s sails” as an anti-obesity target. For example, ghrelin has potent stimulatory effects on appetite in rodents, but ghrelin-null mice are not much leaner than wild-type animals. Given that obestatin and ghrelin derive from the same gene, Hsueh’s team write, ghrelin-null mice would also lack obestatin, counterbalancing ghrelin’s absence.

Still, Tschöp suggested that the relationship between ghrelin and obestatin might not be so simple. Mice lacking the ghrelin receptor are also not particularly lean, he noted, even though obestatin’s effects should be unimpaired. “There is too much we don’t know yet,” he said. Cummings observed that the family ties between the obestatin and ghrelin receptors might explain why certain ghrelin receptor blockers mysteriously increase food intake. “Maybe some of these products inadvertently affect the obestatin more than they do the ghrelin receptor,” he said. An important next step, said Cummings, is to work out how the body independently regulates posttranslational modification of both obestatin and ghrelin from a single precursor. “It would make no sense to turn them both on at once,” he told The Scientist.

The Scientist
December 20, 2005

Original web page at The Scientist

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Seasonal depression may affect hamsters

An Ohio State University study suggests hamsters may suffer from symptoms of anxiety and depression during the dark days of winter, just as some humans. OSU researchers found more symptoms of depression and anxiety in adult hamsters that were housed for weeks in conditions with limited daylight, as they would find in winter, when compared with hamsters who had days with longer daylight. The research also examined whether hamsters that developed prenatally, and then were born during short days, were more likely to suffer from depression and anxiety as adults. The results for those tests suggest hamsters born in winter-like light conditions had increased depressive symptoms as adults.

Overall, the scientists said their research suggests the season during which the hamsters were born, their sex, and the changing of the seasons all might play a role in levels of depression and anxiety. “These results in hamsters may provide some insight into the development of seasonal affective disorders in humans,” said Randy Nelson, psychology professor and co-author of the study with Leah Pyter, an OSU doctoral student in neuroscience. They presented their results Tuesday in Washington, D.C. during the annual meeting of the Society for Neuroscience.

Science Daily
December 6, 2005

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EU plans to cut animal tests

A leading animal rights organization in Europe has given a cautious thumbs-up to a pledge made by industry groups last week to help the European Union expedite the search for new alternatives to animal testing. At a meeting in Brussels hosted by the European Commissioners for Enterprise and Research, Günter Verheugen and Janez Potocnik – along with groups representing the chemical, cosmetic, pharmaceutical, biotechnology, soap and detergents, animal health, and crop protection industries — signed a joint declaration on the “3 Rs”– refining, reducing and replacing the use of lab animals. “We think it is a very promising initiative,” said Marlou Heinen from the Eurogroup for Animal Welfare, an umbrella group for some 19 animal welfare organizations. “But we see it very much as a first step—whether it will be an actual success will very much depend on how this is going to be implemented.”

The ultimate goal of the partnership is to eliminate the use of animals in testing altogether, Verheugen said in a statement. “We do not only wish to reduce animal testing, but also want to bring it to an end in the long run.” So far, the details on how that is going to be achieved are yet to be worked out. The declaration commits the signatories to developing an action plan, which they are due to deliver by early in 2006, said Sebastian Marx from Colipa, the cosmetic industry group. “Then the real flesh will be put on the bones,” he told The Scientist. Colin Humphris, spokesman for the chemicals industry group Cefic, agreed that the partners were “feeling our way” in terms of how the collaboration might work. “But what I think is important is that when you bring together these different sectors, you bring together different programs of work that relate to this area,” he told The Scientist. “There’s some common interest there.”

As many as 10.7 million animals are used annually for experiments in the EU according to figures quoted by the European Union. More than half of these are used in research, human medicine, dentistry and fundamental biological studies. Another 16 percent are used in production and quality control of products and devices in human and veterinary medicine and dentistry, and 10 percent for toxicology and other types of safety evaluation. In 1986, an EU directive insisted that whenever an alternative to an animal experiment exists, it has to be used, and called for support to make such alternatives available. But the timing of the current initiative had a lot to do with another proposed EU directive that could have the opposite effect of the earlier directive.

That new law, under debate at the moment, is REACH (Registration, Evaluation and Authorization of Chemical Substances), a framework designed to gather better information on the chemical substances that reached the market before 1981. REACH would require testing of those old substances to evaluate their health and environmental risks. The trouble is, Verheugen said, the proposal as it stands could require millions more animal tests. “REACH is not ethically acceptable if it leads to such excessive additional use of animals. I will do everything I can to change the current proposal in this respect,” he said in a report on the Euractive news service. Marx said the imminent arrival of the REACH rules was a big impetus for last week’s animal testing declaration. “This is not really a response to the actions of animal rights groups,” he said. “We have new legislation coming and we need to do something.”

The declaration signed last week commits the signatories to making a progress report at about the same time next year. For Heinen, this is another positive sign. “That will enable us and other stakeholders to see that the promises are being met.” A key sign of success will be evidence of extra financial commitment from industry, she said.

The Scientist
December 6, 2005

Original web page at The Scientist

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Male mice produce ultrasonic love songs.

Mickey Mouse may have kept quiet during his early days on the silver screen, but his lab counterparts seem to have a penchant for song. That’s the finding in an analysis of the ultrasonic sounds made by male mice wooing potential mates. For years, animal-behaviour experts have known that mice make vocalizations that are too high in pitch to be picked up by the human ear. Young mice, for example, make ‘isolation calls’ when cold or distressed. And male mice emit ultrasonic sounds in the presence of a potential mate or in response to chemical sex cues, called pheromones, in the urine of female mice.

But until now, scientists had not examined these sounds for musical patterns. Thanks in part to a sophisticated computer program, Timothy Holy and Zhongsheng Guo of the Washington University School of Medicine in St Louis, Missouri, were able to tackle this challenge. Holy began by writing software that shifts the pitch of the male mouse’s sounds, making the sounds deeper so that they can be heard by humans. “No one had ever pitch-shifted the mouse vocalization,” he says. “The first time I played it back it was pretty surprising: it sounded so much like birdsong.”

Holy and Guo then exposed male mice to female mouse urine to elicit mating vocalizations, and recorded the sounds. They analysed the minute details of captured sounds, comparing the pitch from each millisecond with the one immediately preceding it. They looked for patterns in these pitch changes, as well as in the spacing of vocalizations over time. The animals’ high-pitched squeaking has song-like characteristics, the researchers discovered, with distinct pairs of notes arranged in repeating phrases. Holy likens the mouse songs to juvenile bird songs, which lack a complex fixed pattern of musical themes. The findings appear in the journal PLoS Biology. The first time I played it back it was pretty surprising, it sounded so much like birdsong.

The researchers hope to record songs in wild mice, too, and aim to understand whether these contain greater richness and complexity than those produced by lab mice. They also aim to understand whether the mice learn these songs from one another or produce them automatically. If mice do teach one another tunes, they will join an exclusive club of animals: researchers have so far only documented this skill in humans, whales and birds.

A broader group of animals produces unlearned sounds. Some insects, such as cicadas, instinctively produce unlearned routines of clicking sounds as a part of courtship. And many birds, in addition to their learned songs, will instinctively chirp to communicate alarm if a potential predator approaches their nest. Holy notes that the mice he studied each seemed to have a preference for singing certain songs, even though they were all genetically identical. “That’s probably the best evidence we have that it’s a learned behaviour,” he explains. “But I would guess that the degree to which learning plays a role is more limited than in birds.” The complex mouse vocalizations are not necessarily linked to our own gift for music. Birdsong expert Daniel Margoliash of the University of Chicago, Illinois, notes that there is no compelling evidence for learned vocalizations even among our closest primate relatives, suggesting that we evolved our musical skills independently of other species. But the mice’s songs may have something to teach us about the origins of human speech, Holy suggests. He points to evidence that a gene called FOXP2 is essential for both, and adds that further studies could explore this connection.

Nature
November 22, 2005

Original web page at Nature

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Tiny microscope peers into mice brains

A microscope the size of a matchbox is allowing US biologists to peer inside the brains of live animals. Weighing a mere 3.9 grams, the microscope has been used to image blood vessels lying 1 millimetre below the surface of the brains of anaesthetised mice, with a resolution of 1 micrometre (one-thousandth of a mm). The researchers believe they may one day be able to view brain cells in the same way. In future the tiny microscope could be strapped to the head of a conscious, moving animal and beam back a movie of the neurons while the animal engages in a variety of activities, say its creators, led by Mark Schnitzer of Stanford University, US.

“It’s a microscope that fits in the palm of your hand,” says co-creator Ben Flusberg. It could also be a quick, easy way to image diseased or tumour-laden brains, which currently require an MRI scan or large machinery, he says. Or it could pave the way for a mobile, pocket-sized diagnostic tool. But it is not clear whether the device would ever be used in humans because of its invasive nature. “It’s an ingenious device, but whether it’s transferable from mouse to man is not mentioned,” says Britton Chance, a biophysicist at the University of Pennsylvania, US, who focuses on non-invasive imaging. A pin-like probe, 1 mm in diameter, protrudes from the bottom of the device and punches a tiny hole in the head of the anaesthetised mouse. The probe –called a microendoscope – does not enter the brain, but sits on top of the hippocampus. There it images blood vessels directly below it, which are inside the brain, by illuminating tiny sections of the hippocampus one at a time with near infra-red light.

The projected light causes the animal’s blood – previously injected with a fluorescent marker called fluorescein – to emit light of a particular frequency. The probe, which is packed with optical fibres, detects the light and transmits it back to the matchbox-sized device where it is recorded. The image can then be displayed on a connected computer screen. Although blood vessels that lie below the surface of the brain had been imaged before by the group using a microendoscope, the probe had to be connected to a large piece of machinery, which illuminated the brain areas and detected the fluorescent vessels. This meant that the device could not have been used on moving animals or be developed as a portable system for humans. “It was too big,” admits Flusberg.

Schnitzer’s group is the first to cram all this functionality into a device the size of a matchbox, he says. Their secret has been to harness a tiny piezoelectric motor contained within the device to focus laser light on the sections of the brain being examined. They say only now is the device small enough to be strapped to a live animal. “We want to understand what the cells in the brain are doing during animal behaviour,” Flusberg explains. But Chance points out that there is currently no way to work out exactly where inside the brain the probe is. “This gives you an image of a single blood vessel in an unknown place,” she says.

Source: Optics Letters (vol 30, p 2272)

New Scientist
October 11, 2005

Original web page at New Scientist

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Adult human neural stem cell therapy successful in treating spinal cord injury in mice

Researchers at the University California Irvine Reeve-Irvine Research Center have used adult human neural stem cells to successfully regenerate damaged spinal cord tissue and improve mobility in mice. In their study, Brian Cummings, Aileen Anderson and colleagues injected adult human neural stem cells into mice with limited mobility due to spinal cord injuries. These transplanted stem cells differentiated into new oligodendrocyte cells that restored myelin around damaged mouse axons. Additionally, transplanted cells differentiated into new neurons that formed synaptic connections with mouse neurons.

Myelin is the biological insulation for nerve fibers that is critical for maintenance of electrical conduction in the central nervous system. When myelin is stripped away through disease or injury, sensory and motor deficiencies result and, in some cases, paralysis can occur. Previous Reeve-Irvine research has shown that transplantation of oligodendrocyte precursors derived from human embryonic stem cells restores mobility in rats. “We set out to find whether these cells would be able to respond to the injury in an appropriate and beneficial way on their own,” Cummings said. “We were excited to find that the cells responded to the damage by making appropriate new cells that could assist in repair. This study supports the possibility that formation of new myelin and new neurons may contribute to recovery.”

Mice that received human neural stem cells nine days after spinal cord injury showed improvements in walking ability compared to mice that received either no cells or a control transplant of human fibroblast cells (which cannot differentiate into nervous system cells). Further experiments showed behavioral improvements after either moderate or more severe injuries, with the treated mice being able to step using the hind paws and coordinate stepping between paws whereas control mice were uncoordinated. The cells survived and improved walking ability for at least four months after transplantation.
Sixteen weeks after transplantation, the engrafted human cells were killed using diphtheria toxin (which is only toxic to the human cells, not the mouse). This procedure abolished the improvements in walking, suggesting that the human neural stem cells were the vital catalysts for the maintained mobility. This study differs from previous work using human embryonic stem cells in spinal cord injury because the human neural stem cells were not coaxed into becoming specific cell types before transplantation.

Science Daily
October 11, 2005

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EU project will test 20,000 mouse genes

The European Union has approved funding of a three-year research programme designed to test the function of almost every gene in the mouse genome. The €13 million ($16 million) project proposes to generate 20,000 mutations in mouse embryonic stem cells and make them available to researchers worldwide, from a central depository in Heidelberg, Germany. The European Conditional Mouse Mutagenesis Programme (EUCOMM) is not seen as a competitor to the World Stem Cell Hub, the creation of which is expected to be announced this week in South Korea. The hub, under the auspices of the “king of cloning”, Woo Suk Hwang, of Seoul National University, will act as a central bank for human embryonic stem cells. It involves the creator of Dolly the sheep, Ian Wilmut, from the Roslin Institute, UK, and Gerald Schatten of the University of Pittsburgh School of Medicine, US.

The European programme will mutate mouse genes – there are some 25,000 in its genome – in order to study their function, whereas the South Korean hub will be concerned with therapeutic uses of human stem cells. An official announcement about EUCOMM will come later in 2005, when contract negotiations with the EU are complete. Research will start in 2006, using two techniques to achieve specific mutations. A technique called “gene trapping” can be used to randomly mutate genes by inserting a DNA element into the gene. This can be used not only to knock-out the gene but also to identify where it has been knocked out. A second technique called “gene targeting” uses inserted DNA to disrupt a gene but in a specific place. Both techniques should create “conditional mutations” – so they can be switched on or off in different tissues and at different times during development.

European scientists are at the forefront of conditional mutagenesis technology, says Wolfgang Wurst, director of the Institute of Developmental Genetics in Neuherberg, Germany. “This allows us to dissect gene function in vivo (within a living organism) more precisely and allows us to mimic human disease conditions more closely,” he says. The resource is likely to see a rise in the number of transgenic mice used in research. Current figures show that about three million animals are used in research in the UK each year – 84% being rodents. Figures have been steadily rising and new figures from the UK Home Office in the next few weeks are expected to show a further rise.

EUCOMM researchers would like to achieve different kinds of mutations in each of the 20,000 mouse genes, says Steve Brown, director of the MRC Mammalian Genetics Unit in Harwell, UK. “Ninety-nine per cent of human genes are represented in the mouse,” says Brown. “Over the next 10 to 20 years we will create mutations in every gene in the mouse genome and ask: what is the outcome in terms of disease and development?” The system could be used to study motor neurone disease, for example. A US version of the mutagenesis programme – the Knockout Mouse Project – has been mooted but the US National Institutes of Health has yet to make a request for applications, says Brown.

New Scientist
August 30, 2005

Original web page at New Scientist

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Memory loss reversed in mice

Mice with memory loss have had their condition reversed, a discovery that should help refine the search for a cure for Alzheimer’s disease and other dementias. The study also helps clarify the actual cause of dementia, which should give more focus to drug studies. The brains of people with Alzheimer’s and some 50 other forms of dementia are known to have certain characteristic features, including messy bundles of fibres in nerve cells called neurofibrillary tangles. But no one has been sure whether the tangles are a cause or symptom of dementia.

Mice engineered to massively overproduce a protein called tau tend to grow more of the tangles and display the same problems with memory and learning as humans with dementia. Researchers think that it is a certain version of the tau protein, rather than a simple over-abundance, that leads to the tangles. It has been speculated that these tau proteins, rather than the tangles, kill nerve cells. Karen Ashe, a neurobiologist at the University of Minnesota Medical School in Minneapolis, and her colleagues hoped to untangle this mystery. They trained mice to navigate a maze partly submerged in water, and watched for signs of memory loss. By the age of three months, mice genetically engineered to express 13 times too much tau protein couldn’t remember the route to dry land, and had developed tangles in their brains. But surprisingly, when the researchers turned off the switch promoting tau expression, the mice began to gain back some lost memory.

The team reports in Science that the performance of the ‘switched-off’ engineered mice was roughly half as good as their normal counterparts, and twice as good as those that continued to overproduce tau. And their performance improved even through the tangles in their brains remained. The results indicate that some variety of tau proteins, and not the tangles it promotes, is responsible for dementia-related memory loss. But researchers are not yet sure which version of tau protein cause problems in the brain. That’s the next step, says Ashe: “We have to figure out the molecular form of tau that is poisoning the neurons.” That should give drug developers a better understanding of the molecules they should target. But researchers caution that in the context of dementia, this is just half the story.
Alzheimer’s patients also have plaques in their brains made of a protein compound called beta-amyloid. Most think that this also plays a role in causing memory loss. “This is a two-protein disease,” says Ashe.

Nature
August 2, 2005

Original web page at Nature

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Mice gang up on endangered birds

On one of the Earth’s most remote islands, mice have learned, and are apparently teaching each other, how to attack and kill bird chicks that are 200 times their size. Far from exulting in the cleverness of mice, the researchers who discovered this want to eradicate the rodents from the island in order to save endangered albatrosses.

Biologists on Gough Island, a speck in the Atlantic between the southern tips of Africa and South America, first learned of the problem when they found that tristan albatrosses (Diomedea dabbenena) were losing their chicks at an extremely high rate: up to 80% were dying.
Researchers suspected that house mice, which were accidentally introduced to the island, might be the culprits. So husband-and-wife team Ross Wanless and Andrea Angel spent a year on the island videotaping birds’ nests and collecting data. The videos confirm that mice are taking on the chicks, biting them over and over until they die from loss of blood or infection. Wanless, an invasive-species biologist from the Percy FitzPatrick Institute of African Ornithology at the University of Cape Town, South Africa, vividly recalls watching the first videos. “It was carnage. Chicks half alive, with massive gaping wounds and guts hanging out.”

The mice are able to defeat the much larger birds by biting the same spot over and over. They take advantage of the fact that the birds, which have evolved in an area that has been without land predators for millions of years, have no defensive response against such attacks. Wanless was surprised by the results. Such behaviour is unprecedented in mice, he says. And, oddly, the attacks only take place on some of the island’s peaks, despite the fact that the mice live everywhere on the island.

The research duo chose two sites for further inspection that had radically different death rates for chicks. They found the same vegetation, altitude, slope, numbers of mice and albatross nests at each site. But one group of mice attacked chicks and the other did not. From this the team infers that the attack is a learned behaviour. The transmission of learned skills from one generation to the next is a relatively rare phenomenon, and not one seen in mice in the wild before. The researchers note that it is particularly surprising in this case because only a few mice from each brood would be expected to live through a winter. Wanless and Angel are now determined to save the albatrosses by removing the mice. But they warn that similar attacks might be threatening other bird species. “This is probably not unique to Gough,” says Wanless. “It is just that nobody has looked.”

Nature
August 2, 2005

Original web page at Nature

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Rat olfaction molded early

The odors that newborn rats are exposed to appear to govern the development of synapses that carry information into the rat olfactory cortex, the seat of odor perception, researchers report in Neuron this week. Kevin Franks and Jeffry Isaacson of the University of California, San Diego, found that in newborn rats, early olfactory experiences caused changes in the relative amounts of two types of glutamate receptors in lateral olfactory tract fibers. Specifically, they observed a decrease in the number of NMDA receptors, which are believed to be important in synaptic plasticity and long-term changes, relative to AMPA receptors, which mediate fast synaptic transmission. The researchers suggest this phenomenon might be associated with “olfactory imprinting,” the strong attachment to maternal odors that occurs early in mammalian development.

“The ability of the animal to smell caused downregulation in the number of NMDA receptors,” Isaacson told The Scientist. “Very early in rat development, there is quite robust NMDA receptor-mediated, long-term potentiation from the sensory synapses into the cortex, but later in life, after the animal has had time to smell, the loss of NMDA receptors makes it difficult to induce any long-term changes in the strength of the synaptic transmission.” To study the synaptic modifications that occur during development, the authors took advantage of the layered architecture of the rat’s olfactory cortex. “The stratification makes this a nice experimental model to use,” said Kurt Illig of the University of Virginia, who did not participate in the research. “The authors were able to selectively stimulate different types of cells and look at the development of the responses for each of those layers independently in an in vitro preparation. The loss of NMDA receptors they observed could be a mechanism by which early olfactory experience shapes the cortex to respond to particular odors.”

To test for the role of sensory experience in the synaptic changes, the authors occluded one of the nostrils in newborn rats, depriving one side of the brain of olfactory stimulation, and compared the two sides of the brain in each animal. “This is a great technique because the olfactory information in the brain is ipsilateral,” said Isaacson. The results pointed to the existence of a critical period during which sensory synapses are especially plastic, a phenomenon that also has been shown in the visual, auditory, and somatosensory systems. “Olfactory deprivation caused loss of NMDA receptors in young rats, but not in rats 2 months old or so,” he said. “There have been very few researchers who have looked at how experience can modify the olfactory cortex,” said Ben Philpot of the University of North Carolina at Chapel Hill, who wrote a related preview. “This work shows that you can have changes with olfactory experience in the cortex very early on.”

Philpot added that the changes in the receptor levels may also be involved in the pruning back of exuberant projections from the olfactory bulb to the olfactory cortex. “This would be a nonexclusive possibility, equally exciting,” he said. According to Takao Hensch of the Riken Brain Science Institute in Saitama, Japan, who was not part of the research team, the results bode well for identifying a critical period in the olfactory system. “What Isaacson et al. will need to show in the future is that the NMDA-mediated events at lateral olfactory tract synapses indeed have behavioral consequences. Does nostril occlusion delay the critical period for imprinting, as they would suggest?”

E-mail address The Scientist Daily
July 19, 2005

Original web page at The Scientist

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Pet rodents linked to multidrug-resistant salmonellosis outbreak

Handling pet rodents is a potential health risk, according to a report published in the May 6 issue of the Morbidity and Mortality Weekly Report. Public health practitioners are urged to consider pet rodents as a potential source of salmonellosis. During 2004, the Minnesota Department of Health’s Public Health Laboratory notified the CDC, which publishes the MMWR, about the isolation of multidrug-resistant Salmonella Typhimurium from ill hamsters from a Minnesota pet distributor. The report describes two of the first identified human cases associated with the outbreak, summarizes the multistate investigation of human Salmonella Typhimurium infections associated with exposure to rodents purchased at pet stores, and highlights methods for reducing Salmonella transmission from pet rodents to their owners. This is the first documented salmonellosis outbreak associated with pet rodents.

Veterinarians, breeders, and distributors are advised to consider submitting specimens to clinical laboratories for Salmonella isolation if substantial diarrhea-associated morbidity or mortality occurs among pet rodents intended for sale. Heightened infection-control practices by pet stores and distributors, including routine sanitizing of animal transport containers and cages, may reduce transmission. These strategies could reduce the need for antimicrobials to prevent disease in rodents.

JAVMA
July 5, 2005

Original web page at JAVMA

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Rabbit Hemorrhagic Disease

On 7 Jun 2005, Rabbit Hemorrhagic Disease (RHD) was confirmed at a private residence in Vanderburgh county, Indiana by the Foreign Animal Disease Diagnostic Laboratory (FADDL) on Plum Island, NY. Specimens from these rabbits were positive for RHD antigen on ELISA, electron microscopy, and PCR. A FAD (Foreign Animal Disease) investigation was initiated on 3 Jun 2005 on a premises that raises rabbits primarily for sale to reptile owners as a food source for snakes.

The investigation revealed that many of the 200 rabbits on the premises suddenly died during the past 10 days. Less than a dozen rabbits had recently been purchased from Kentucky and introduced into the herd. An epidemiologic investigation has begun in Kentucky. The remaining rabbits are quarantined and will be euthanized and disposed of in accordance to State regulations. Cleaning and disinfection of the area will follow.

The Indiana epidemiological investigation is ongoing. APHIS, Veterinary Services (VS), the Indiana Board of Animal Health, and the Kentucky Department of Agriculture are working together to address this situation. VS (Veterinary Services) will assist the affected State in the euthanasia, cleaning, and disinfection of the premises. VS will continue to investigate reports of suspect RHD as part of its foreign animal disease surveillance program and will continue to diagnose suspect cases at FADDL.

The last known positive RHD case in the US occurred in a captive exotic animal facility in Flushing, New York in December 2001. Rabbit hemorrhagic disease (RHD) is a highly infectious viral disease of the European rabbit (Oryctolagus cuniculus). This is the species from which all U.S. domestic and commercial rabbits are derived. American cottontail rabbits and jackrabbits are not susceptible to infection. RHD is not known to harm humans or other animals. Once the disease is introduced into a rabbitry, it can spread rapidly, causing a high percentage of the rabbits to die. There is no treatment for the disease.

RHD damages the liver, intestines, and lymphatic tissue and causes terminal massive blood clots. The incubation period is about 24 to 48 hours. Predominantly, young adult and adult rabbits die suddenly within 6 to 24 hours of the onset of fever with few clinical signs. Fever may be as high as 105 F (40.5 C) but often is not detected until rabbits show terminal clinical signs. Most animals appear depressed or reluctant to move in the final hours and may show a variety of neurologic signs, including excitement, incoordination, paddling, and opisthotonos (abnormal position of the head due to spasms of the muscles at the top and back of the neck). Some affected rabbits may have a foamy nasal discharge. The death rate for RHD ranges from 50 to 100 percent.

RHD is caused by a highly contagious virus. The disease can be transmitted by contact with infected rabbits, rabbit products, rodents, and contaminated objects, such as cages, feeders, and clothing. The virus also may be carried short distances through moisture in the air. The risk of spread of RHD is higher when confined rabbits are in close contact with each other. Infected rabbits that recover may become carriers of the virus and may shed virus for at least 4 weeks.

To protect against introducing RHD into the U.S. rabbit population, owners and producers should avoid contact between their rabbits and imported rabbit meat, pelts, or other possibly contaminated objects from RHD-affected countries. To prevent the spread of the disease if it enters the United States, rabbit owners should prevent contact between healthy rabbits and infected rabbits and contaminated objects (e.g., cages, feeders, and clothing). Rabbits that appear healthy can be in the early stage of disease and later spread the disease. Recovered rabbits also appear healthy but can be carriers for at least 4 weeks and spread the disease to other rabbits. Owners should be cautious and isolate new rabbits and rabbits returning from shows for at least 5 days. If rabbits were exposed to RHD, isolation may help prevent spread to other rabbits. Clinical disease usually will be noticeable within 48 hours of infection.

If RHD is suspected, to prevent spread of the virus, rabbit owners should clean and disinfect all equipment. After thorough cleaning, rabbit breeders should use one of the following disinfectant solutions on equipment to inactivate the virus: 2-percent 1-Stroke Environ (Steris Corporation, St. Louis, MO), 0.5-percent sodium hypochlorite, or 10-percent household bleach.

No vaccine is legally available for use in the United States. Vaccine has been used in other countries. Vaccination often reduces the number of rabbits dying from RHD, but will not eradicate the disease. Rabbits vaccinated against RHD may become infected but not show signs of disease, thereby allowing spread of the virus as a carrier.

ProMed Mail
June 21, 2005

Original web page at ProMed Mail

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Strange new rodent discovered as Asian snack

A weird species of rodent, totally new to science, has been discovered on sale in a southeast Asian food market. The rock rat – or kha-nyou as it is known in Laos – is unlike any rodent seen before by scientists. “It was for sale on a table next to some vegetables,” says conservation biologist Robert Timmins, “And I knew immediately it was something I had never seen before.” People in the Khammouan region of Laos know of the species, and prepare it by roasting it on a skewer, says Timmins, of the Wildlife Conservation Society, based in New York City, US.

Timmins and his team have subsequently trapped the animal with the help of local people, but have never seen it alive either in the wild or in the market. Relatively little is yet known of how it lives or the full extent of its habitats. The creature looks something like a cross between a large dark rat and a squirrel, but is actually more closely related to guinea pigs and chinchillas. The long-whiskered rodent has a thick, furry tail, large paws, stubby limbs and is around 40 centimeters from nose to tail. Initial evidence suggests it gives birth to a single young at a time. The discovery was reported in the journal Systematics and Biodiversity. What makes Laonastes aenigmamus so unusual is that it is not closely related to any other rodents. The researchers behind the find have had to create a whole new family, the Laonastidae, to accommodate it.

Although new rodents are discovered by scientists at the rate of about one a year, new mammal families are much rarer, Timmins told New Scientist. The last new mammal family was created in 1974 with the discovery of the bumblebee bat. “To find something so distinct in this day and age is just extraordinary,” he says. The species may be the primitive ancestor to a large group of mostly African and South American rodents known as the Hystricognathi.
This group includes mole rats, guinea pigs, capybaras, porcupines and chinchillas. Laonastes may have diverged from these species tens of millions of years ago, Timmins says. Today its closest living relatives are found in Africa. “The discovery is particularly interesting because it may throw new light on theories about the evolution and past distribution of Old and New World rodents,” says rodent expert and study co-author Paulina Jenkins of the Natural History Museum in London, UK.

The researchers have too little information on the population size and distribution of Laonastes to currently confirm whether or not it is an endangered species, says Jenkins. However, evidence suggests its habitat is confined to rocky limestone outcrops in and around the protected Khammouan National Biodiversity Conservation Area, and is therefore not likely to be very widespread.

New Scientist
June 7, 2005

Original web page at New Scientist

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Sperm fusion protein identified

A team of researchers has identified a sperm protein that is essential for the fusion of the sperm and egg membranes during fertilization, according to a report in this week’s Nature. They called the protein “Izumo,” after the Japanese shrine dedicated to marriage.

Masaru Okabe of Osaka University and coworkers used the fusion-inhibiting monoclonal antibody OBF13 and gene-cloning techniques to identify an antigen on mice sperm involved in fusion. “The antigen Izumo is a novel protein of the immunoglobulin superfamily,” Okabe, the senior author of the study, told The Scientist. Proteins belonging to this family are membrane-anchored, and are usually involved in cell-cell adhesion.

Okabe has been seeking the sperm protein since 1987, when his group reported that OBF13 bound to an unknown molecule on the sperm head. Further studies revealed that the antibody did not prevent sperm binding, but did inhibit fertilization. This gave the researchers a clue as to the protein’s involvement in fusion. “For the next ten years, I tried very hard to find the antigen [that OBF13 bound], but I couldn’t clone the gene,” explained Okabe. “Then, I rested for five years, until Noakazu Inoue, the first author of the paper, joined our group. Inoue tried the procedure again, with newer and more sensitive techniques, and succeeded.”

In recent years, Okabe and his team have also studied egg proteins. In 2000 they published a paper in Science, reporting that CD9—a protein on the egg surface—was essential for sperm-egg fusion. A group in France also published a report presenting similar results in the same issue. “We had this egg protein involved in fusion, but on the sperm side, nothing was clear,” said Okabe. “With the discovery of Izumo, the mechanism of sperm-egg fusion is now ready to be examined.” In their latest paper, the researchers established the role of Izumo in fertilization by producing a knockout mouse line. The experiments rendered unambiguous results. The mice were healthy, but the males were sterile. Their sperm bound to, and penetrated the zona pellucida but could not fuse with the egg.

Paul Primakoff of the University of California at Davis, who was not involved in the research, welcomed the paper, which deals with a field in which there had not been breakthroughs lately. “The fertilization process is difficult to study for many reasons,” said Primakoff, “primarily because it’s hard to get the number of eggs necessary for the experiments, but also because there aren’t many people working in the field.” He added that no one has worked out a rapid screening method to identify proteins involved in the fusion process, which makes its study particularly hard. “Now we can start thinking about the mechanism of how this protein acts, and to formulate models on how the fusion process might occur.”

“This paper will provide a jolt of energy to the field,” said Richard Schultz of the University of Pennsylvania, who did not participate in the study. “Now there’s a candidate molecule that is out there that might help us understand better how membranes actually fuse. I think it’ll generate a lot of excitement in the field. Schultz, who wrote a related News and Views article, pointed to the potential of Izumo as a target for non-hormonal contraception, based on Okabe’s finding that human sperm also contains Izumo, and that when exposed to the antibody, sperm becomes unable to fuse with the egg.

Okabe is expecting accompanying proteins to be involved in fusion. “We think that Izumo is not the only factor affecting fusion. We just opened the door for studying the fusion mechanism,” he said. “Now we have to find other factors which are also involved in fusion, using Izumo and CD9.”

The Scientist
March 29, 2005

Original web page at The Scientist

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Severed optical nerves can be made to grow again

Damaged optic nerves – which run from the eye to the brain – have been regrown for the first time by scientists working with mice. The researchers believe the technique might one day restore sight to people whose optic nerves have been damaged by injury or glaucoma. It could even help regenerate other nerves in the body, they say.

A team led by Dong Feng Chen, at the Schepens Eye Research Institute in Boston, US, combined two genetic modifications to regrow the optic nerve after it was damaged. First they turned on a gene called BCL-2, which promotes growth and regeneration of the optic nerve in young mice. This gene is normally turned off shortly before birth. They then bred those animals with other mice carrying genetic mutations that reduce scar tissue in injured nerves. The researchers crushed the optic nerves shortly after birth, and found that in young mice – less than 14 days old – between 40% and 70% of the injured optic nerve fibres regrew to reach their target destinations in the brain. No regrowth was seen in injured mice without the genetic modifications. That suggests the mice may have regained some vision, Chen told New Scientist, although the study cannot prove it did.

However, the approach did not work on mice more than two weeks old. This may be because the effect of BCL-2 begins to weaken, and scarring – which takes time to set in – starts to inhibit healing. The study could offer some insight into why mammals tend to lose their ability to regrow nerves after a certain age – a phenomenon that remains largely a mystery, suggest the authors. “Unfortunately, the fountain of youth doesn’t last forever,” says Jerry Silver, a nerve regeneration expert at Case Western Reserve University in Ohio, US, who is not part of the team. “You can only give them two extra weeks” before the regeneration stops. But Silver was impressed by the breakthrough, saying the study had the potential to become a “classic” in the field.

Chen says the relatively simple structure of the optic nerve may shed light on how to repair damage to more complicated systems, such as the spinal cord. To apply the genetic technique to humans, a form of gene therapy would have to be developed, or drugs developed that manipulate gene expression. Chen and her colleagues have applied for two patents on the methods they have developed to stimulate neural cell regeneration and prevent degeneration.

Journal reference: Journal of Cell Science (March 2005 issue)

New Scientist
March 15, 2005

Original web page at New Scientist

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Males can grow eggs cells, too

In mice, female germ cells can develop into viable ova in male testes, Japanese researchers report this week. Their paper in PNAS suggests that sex determination occurs early in embryonic development before genomic imprinting takes effect. “[Our] paper indicated that once the direction of the sex is determined in germ cells, the following influence from the environment seems not to be significant,” coauthor Masaru Okabe told The Scientist in an E-mail. The research showed that female germ cells encompassed by male tissue with XY gene imprinting continue their programmed development into eggs. Okabe, from Osaka University’s Genomic Information Research Center, and colleagues combined male embryos with female embryos minus the zona pellucida in vitro to create XX-XY chimeric embryos.

Growing the embryos to term, researchers found that the seminiferous tubules of 3-week-old male mice produced meiotic XX germ cells with a normal zona pellucida structure that could fuse with sperm. The so-called “testicular eggs” were smaller and grew more slowly than normal ovarian eggs, and they were present only in anterior and posterior parts of the testis nearby resident Sertoli cells. Like normal ova, the testicular eggs were heavily methylated and in some cases expressed SCP3, a primary meiotic complex protein. Methylation patterns in cells are characterized as sex specific, according to the authors, and have been shown to turn off gene expression. “It is difficult to elucidate the role of genomic imprinting in germ cell development, but we consider the imprint as a result of sequential chain events after the initial sex determination of germ cells,” Okabe told The Scientist.

Previous research has shown that XX spermatogonia-like cells are normally found within the testis but disappear a few days after formation for unknown reasons. Okabe says sex determination in germ cells likely occurs before genomic imprinting because inside the testes, genomic imprinting did not always follow cues from the environmental conditions in which the germ cells were found. Earlier work in sex-reversed mice by Anne McLaren from the United Kingdom’s Cambridge University showed that large, egg-like cells could develop in the testes. The new findings published in PNAS lend proof such cells are oocytes by showing they can fuse with sperm, although, Okabe pointed out, they were too small or immature to develop.

“One of the intriguing results of this excellent paper is the failure to find any XY growing oocytes in the chimeric testes,” McLaren told The Scientist by E-mail. “XY germ cells that are not in a testis have been shown to enter meiosis about 13 days after fertilization, and thenceforth pursue the oogenesis pathway, just like XX germ cells.” “The existence of [these] oocytes in the testes suggests that we can separate folliculogenesis from oocyte maturation, which have traditionally been thought to be one pathway,” Jana Koubova, a researcher from the Massachusetts Institute of Technology in Cambridge, told The Scientist in an E-mail.

“Surprisingly, the egg does not seem to need the follicle to develop,” said Koubova, who wasn’t involved in the study.

The Scientist
March 15, 2005

Original web page at The Scientist

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Of rats and men: superwarfarin toxicity

He lay in a heap with face contorted, yet his friends delayed seeking medical help; one would later boast, “I did him in”. The autopsy showed haemorrhage in the left cerebral hemisphere and stomach, although a final report strangely omitted the gastric haemorrhage–possibly to conceal the fact that one of the biggest mass murderers of all time, Joseph Stalin, had been poisoned with warfarin. 20 years earlier, in 1933, Karl Link, a chemist at the Wisconsin Alumni Research Foundation, began work on identifying the active ingredient in mouldy sweet clover that had led to a haemorrhagic illness in local cattle. This compound, coumarin, responsible for the clover’s sweet smell and bitter taste, was eventually modified into a stable odourless anticoagulant and patented in 1948 as a rodenticide, taking the name warfarin from its founding company. Although rats would soon develop resistance, more potent derivatives of warfarin were rapidly developed that led to greater absorption and coagulation inhibition, which led to them becoming the most widely used rat poisons–the superwarfarins.

Jiri Pavlu and colleagues report in The Lancet on a young woman who presented with abdominal pain, gum bleeding, and very prolonged clotting times, and who required vitamin K at a dose ten times greater (ie, 100 mg daily) than that used in other coagulopathies. This observation implied antagonism of vitamin K metabolism, which was eventually confirmed when brodifacoum, a superwarfarin, was detected in her blood. But how she had been poisoned remains a mystery. Poisoning with warfarin or superwarfarins, whether accidental or intentional, is a growing public-health problem. Warfarin over-anticoagulation represents one of the commonest causes of hospitalisation due to an adverse drug effect, and about 10 000 cases of superwarfarin poisoning are reported a year in the USA alone, with a mortality of up to 20%. In 1983, nearly 200 infants died of internal bleeding attributable to use of talc contaminated by warfarin.

Patients often fear treatment with warfarin despite it being 100 times less potent than superwarfarin rat-poison. The darker side of warfarin therapy appears to have remained in the public mind despite some 50 years of effective service in the long-term prevention of devastating arterial and pulmonary embolic events. Anticoagulation with warfarin in patients with atrial fibrillation is over twice as effective in the secondary prevention of stroke than any other intervention, including carotid endarterectomy or smoking cessation, and will prevent 20 ischaemic strokes for every haemorrhagic one it causes. Yet a certain anxiety about warfarin is also held by prescribers, reflecting the wide variability within and between individuals in their response to long-term warfarin treatment. Long-term warfarin treatment doubles the rate of major haemorrhage, and not surprisingly therefore is only prescribed in about 25% of patients with atrial fibrillation.

There are two ways out of this quandary for apprehensive prescribers of long-term anticoagulants: to await the development of a more reliable anticoagulant, or to identify and predict the factors that underlie the wide variability in warfarin potency and risk of toxicity. The first option recently had a setback with reports that the most promising new oral anticoagulant, the direct thrombin-inhibitor ximelagatran, is associated with cardiac and liver toxicity, prompting a delay in its licensing by the US Food and Drug Administration. Other drug strategies have been proposed, including inhibition of other stages of vitamin K metabolism and use of RNA-aptamers to inhibit specific components of the coagulation cascade (complete with the attraction of a selective antidote with complementary RNA strands, or co-aptamers). However, it is unclear how many of these will stand up as safe oral agents available in fixed doses.

The second strategy of risk-profiling for a patient has made significant headway recently with the recognition of genetic markers of warfarin sensitivity or resistance. Dose requirements and the likelihood of toxicity are dependent on allelic variants of pharmacokinetic (cytochrome P450 2C9) and pharmacodynamic factors, including the immediate target of warfarin, vitamin-K-epoxide reductase (VKOR), and proteins involved in the vitamin K mediation of coagulant activation. Indeed, mutations in the VKOR protein underlie the rare combined deficiency of vitamin K dependent clotting factors type 2 (which like warfarin toxicity presents with frequent intracranial haemorrhages) and warfarin resistance in human beings and rats. Moreover, predicting patients at risk of the most feared complication of warfarin therapy–intracranial haemorrhage–might be possible through genetic (APOE type 2) or MRI markers, such as extensive small-vessel white-matter disease.

Once haemorrhage secondary to warfarin or superwarfarin occurs, medical attempts at reversing the block in coagulant activation suffer from relative resistance to vitamin K, and the need for large volumes of fresh frozen plasma. A novel therapeutic modality is provided by recombinant factor VIIa, which bypasses some of the vitamin-K-dependent steps of clotting, although cotreatment with vitamin K is still required to restore components of the common pathway. Large trials are required to see whether outcome is significantly better than the current prognosis of warfarin-associated intracranial haemorrhage–50% mortality at 3 months.

Source: Pankaj Sharma, Paul Bentley

The Lancet
March 15, 2005

Original web page at The Lancet