News this Week

Science  21 Jun 2002:
Vol. 296, Issue 5576, pp. 2116

    Did Bioweapons Test Cause a Deadly Smallpox Outbreak?

    1. Martin Enserink

    WASHINGTON, D.C.—A preliminary report that a 1971 smallpox outbreak in the former Soviet Union was triggered by a secret bioweapon field test has sparked a heated debate—and some nasty backbiting—among the small circle of bioterrorism experts. The outbreak shows that an aerosol attack with smallpox could actually kill, and it suggests that the Soviets turned an extremely deadly smallpox strain into a weapon, said Alan Zelicoff, a physician and smallpox expert at the Sandia National Laboratories in Albuquerque, New Mexico, at a meeting* here last weekend. “I've never seen anything quite so disturbing,” said Zelicoff.

    If true, the allegations suggest that new drugs and vaccines against the strain are needed, some experts say. However, one top U.S. bioterrorism official, Donald A. Henderson, immediately questioned the idea that a test triggered the outbreak. Henderson, who led the worldwide smallpox eradication campaign and now advises the government on bioterrorism policy, accuses Zelicoff of seeking the media spotlight with a “half-baked report.” But Kenneth Alibek, a former top manager at the Soviet bioweapons program who defected to the United States in 1992, backed most of Zelicoff's account in an interview with Science. Zelicoff, meanwhile, has urged Russian officials to investigate the incident and send the strain to the United States so researchers can find out how dangerous it is.

    The epidemic in the city of Aralsk, Kazakhstan, on the northern shore of the Aral Sea, struck 10 people, killing three. At the time, the Soviet Union swept it under the rug and didn't report it to the World Health Organization—a violation of international agreements that in itself raises suspicions, Zelicoff says. News about it never reached the West until a classified official account, written in the 1970s, was sent to the Monterey Institute of International Studies in California last year by a Kazakh scientist. The report claimed the outbreak had a natural origin. But after scrutinizing the document and interviewing two of the surviving victims by phone, Zelicoff and his Monterey Institute colleagues reached a different conclusion.

    The Soviet report concluded that the first patient most likely contracted smallpox while on a 2-month excursion on the Lev Berg, an ecological research ship. She probably picked up the virus during visits to Uyaly or Komsomolsk, two cities where the boat docked during its voyage (see map), then brought it home to Aralsk. But smallpox's incubation period makes that theory problematic, Zelicoff argues; moreover, the young woman never disembarked at any of the ports of call. A much more plausible explanation, he says, is that she was infected when the ship passed close to Vozrozhdeniye Island, at the time a top-secret outdoor testing site for bioweapons.

    Fateful voyage.

    Sailing too close to Vozrozhdeniye Island, a bioweapons test site, caused a 1971 smallpox infection aboard the Lev Berg, says Alan Zelicoff (top)—not a visit to one of several port cities.


    Zelicoff suggests that the strain was unusually infectious, because three of the 25 people who were vaccinated against smallpox and were close to a vaccinated patient got sick themselves—an unusually high percentage. What's more, three patients who had never been vaccinated developed the fatal hemorrhagic form of smallpox, which in other outbreaks occurred in fewer than 2% of patients. “That could be due to chance,” says Zelicoff, “but boy. …”

    At the meeting, Henderson immediately went on the attack. Ultraviolet light would quickly kill the virus in an aerosol cloud wafting over the Aral Sea, he argued—and if somehow it had survived, it would have infected more than just a single crew member aboard the ship. Zelicoff—who was initially denied a chance to respond because time was running out, creating an uproar in the audience—countered that aerosol tests would have been carried out at night to reduce UV exposure, and that the woman was particularly vulnerable because she spent much more time on deck than other crew members. Zelicoff accuses Henderson of downplaying the findings because they are a strong argument for keeping the smallpox virus alive to study, whereas Henderson believes the world would be better off if the remaining caches, now tightly guarded in the United States and Russia, are destroyed (Science, 15 March, p. 2005). Henderson vehemently denies such a bias.

    Alibek, in contrast, supports Zelicoff's analysis of the incident. After he joined the Soviet bioweapons program in 1975, two sources told him that a fatal smallpox incident had happened at Vozrozhdeniye Island a few years before, he says. (He didn't include the incident in his 1999 tell-all bestseller Biohazard because he didn't know any further details, he says.)

    However, Alibek does not believe that the test involved a hitherto unknown strain but rather India-1967 (also known as India-1), a strain that the Soviets have long been suspected of using in their bioweapons program and whose DNA was sequenced in the early 1990s. Smallpox researcher Peter Jahrling of the U.S. Army Medical Research Institute of Infectious Diseases in Fort Detrick, Maryland, is also “not completely convinced” that the Aralsk strain is an unknown, exceptionally virulent strain, in part because of the small numbers on which Zelicoff based his conclusions.

    Such questions could be resolved by studying the strain or tissue samples from the 1971 outbreak, which Zelicoff is convinced are stored somewhere in Russia. But when he tried to enlist his counterparts at VECTOR, the biodefense lab in Siberia where the Russian smallpox isolates are kept, they initially denied any knowledge of the incident, he says. Only after announcing that he would go public did they agree to look in their freezers.

    Zelicoff and Jahrling find this reticence troubling—especially because U.S. financial support keeps VECTOR and several other former Soviet bioweapons labs running, and mutual visits have fostered close ties between the former enemies' scientists. “These people are my friends,” says Zelicoff, “and yet it appears that they are lying.”

    • *“The Scientific Basis for Vaccination Policy Options,” Institute of Medicine, 15 June.


    Moratorium Replaces Ban as U.S. Target

    1. David Malakoff

    Biomedical research advocates appear to have won a major victory in the U.S. Senate. Senator Sam Brownback (R-KS) last week announced that he was abandoning his efforts to persuade the Senate to pass a bill outlawing all human cloning—including some types of research aimed at developing new medical treatments. Instead, Brownback says he will work to win congressional approval for a 2-year moratorium on such work. But critics say even that step would cause unacceptable delays for studies that could result in important medical benefits.

    Science advocates are pleased with the latest turn of events, but they don't plan to pack up and go home. “We've made great progress, but there is a very long way to go,” says Kevin Wilson of the American Society for Cell Biology, one of many research groups opposing Brownback's bill. And Brownback's allies, who just months ago seemed likely to prevail, promise that “the issue isn't going to go away. There is going to be a sort of guerilla campaign now,” says Nigel Cameron of the Council for Biotechnology Policy, a conservative think tank in Reston, Virginia.

    For months, senators and science lobbyists have been preparing for what was expected to be an emotional and historic debate over how the government should regulate cloning, an array of techniques that can produce genetically identical embryos. The predebate tension was heightened by reports that some scientists were on the road to cloning humans. Last summer, the House of Representatives passed legislation that would make it a criminal offense to engage in either reproductive cloning or so-called therapeutic cloning, in which scientists would transplant the nucleus from an adult cell into an embryo to produce genetically matched cells that might be useful for medical treatments. Brownback sponsored a similar bill in the Senate. But biomedical researchers and patient groups felt that Brownback's bill went too far. Instead, they supported a competing proposal that would ban reproductive cloning but allow related research to continue with greater regulation (Science, 10 May, p. 997).

    Strategic retreat?

    Senator Sam Brownback now says he'll settle for cloning moratorium.


    Brownback, who had the backing of several conservative groups and President George W. Bush, once appeared to have the votes to pass his bill. But a broad coalition of research and patient groups fought back with an arsenal that included television ads and Capitol Hill visits from Hollywood stars, Nobel Prize-winning scientists, and children suffering from currently incurable conditions. Their message: Don't lump therapeutic research in with the reproductive cloning ban. The tide turned in their favor last month after Senator Orrin Hatch (R-UT), a leading antiabortion conservative, announced that he would oppose Brownback's bill.

    Last week, Senate leaders seemed close to a deal to bring the dueling bills to a vote. But negotiations collapsed after neither side could show that it had at least the 60 votes needed to overcome procedural hurdles and bring their bill to a vote. As a result, Senate Majority Leader Tom Daschle (D-SD) put the gridlocked issue aside.

    The move angered Brownback, who told reporters last week that opponents— including Daschle—had set ground rules that were “stacked … against me.” He has since moved—so far unsuccessfully—to attach pieces of his bill, including a cloning moratorium and a ban on cloning-related patents, to unrelated bills before the Senate.

    Brownback's opponents have vowed to block a moratorium. “A moratorium of a year or two may not seem like much … but it could mean the difference between life and death for a patient with Parkinson's disease,” says Senator Edward Kennedy (D-MA), alluding to the high hopes that some patient groups have for therapeutic cloning. “From the science community's perspective, a moratorium equals a ban,” adds Wilson. Some observers say that Brownback's tactics reflect his growing desperation. “The fact that he has fallen back to the idea of a 2-year moratorium suggests that he can't find the votes he needs,” says Pat White of the Federation of American Societies for Experimental Biology.

    The current stalemate doesn't bother White and other science lobbyists. “[Having] no bill is better than [passing] Brownback's bill,” says one. However, the inaction also leaves in limbo the one issue on which all sides can agree—the need to ban human reproductive cloning.


    New Effort Aims to Thwart Dirty Bombers

    1. Richard Stone

    CAMBRIDGE, U.K.—Russia and the United States have agreed to join forces on an unprecedented effort to hunt down stray radioactive materials—the potential stuff of dirty bombs—across the former Soviet Union. Under the agreement, expected to be announced next week, Russia will provide information on “orphaned” sources that could pose a threat, and the U.S. Department of Energy (DOE) will provide initial funding of roughly $40 million over the next 2 years to track them down. Lending extra urgency to the initiative—which will be organized and managed by the International Atomic Energy Agency (IAEA) in Vienna, Austria —are revelations about the legacy of a secret Soviet research program to spray radioactive cesium on agricultural fields.

    Because there are “literally millions” of radiological sources that could be used in a dirty bomb, “it is crucial to focus limited resources on those that could pose the most dangerous potential terrorist threat,” says Matthew Bunn of Harvard University's Belfer Center for Science and International Affairs. “Russian openness on data about how many sources of what types were produced, and where they went, will be crucial to success.”

    The hunt is on.

    These hot Georgian generators raised dirty-bomb fears.


    Vague fears that a terrorist might try to concoct a dirty bomb from radiological materials and conventional explosives gained a measure of credibility last week, when the U.S. government trumpeted the news that it had foiled an alleged al Qaeda plot to steal radioactive materials from an undisclosed U.S. facility. Far more worrisome to many observers, however, is the possibility that terrorists could obtain stray radioactive materials in former Soviet republics. The magnitude of this threat is unknown, mainly because Russia has not been forthcoming about the Soviet nuclear legacy out of fears that it could be held liable for materials outside its borders after the Soviet Union dissolved, says IAEA's Abel Julio González. At the same time, the U.S. government has focused on the threat posed by former Soviet fissile materials.

    But the demands for better information grew urgent after the 11 September attacks. “The U.S. got very nervous” about radiological sources, says Kenneth Luongo, executive director of the Russian American Nuclear Security Advisory Council in Washington, D.C. According to Bunn, DOE launched a series of classified studies on the impact of potential radiological terrorist attacks and assessments of which materials posed the most serious threats. Ratcheting up anxiety levels was the recent discovery of Soviet-made thermoelectric generators packed with strontium-90 that IAEA and the Republic of Georgia recovered last February near the breakaway Abkhazia region (Science, 1 February, p. 777).

    A newly recognized threat could come from loose cesium chloride. The radioactive form of this salt has a variety of uses, including sterilizing medical supplies, and is kept under strict control because it is manufactured as a talcumlike powder that's easy to disperse. González told Science that IAEA has learned from reliable Russian sources that the Soviet Union ran a secret program in which farmers spread radioactive cesium chloride powder on their fields. Experts speculate that the purpose of the program, code named Gamma Kolos (kolos is Russian for “ear,” as in ear of corn), was to see if the powder would alter germination rates or induce beneficial mutations in corn. At least four trucks packed with the substance turned up recently in Moldova, a tiny and impoverished former Soviet republic, and recent photos of similar trucks have surfaced in the Republic of Georgia. The cesium-137 in each truck has an activity of about 3500 curies—“a very significant amount,” notes González.

    As scary as that sounds, Bunn and others warn that stepped-up efforts to track down radiological sources should not come at the expense of securing “hotter” fissile materials that could be used to create far more devastating nuclear bombs. “Radiological terrorism could be expensive to clean up, but it would not mean tens of thousands of people dead and the heart of a major city incinerated in a flash,” Bunn says. But the dirty bombs are easier to produce—and the threat is finally being taken seriously.


    Gruss Takes Max Planck Helm

    1. Philipp Weis,
    2. Gretchen Vogel*
    1. Philipp Weis is a science writer in Berlin.

    HALLE AN DER SAALE, GERMANY—Peter Gruss's honeymoon period as president of Germany's premier research organization, the Max Planck Society (MPG), could not have been shorter. He spent his first full day on the job waiting to hear whether he would have to rein in the organization's budget. But discussions on Monday between the federal and state governments eased what Gruss called his “first worry”: The society got a 3% budget increase. MPG was spared, at least for now, from the nationwide belt-tightening that most German agencies are expecting in response to the sluggish economy.

    Observers had already predicted that flat or even declining budgets would threaten Gruss's main goal: to keep MPG in the top ranks of global science. He wasted no time confronting the government over the matter in his inaugural speech at MPG's annual meeting last week. Although he acknowledged that German scientists can't expect a windfall similar to the 5-year budget doubling that the U.S. Congress has promised the National Institutes of Health, Gruss said a budget cut would send exactly the wrong signal to German scientists and to the rest of the world. “We can save anywhere except in shaping our future,” he said.

    Gruss, a prominent developmental biologist who has been a director at the Max Planck Institute for Biophysical Chemistry in Göttingen since 1986, has little political experience, but he has not been reluctant to jump into political debates. In remarks to the press last week, he strongly criticized Germany's compromise law on the use of human embryonic stem cells passed by parliament earlier this year (Science, 8 February, p. 943). Although the law allows basic research to go forward by permitting researchers to work on existing stem cell lines, he pointed out that the compromise rules out any therapeutic applications because these lines have been exposed to mouse cells and would not be safe for implanting in humans. He has also said that he would welcome final passage of a controversial immigration law in Germany, saying it would remove obstacles to recruitment of top foreign scientists.

    Outgoing MPG president Hubert Markl leaves the organization in the hands of one of his former students. At the 14 June handover ceremony, Markl joked that Gruss “showed good judgment at an early age” when he chose a different field after hearing Markl's lectures in zoology.

    Markl's 6-year term was dominated by rapid expansion. The society founded more than a dozen new institutes in the former East Germany, bringing the total up to 80. Now, with 18 institutes and one research station in the new Länder, the “buildup of the east” is all but completed, Markl said at a press conference. East-West divisions are gone, he said, and “the Max Planck Society is now a unified organization.” Markl also earned praise during his term for encouraging the society to examine the darker periods of its history. Last summer, he offered the first explicit apology to victims of abuses during the Nazi era by scientists of the Kaiser Wilhelm Society, the forerunner of MPG (Science, 15 June 2001, p. 1979).

    Gruss told the MPG meeting that one of his top priorities will be strengthening connections with German universities, especially through the new International Max Planck Research Schools, which are jointly funded and run by Max Planck institutes and cooperating universities. Establishing the interdisciplinary graduate schools “is one of the best things Markl did” as president, says Wieland Huttner of the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden. Gruss said he hopes to strengthen the existing 19 schools and launch seven new ones already in the planning stages.


    Plant a Few Cells, Sprout a Thymus

    1. Jennifer Couzin

    Shriveling as it ages, wedged between the heart and the thyroid, the raisin-sized thymus is an unlikely warrior. Still, without one, people wouldn't last long. The thymus attracts a blank slate of stem cells from the bone marrow and transforms them into infection-fighting T cells. Now, two teams have found that a tiny subset of cells from a mouse embryo can be grown into a full-blown thymus and beget a healthy immune system in the recipient mice. The finding suggests that it might be relatively easy, as far as regenerating organs goes, to give a failing thymus a boost.

    “What's interesting is the ability to generate an organ from a small population of cells,” says Nancy Manley, a developmental biologist at the University of Georgia, Athens, who was “totally floored” when she first heard about the work.

    Both groups, one at Monash University Medical School in Melbourne, Australia, and the other at the Centre for Genome Research at the University of Edinburgh, U.K., are quick to say that they haven't found the putative “thymic stem cell”: a single cell that, by itself, can produce a fully functioning thymus. Both used hundreds of cells to jump-start the thymus-growing process. However, the teams say the work strongly suggests that such a stem cell, whose existence is a source of passionate debate in the thymus world, is buried somewhere amid the cells they isolated.

    The Monash group, led by immunologists Richard Boyd and Jason Gill, and the Edinburgh group, led by developmental immunologist Clare Blackburn, started with cells that met two criteria. They had to belong to the set of functional cells in the thymus, called thymic epithelial cells. And they had to flourish early in development but fade from the picture later on—a pattern a stem cell would follow. Both groups used monoclonal antibodies, which bind to specific molecules on a cell surface, to home in on epithelial cells that fit the profile. These cells were marked by two proteins, MTS20 and MTS24.

    Organ transplant.

    A class of young thymus cells in a mouse embryo (stained red) grow into a working thymus when grafted onto another animal's kidney (top).


    In mouse embryos, so-called MTS20/24 cells make up about a third of the cells in the developing thymus. The researchers grafted clusters of these cells onto the kidneys of adult mice and watched what happened. Whether 40,000 or 1000 MTS24 epithelial cells were implanted made little difference, says Gill: “We put them underneath a kidney from a mouse, then pat it on the head and let it run around for 4 or 8 or 6 weeks, then we open up the mouse and lo and behold, there's a plump, juicy, functioning thymus.” Gill and his colleagues, who grew the organs in mice that had their own thymuses, found a host of T cell types within the new tissue.

    Blackburn and her colleagues implanted even fewer cells, less than 500, into four “nude” mice that lack a thymus altogether. This allowed them to test the new-grown thymus's powers by examining distant lymph nodes for T cells. Three months after implantation, the researchers determined that the mice had relatively healthy immune systems, although they produced half as many T cells as normal mice. But when the researchers dug around the kidney to find the thymus, they saw nothing. They suggest that the cells differentiated and produced a range of T cells but then died off. Blackburn believes that 500 cells might be below the critical mass needed to sustain a thymus; the group reports finding full thymuses in mice that had received larger implants. Blackburn's work will be published in the June issue of Immunity; the Monash team's results appear in the 17 June online edition of Nature Immunology.

    Still to be proven, however, is that the thymic stem cell exists. If MTS20/24 cells are largely homogeneous, that would bolster the case that the long-sought thymic stem cell exists and is among them. But the thymic cells huddled in the original implanted cluster might have been more diverse than they appeared. If so, more than a single cell type might be needed to grow a new thymus. Howard Petrie, an immunologist at Memorial Sloan-Kettering Cancer Center in New York City, also questions whether all the cells in the Monash thymus arose from the original cluster; they might have recruited others from the recipient's body. Still, Petrie and others embrace the therapeutic potential of stimulating the thymus in those with weak immune systems—an enormous population that includes the elderly, patients undergoing chemotherapy, and people with immune diseases, including AIDS.


    Nerves Tell Arteries to Make Like a Tree

    1. Greg Miller

    By the time an embryo's heart beats for the first time, an extensive tree of arteries is already in place. Its delicate branches—which will ultimately stretch tens of thousands of kilometers in a full-grown human—ensure that no bit of tissue goes wanting for oxygen and nutrients.

    How arteries shape themselves into such fine patterns has been an open question. Now a study shows that arteries follow the lead of another of the body's branching specialists: nerves. In the 14 June issue of Cell, developmental neurobiologists Yoh-suke Mukouyama and David Anderson of the California Institute of Technology (Caltech) in Pasadena and colleagues report that embryonic nerves form a template that directs the growth of arteries. The team also identifies a molecule released by the nerves that apparently signals the arteries to fall in step.

    “This is the most elegant paper I've read in years,” says angiogenesis researcher Judah Folkman of Harvard University Medical School and Children's Hospital in Boston. “They answer one question after another.”

    Blood vessels and peripheral nerves tend to snuggle closely together. This arrangement has advantages: Arteries supply neurons with oxygenated blood; nerves tell blood vessels when to dilate or contract and help direct immune responses. However, few studies have examined how this relationship develops.

    The Caltech researchers labeled nerves and blood vessels in the skin of embryonic mice. They found that arteries, but not veins, align closely with nerves. Snapshots of the skin at several time points revealed that the nerves appear first. Soon after, primitive vessels—which have yet to don the molecular trappings of arteries—align with the nerves. This hinted that the nerves might be calling the shots.

    Developmental tango.

    Branching arteries (red) follow the lead of neurons (green) in embryonic mouse skin.


    The team then turned to mutant mice lacking a gene important for guiding axons, the long tendrils extending from neuron bodies. Peripheral nerve axons in these mice tend to clump together and have fewer fine branches, and Mukouyama and Anderson's team found that the mice's arteries had the same pattern. Apparently, arteries follow axons even when the axons go astray.

    It makes sense that the development of nerves and arteries is linked, says George Yancopoulos, a molecular geneticist at Regeneron Pharmaceuticals in Tarrytown, New York. “It's easy to speculate that if you're going to have two branching systems that integrate into the various tissues of the body, when one system comes up with a solution, it's very economical to have the second system just follow along.”

    Nerves also appear to secrete a molecule that tells embryonic blood vessels to become arteries in the first place. The team found that neurons and accessory cells called Schwann cells make a molecule called vascular endothelial growth factor (VEGF), which is found in a variety of tissues and has been shown to spur blood vessels to sprout and grow. In test tube experiments, the team found that VEGF compels undifferentiated blood vessels to take on characteristics of arterial cells. Anderson hypothesizes that VEGF secreted by nerves first attracts primitive blood vessels, then tells them to become arteries.

    Although people have long noted the anatomical similarities between arteries and nerves, there has been little evidence that their development is coordinated, says molecular geneticist Peter Carmeliet of the University of Leuven in Belgium: “Until now there have been no molecular clues.”

    The research could also help explain a number of disorders that have baffled physicians, according to Folkman. Children with Möbius syndrome, for example, fail to develop several cranial nerves and have improperly formed arteries. The link between nerve and artery development could be a step toward an explanation, Folkman says.


    Act Seen as First Step in Protecting Species

    1. Wayne Kondro*
    1. Wayne Kondro writes from Ottawa.

    OTTAWA, CANADA—Canada's House of Commons last week approved the country's first law to protect endangered species. But although federal officials say the legislation, which relies on incentives rather than punishments, sets a new standard for cooperation between public and private sectors, environmental groups grumble that the approach leaves much to be desired.

    “We want to get ahead of the curve, with stewardship programs,” says Environment Minister David Anderson. But that approach “is just a starting point,” complains the International Fund for Animal Welfare's national director, Rick Smith. “It will still leave the majority of species in the country without mandatory protection.”

    The parliamentary vote caps a decade-long debate over how best to protect Canadian plants and animals and their habitats (Science, 24 August 2001, p. 1417). In addition to first-ever mandatory protection on federal lands, the Species at Risk Act will offer incentives and compensation to landowners and industry to do the right thing, says Anderson. Those inducements will amount to $29 million this year and an expected $38 million next year. The government can wield a big stick if necessary, he adds, including arrests and fines of up to $650,000.

    Wise decision?

    A new Canadian law will try to help endangered friends of this great owl.


    Despite giving private landowners financial incentives to cooperate, the new bill seemed headed the way of its predecessors until Anderson struck a compromise with all sides. He appeased the rural caucus of his own Liberal party by promising that property owners will receive adequate compensation if their lands are declared protected areas because of their value to at-risk species. He mollified the environmental caucus with a pair of olive branches. The first gives slightly more power to scientists on the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). If the committee declares a species endangered, its decision will now be final unless politicians vote within 9 months to overturn it and put their reasons in writing. The second makes habitat protection mandatory on federal land (about 6% of Canada's land mass) and waters, and on all land north of the 60th parallel not governed by aboriginal land-claim agreements.

    For private lands, the bill relies on a different model of governance from what he calls the “coercive command-and-control approach” of the U.S. Endangered Species Act, says Anderson. Pressure from U.S.-linked environmental groups for more stick and less carrot “have not been helpful to the debate,” he adds.

    Others hold a decidedly less rosy picture of the bill. “I really don't think it will do the job,” says ecologist David Schindler of the University of Alberta in Edmonton, who says that the legislation defers to the provincial governments, which have a mixed record of species and habitat protection. “I would be surprised if we saw any slowdown whatsoever in the rate at which new species are added to the list.”

    Environmentalists calculate that the bill leaves about two-thirds of the 402 species within various COSEWIC risk categories without any form of mandatory habitat protection. “If we're going to save species, we have to save spaces for them, and the government is only delivering on federal lands and for aquatic species,” says Kate Smallwood, endangered species program director of the Sierra Legal Defense Fund. Migratory birds are at special peril, she says, “unless the nest and its habitat are in a post office, military base, an airport, a Coast Guard station, or a national park.” Anderson disagrees. An existing agreement with the United States already obliges Canada to protect migratory fowl habitat, he points out.

    The bill's final parliamentary hurdle—Senate approval—is a low one, because only once in the past decade has the upper chamber overturned legislation. And that's fine with an exhausted Anderson. “I'll be glad to have it over with,” he says.


    Amgen Splits With Lab, But Its Money Lingers

    1. Wayne Kondro*
    1. Wayne Kondro writes from Ottawa.

    OTTAWA, CANADA—Amgen has decided to sever its ties to the University of Toronto (UT)-based research institute it has funded for nearly a decade. But in an unusual twist, it's going to continue paying millions of dollars a year for work to which the university will hold all intellectual property rights.

    The Amgen Institute was created in 1993 by the California biotechnology company, and the agreement was updated in 1999 to run through 2008. But soon thereafter the company installed a new management team, which last month decided that the institute's basic research into the functions of similar genes in mice, Drosophila, and Caenorhabditis elegans didn't fit into its new corporate strategy to focus on applied research. Earlier this month it negotiated a settlement with the university, and last week the lab set up shop under the umbrella of UT's network of teaching and research hospitals.

    UT officials say that they are precluded from discussing the terms of the settlement. But both sides agree that the company negotiated an end to paying indirect research costs—administrative overhead, utilities, and the like—in exchange for renouncing any commercial claims to discoveries. “We have a contract with them to 2008 [that includes an annual payment of $6.5 million], and we are living up to that contract,” says Amgen spokesperson Jeff Richardson. “We're ending our affiliation with them, but if someone were to pay me until 2008, I would think they were still supporting me.”

    The institute's director, molecular geneticist Tak Mak, says that he's grateful for Amgen's support but that “we are happy to be again on our own, concentrating on the science.” Although he says that the settlement “is not consistent” with Amgen's previous level of support, he acknowledges that there's little he can do about it: “If you're the boss, you can call the shots.”

    The institute is now part of the new Advanced Medical Discovery Institute within Toronto's University Health Network. UHN research vice president Christopher Paige says the settlement buys the university enough “breathing room” between now and 2008 to raise an estimated $65 million needed to maintain the institute's current level of operations, including a half-dozen or so principal investigators and as many as 90 technicians, students, and support staff. One investigator, Josef Penninger, had previously announced that he was moving his lab to his native Austria.

    Both parties agree on one thing: The new setup gives scientists more freedom to pursue their research and disseminate the results. “Having those researchers in a university setting doing proprietary research did not allow them to speak with their colleagues about what they were doing,” says Richardson. “Now they have academic freedom, and they really didn't have that before.”

    Mak says that he's relieved to be shedding 10 years of corporate ties and that having UHN own the intellectual property rights to any discoveries “allows us to be more free in terms of giving away animals and reagents, without six lawyers signing off.” Mak has been criticized in the past by colleagues for being unresponsive to such requests (Science, 23 June 1995, p. 1715).


    Jupiter's Brother Joins the Family

    1. Richard A. Kerr

    Last week American astronomers announced the discovery of a new yet familiar-looking planetary system—not “a sibling of the solar system,” but “a first cousin.” The distant relation made front-page news, but in the hour before the Americans' televised press conference got under way at NASA headquarters, European astronomers were discreetly spreading the word via e-mail that they had uncovered a nearer relation: an exoplanet that more closely resembles Jupiter in a planetary system far more like our own, “a younger brother” of Jupiter, as one of the discoverers put it. The find marks the true beginning of an expected string of discoveries of planetary systems in which Earth-like planets might be hiding.

    Both discoveries came as astronomers searched for telltale stellar wobbling, a sign that the gravity of a massive unseen planet is tugging the parent star back and forth. Before last week, 76 exoplanets had been discovered, but none resembled Jupiter, the solar system's most massive planet. All either were “hot Jupiters” orbiting closer to their stars than Mercury does to the sun or were on wildly elongated orbits.

    Do the numbers.

    When the two Jupiter-like planets are compared, the one orbiting HD 190360 announced this week is closer to Jupiter in mass and orbital eccentricity.

    View this table:

    Last week astronomer Geoffrey Marcy of the University of California, Berkeley, and his colleagues announced their “near analog to our Jupiter” (Science, 14 June, p. 1951). It is a body at least 4.3 times the mass of Jupiter. It orbits the star 55 Cancri at a distance of 6.0 times Earth's orbital distance (6.0 astronomical units, or AU). And it is in an orbit “just a little out of round,” having an eccentricity of 0.16 (Jupiter's eccentricity is 0.05).

    Unfortunately, planet 55 Cancri d hangs out in a distinctly un-Jovian neighborhood. The American team had already found one hot Jupiter orbiting 55 Cancri and announced a second last week. Like all hot Jupiters, these must have formed farther out and drifted inward, driving everything before them into the star and vaporizing any inner, Earth-like planets. “It's not like our solar system,” says exoplanet searcher William Cochran of the University of Texas, Austin, “which is what people are looking for.”

    Now astronomer Michel Mayor of the Geneva Observatory in Sauverny, Switzerland, and his colleagues believe they have discovered a true Jupiter orbiting the star HD 190360. The new planet, which they formally announced this week at a meeting* in Washington, D.C., has a minimum mass just 1.1 times that of Jupiter and orbits at 3.7 AU. In the solar system, that would put it outside Mars between the asteroid belt and Jupiter. And its eccentricity is less than 0.1, indistinguishable from Jupiter's. Best of all, Mayor's HD 190360 has no hot Jupiters. In Doppler-shift observations, its planetary system looks nearly identical to what alien observers would see if they looked at ours. Astronomers are welcoming both discoveries as the vanguard of a coming Jupiter bonanza. “I think it's great,” says astronomer David Trilling of the University of Pennsylvania in Philadelphia. “In the next few years, there will be dozens and dozens more.”

    • *“Scientific Frontiers in Research on Extrasolar Planets,” 18 to 21 June, sponsored by NASA and the Carnegie Institution of Washington.

  10. JAPAN

    New Program to Aid Smaller Universities

    1. Dennis Normile

    TOKYO—Like most of Japan's smaller universities, Fukui University doesn't have a big research budget. But a new program to help universities build up strengths in specific areas will give scientists there a chance to compete against the scientific heavyweights at Tokyo, Kyoto, and Tohoku universities for precious government funding.

    Last week the Ministry of Education launched its 21st Century Centers of Excellence program and invited all universities, public and private, to compete. The $160-million-a-year program will concentrate resources, in contrast to Japan's traditional approach of scattering small grants across the academic research enterprise. Another novel wrinkle is that applications must be submitted by the president of the university, rather than by a research group or individual scientist. The idea is to ensure the institution's commitment to the project. “From now on, universities will have to begin thinking strategically,” says Yoshihide Akatsuka of the ministry's University Reform Office.

    The ministry hopes to fund 20 or so centers in each of five areas: life sciences, chemistry and materials sciences, electronics and information sciences, humanities, and the deliberately vague category of interdisciplinary studies. A separate review committee in each area will select centers based on both the track record of its investigators —especially publications and awards—and the likelihood of achieving “epochmaking results.” Grants will range from $800,000 to $4 million a year for 5 years.

    Akatsuka admits that the amounts might not be large enough to build a new program. But Yukinori Urushizaki, an administrator overseeing research support at Fukui University, says that winning a grant “would be a considerable boost” to the image of the university, located 350 kilometers west of Tokyo. “We are different from the big universities … we have a limited budget for research,” Urushizaki adds.

    The program's funding levels are “not so attractive to the major universities,” acknowledges Keiichi Kodaira, president of the Graduate University for Advanced Studies in Hayama, near Tokyo. But that doesn't mean his institution and other research heavyweights won't compete. Kodaira, a member of an advisory committee that vetted the program, predicts that “many [large universities] will apply simply because if they don't, their [programs] will be regarded as below standard.”

    The first batch of centers is expected to be selected by the end of September. Ministry officials hope to receive enough new money in the next fiscal year, which begins 1 April, to add five more areas and another 100 centers. The program's long-term status, however, has not yet been determined.


    Demolition Crew Gets a Hand From Chaperones

    1. Jean Marx

    The body's cells react rapidly to the ups and downs of the hormones that control their activities. But that presents a puzzle: The biochemical machinery that responds to certain hormones is so large and seemingly cumbersome that researchers have long wondered how it manages to react quickly to changes in hormone concentrations. New results, described on page 2232 by Brian Freeman and Keith Yamamoto of the University of California, San Francisco (UCSF), suggest a solution to this long-standing conundrum—one that might provide a new role in regulating gene expression for the proteins known as chaperones.

    Donald DeFranco of the University of Pittsburgh School of Medicine calls the new results “novel and important” and predicts that “this will be an ‘impact’ paper.” There is, however, at least one competing view of which molecules to thank for a cell's rapid-response capabilities.

    The hormones for which the conundrum arises include the thyroid hormone thyroxine and the steroids, such as the sex hormones and the glucocorticoids that regulate cell metabolism. When one of these hormones binds to its receptor, the resulting complex moves to the nucleus, where it, together with several other proteins, binds to regulatory sequences on the DNA. This, in turn, activates or inhibits appropriate genes. The mystery has been how the receptor can, while buried deep in this regulatory complex, detect when hormone levels fall off—a feedback needed to tell the regulatory complex to stop overseeing the genes.

    Going in circles.

    From the upper right, when a steroid receptor (purple, top) binds a hormone (yellow), it kicks off chaperones (blue, green, and red). It then settles on DNA and draws in several other proteins (green and orange) to form a regulatory complex. Disassembly of this complex is triggered by chaperones that the energy compound ATP aids, ultimately returning the receptor to its initial state.


    The new results by Freeman and Yamamoto indicate that two so-called chaperone proteins, named p23 and Hsp90, help disassemble regulatory complexes shortly after they form on the DNA. When thus released, the receptor can, as Yamamoto puts it, “quiz the cell: Is the hormone around?” If it is, the regulatory complex can be reformed, but if not, the response can be terminated.

    Freeman and Yamamoto were inspired to undertake the current experiments partly by a finding from Gordon Hager's team at the National Cancer Institute in Bethesda, Maryland: Despite their size, steroid hormone regulatory complexes get on and off DNA very quickly (Science, 18 February 2000, p. 1262). “Here we are building these immense protein machines, and they are turning over within seconds,” Yamamoto says.

    Chaperones, which help proteins fold into their proper three-dimensional shapes, were known to facilitate the binding of steroid hormones to their receptors. But earlier hints from Yamamoto, DeFranco, and others that the proteins might also act later to break down the resulting regulatory complexes were met with skepticism. Disassembly of the complexes takes place in the cell nucleus, and chaperones' actions were thought to be limited to the cell cytoplasm.

    The current work appears to demonstrate chaperones' role in ending a receptor's effects on transcription. For example, in vitro experiments revealed to Freeman and Yamamoto that p23 both triggers the release of the thyroxine-thyroid hormone receptor complex from the DNA to which it binds and also decreases the gene transcription normally caused by the complex. And in living cells, the investigators showed that p23 binds to glucocorticoid-induced regulatory complexes—which it must do in order to trigger their breakup—and blocks transcription induced by the hormones. Hsp90 had similar, but weaker, effects. What's more, the UCSF researchers found that p23 blocked transcription by two very different regulatory complexes—ones not containing steroid hormone receptors—suggesting a more general role for chaperones in controlling gene activities.

    DeFranco says that these experiments provide “by far the clearest and most striking evidence” that chaperones help disassemble regulatory complexes containing hormone receptors. But Hager is not so sure. Some cellular actor is needed to release receptors from the complexes, he says, but his work, including results that appear in the May issue of Molecular and Cellular Biology, indicates that the job is performed by the so-called Swi/SNF complex, which alters the way that the DNA and its associated proteins are wound together. Hager notes that both Swi/SNF and the chaperones might have a role, depending on the circumstances. He and Yamamoto are collaborating to see if they can reconcile their differences.


    Plasticity: Time for a Reappraisal?

    1. Constance Holden,
    2. Gretchen Vogel

    Long-standing biological dogma—that a cell, once committed, can't alter its fate—has been challenged by recent research. But now scientists are taking a more critical look

    Although not quite the cold fusion of biology, it ignites similar passions. Stem cell “plasticity”—the ability of cell types from adult tissues to take on surprising new identities—can inspire debates as heated as any about that infamous physics claim. In hallways during conference coffee breaks, in strongly worded journal commentaries, and in behind-the-scenes conversations, the stem cell community is picking apart, and sometimes battling over, the evidence: Can cells from one type of tissue be induced to look and act like cells from a different tissue? Do these switches happen naturally? And could such transformations be used to treat deadly diseases?

    A host of recent papers has suggested that stem cells from various adult tissues can, indeed, be reprogrammed—in defiance of age-old dogma that once a cell has become specialized, it can't backtrack or adopt a new identity. But several years after the first reports, some of the results are proving hard to replicate. “We have gone into so many traps recently with stem cells that I don't know any longer what's true,” says Ole Isacson of Harvard University. The political stakes are enormous. If adult stem cells turned out to be so multitalented, they would offer a less controversial alternative to the use of embryonic stem (ES) cells. Ideally, a relatively easily accessible tissue such as skin or bone marrow could be cultivated into neurons, whose own stem cells are hard to reach, or blood, for which stem cells have proved hard to grow in culture.

    Most scientists say no evidence yet suggests that adult stem cells can match the versatility of those derived from embryos, although work by marrow stem cell researcher Catherine Verfaillie of the University of Minnesota, Twin Cities, on a possible new type of cell might come close. And those cases in which plasticity seems to have occurred are so rare that the phenomenon might never be of practical use. Nonetheless, opponents of ES cell research have seized on any suggestion of plasticity in adult stem cells, often hyping it to make their case that ES cells are unnecessary (Science, 8 June 2001, p. 1820). Says Ihor Lemischka of Princeton University, “It's dangerous to talk about plasticity. … Anything that is reported gets immediately translated into arguments pro and con in the White House.”

    Too good to be true?

    Studies in mice have yielded evidence, now being reassessed, that stem cells from a variety of tissues can produce progeny in different organs. Bone marrow, which has several types of stem cells, seems particularly versatile.


    New paradigms?

    Decades of experiments have shown that as animal cells divide during development, they take on new roles in an orchestrated way with little sign of haphazard fate-switching. That's why developmental biologists have been so surprised by recent claims of “transdifferentiation” or “plasticity,” as the as-yet-unexplained observations of fate-switching are alternatively called.

    Some scientists are quite happy to question dogma. For instance, Darwin Prockop of Tulane University in New Orleans, Louisiana, who works with bone marrow-derived stem cells called mesenchymal cells, says, “Our lab is betting on the idea that these cells will do the whole job”—that is, supply a variety of cell types to treat neurological and other disorders. Others are more cautious: “I am not going to take a handful of papers” generated in the past few years and use them as a basis to dismiss decades of careful work, says Lemischka.

    Plasticity was such a hot phenomenon in the late 1990s that journals, including Science, were snapping up and publishing partial—or what are now seen as questionable—results. “A lot of the big journals were willing to take extremely marginal stuff,” opines Markus Grompe of Oregon Health & Science University in Portland.

    As the political stakes have gone up, scientists have begun to pay closer attention to the multiple ways cells can fool them. For instance, cells in culture can mutate and develop markers characteristic of other lineages. Cells injected into foreign tissue can take up local DNA—and thus appear to have changed identity—without actually becoming transformed. An introduced macrophage can show the markings of a cell it's eaten. Or local residents might be mistaken for visitors. Margaret Goodell of Baylor College of Medicine in Houston, for example, discovered that what she thought were muscle stem cells turning into blood-forming cells were actually hematopoietic cells already residing in muscle.

    Wake-up call.

    Austin Smith of the Univeristy of Edinburgh led one of two teams that have raised the specter of cell fusion. Pictured here: chimeric mouse embryo with fused cells (blue).


    Hints that the picture might be a lot more complicated than some have assumed came last March, when research suggested that some apparent reprogramming of adult cells might instead be a case of cell fusion (Science, 15 March, p. 1989). Austin Smith of the University of Edinburgh, U.K., and Naohiro Terada of the University of Florida College of Medicine in Gainesville showed that when they cultivated adult bone marrow or brain cells with ES cells, the two cell types occasionally formed hybrids that looked like ES cells—but that had twice the usual numbers of chromosomes and were thus likely to be genetically unstable. The papers challenged only those plasticity observations that involve co-culturing two types of cells. And the fusion danger might be limited to the highly volatile embryonic cells. Nonetheless, the findings were a wake-up call to several teams. “The fusion possibility never even entered our minds,” Goodell admitted at a stem cell meeting held in March in Keystone, Colorado.

    The fusion scare has given further impetus to efforts to establish rigorous standards for demonstrating plasticity. Irving Weissman of Stanford University—one of the most outspoken skeptics in the field—with David Anderson of the California Institute of Technology in Pasadena and Fred Gage of the Salk Institute for Biological Studies in La Jolla, California, laid out in the April 2001 issue of Nature Medicine a set of requirements. First, the cells must be properly identified at the outset, because a single alien cell in ostensibly purified culture could produce misleading results. Putting out a few new proteins does not count, they said; instead, the cells must contribute to the functions of the host tissue: passing electrical signals in the nervous system, or filtering impurities from blood in the liver. This means that a single well-characterized donor cell has to be shown capable of creating a “robust” population and not just a scattering of cells in the new tissue. The trio also argued that the plasticity should be a natural phenomenon, which means cells must perform in the host tissue without having been altered in culture.

    Others in the field agree that cells need to be better characterized and that they must demonstrate functionality. But beyond that, agreement breaks down on how many hoops these cells must jump through. The “gold standard” for demonstrating functionality was set by experiments done since the 1960s in which transplants of single blood stem cells recreated an entire blood system in lethally irradiated mice. But the blood system is one of the only tissues where such a dramatic demonstration of functionality is possible. Demonstrating that a particular neuron is functional in a brain, on the other hand, is difficult even for neuroscientists, notes Helen Blau of Stanford University, who did some of the earliest work in the 1980s suggesting that cells could switch lineages. “Yes, it's a good goal,” she says. “But it's not an easy goal.”

    As for the “no-alteration-in-culture” requirement, Jonas Frisén of the Karolinska Institute in Sweden says that is relevant only if the goal is to study normal physiology. “If you're studying what is possible, then it's absolutely OK to culture.” In fact, he adds, cells that are creatures of the test tube could prove useful in treating disease.

    Do studies pass muster?

    None of the studies so far purporting to demonstrate plasticity measures up fully against Weissman, Anderson, and Gage's rigorous criteria. For example, Diane Krause of Yale University and colleagues performed a complex two-stage study in which they injected dye-tagged male mouse blood stem cells into irradiated females. Then, to ensure that they were dealing with one particular type of cell, the researchers killed these mice and pulled out tagged cells that had homed in to the bone marrow. They then implanted a single cell into each of a second group of female mice. The researchers reported in the 4 May 2001 issue of Cell that some of the progeny of these cells became incorporated into various tissues including lung, skin, intestine, and liver, as well as bone and blood. Thus, Krause met two requirements: She used cells that had not been altered in culture, and she showed that a single blood stem cell can give rise to multiple cell types.

    Voice of caution.

    Stanford's Irving Weissman is probably the most outspoken critic of plasticity claims.


    But Krause is now checking for evidence of cell fusion. And Eric Lagasse of StemCells Inc. in Palo Alto, California, is skeptical, noting that the cells plucked from the first transplant recipient were “not very well characterized.” Verfaillie adds that the studies “still don't really show significant contribution to any organ. They're just small groups of cells without any function.” Finally, the experiment hasn't yet been replicated. Weissman reported at a June meeting in Stockholm at the Karolinska Institute that when he and his team tried to repeat the result with carefully screened blood stem cells, they found only the expected bone and blood derivatives, six liver cells (too few to be significant, he says), and one brain cell. And the brain cell, a large type called a Purkinje cell, had twice the normal complement of DNA—meaning that it could have been a local cell that fused with one of the tagged cells. “There is no evidence that purified [blood] stem cells can contribute to any other tissue,” he says—although Krause counters that her team's precursor cell might be different from the one Weissman tested.

    Failure to replicate has put one of the earliest, most provocative studies (Science, 22 January 1999, p. 534) on shaky ground as well. Christopher Bjornson of the University of Washington, Seattle, Angelo Vescovi of the National Neurological Institute in Milan, Italy, and their colleagues reported that cultured mouse neural stem cells appeared to produce a variety of blood cell types after being planted in irradiated mice. When Derek van der Kooy of the University of Toronto repeated the protocol, using a population of neural stem cells derived from a single progenitor cell, he failed to generate any blood cells, he reported in the February issue of Nature Medicine.

    Two years ago, a study by Karolinska's Diana Clarke, Frisén, and their colleagues also seemed to support the idea that brain cells have broad potential. But it, too, is meeting skepticism. The Karolinska team purified neural stem cells in culture, allowed them to grow into cell clumps called neurospheres, and inserted them into the blastocyst of a developing mouse embryo. These cells, marked with a blue dye, contributed to development in all three germ layers in the animal, the team reported in the 2 June 2000 issue of Science (p. 1559). Although Frisén concedes that some of the apparently reprogrammed cells might be fused, he believes fusion events are so rare that they can't account for the extensive presence of the blue-dyed cells. A more serious limitation of the study, says Rudolf Jaenisch of the Massachusetts Institute of Technology, is that the pups were allowed to survive only through embryonic day 11—too early to tell whether the brain-derived cells were really functional. Frisén says his team is working on follow-up experiments in which the pups will develop to birth.

    In the opinion of many scientists, the published study that comes closest to demonstrating true plasticity is by a group headed by Lagasse at StemCells, a company started by Anderson, Gage, and Weissman, that is working to develop potential applications for neural and hematopoietic stem cells. As reported in Nature Medicine in November 2000, the researchers purified male mouse hematopoietic stem cells based on no fewer than 13 cell-surface markers. Then they injected the cells into the bloodstream of irradiated female mice that suffered from a genetic liver disorder. The transplant not only led to the restoration of the blood systems in four of the nine mice, it also seemed to cure them of their liver disease. When the scientists killed these mice 7 months later, between a third and a half of their liver tissue was derived from donor cells.

    Optimistic skeptic.

    Fred Gage of the Salk Institute wants to raise the bar, but he predicts that some studies will hold up to rigorous scrutiny.


    But even this experiment falls short. Because groups of cells rather than single cells were injected into the mice, the work does not prove that the same precursor cell can generate both blood and liver cells. Grompe, one of the co-authors, also notes that the impressive percentage of liver is a bit misleading. When they looked carefully, he says, the researchers realized that the new liver tissue had grown from just a few progenitors. That's promising news for eventual medical applications, as it means that just a few cells might do the trick. But it works against the idea of plasticity, as it implies that only a very tiny proportion of blood cells managed to become liver. The team is now trying to replicate the study to rule out the possibility of fusion.

    Best evidence yet?

    Ironically, says Grompe, raising the bar might have a downside: Even scientists who have taken time to do careful controls might be having a harder time publishing. Exhibit A is Verfaillie, who finally has a full report of her work published online by Nature on 21 June. In work reported at several meetings, which earns kudos from some of the field's staunchest skeptics, Verfaillie has been examining the properties of what could conceivably be a “universal” stem cell—a type never before identified. Verfaillie grew the cells from populations of mesenchymal cells, the marrow cells that generate bone and muscle, from three species: mice, rats, and humans. Because she's not absolutely sure her cells qualify as stem cells, Verfaillie calls them multipotent adult progenitor cells (MAPCs).

    Her team stumbled on these cells. “We were planning to grow mesenchymal stem cells,” she explains. But “we came up with a culture system that for reasons I still don't understand appears to select for [more primitive cells].” The researchers found that after about 30 population doublings in culture, the cell population changed and took on a striking similarity to ES cells. But, at least in preliminary studies, MAPCs appear to have a big advantage over ES cells: Injecting them into live animals does not seem to cause tumors, as often happens with embryonic cells because of their mercurial nature.

    Verfaillie's team has put these tiny cells—about one-third the size of mesenchymal cells—through quite a few paces, as she described at the March Keystone meeting. She has demonstrated in petri dishes that they, like mesenchymal cells, can make bone, cartilage, and fat. But unlike mesenchymal cells, they can also be cultured to produce the endothelial cells that line blood vessels, making little vascular tubes in the dish. The team has also reported (in the May issue of the Journal of Clinical Investigation) the creation of cells that look and function like liver cells—even producing urea. Other cells have been cultivated in vitro to show some of the characteristics of nerve cell precursors.

    Going a step further than most researchers, Verfaillie has tracked the fate of individual cells. Researchers commonly use the green fluorescent protein gene to track the progeny of a group of cells. Instead, her group has used a retrovirus, which is inserted in a unique spot in every cell's genome and thus can track the specific parentage of a single cell.

    Moving beyond test tube studies, Verfaillie's team has made chimeric mice by injecting single mouse MAPCs into 12 mouse blastocysts, she reported at Keystone. This technique creates a mosaic animal—called a chimera—made up of cells derived from the original blastocyst as well as the progeny of the injected MAPCs. Four of the injected blastocysts grew into chimeras, and in two of the animals, Verfaillie reported, 45% of the body tissues tested expressed the MAPC genome. Moreover, these cells showed up in every organ, suggesting that they are capable of turning into all three embryonic germ layers: the mesoderm, the ectoderm, and the endoderm. Verfaillie doesn't yet know whether these cells will also contribute to germ line (egg and sperm) cells—a defining characteristic of ES cells.

    In the final experiments Verfaillie reported at Keystone, the group infused MAPCs into young mice. The marked cells eventually showed up in lung, gut, and other tissues but were not seen in the skeleton, heart, or brain.

    Verfaillie has yet to prove that her cells can fully function in the new roles they assume. For example, she observes, “we've shown they can fit into liver and make liver [products] but haven't [yet] shown they can rescue a mouse.” In further experiments, she will see whether MAPCs spring into action in the heart or brain in response to injury.

    New candidate.

    Catherine Verfaillie has cultivated cells that can be transformed into apparent lung (left), duodenum (center), and liver cells (right) in young mice.


    Reaction to her reported work has been enthusiastic. She has shown that “the cells are stable and can contribute to a very broad spectrum of mature cell populations,” says blood researcher John Dick of Toronto's Hospital for Sick Children. But her work still does not provide rock-solid evidence for plasticity, even she concedes. The missing piece, as Dick explains, is that “there's no way of knowing what the founder cell looks like”—that is, what cell gives rise to a MAPC. One possibility, says Gage, is that MAPCs are adult cells that really do show plasticity, “dedifferentiating” in culture to become multipotent. A less likely hypothesis, says Anderson, is that Verfaillie has hit upon a rare “highly multipotent” cell, a kind of universal stem cell, that could be hiding all over the body. But the fact that cells must be cultured at length before MAPCs appear “tends to argue” that they are an artifact of tissue culture, he says.

    In a forthcoming paper in Experimental Hematology, Verfaillie describes cultivating MAPCs from mouse muscle and brain as well as bone marrow—a development that could fit with either theory. Verfaillie holds out hope that hers “could be the ultimate study that explains the results everybody else is getting.”

    Politicians are already keenly interested in Verfaillie's work—as a way to put ES cells out of business. Several members of Congress sought her out last winter, she says, after the press got wind of a patent application she had filed. She wrote back telling them it's too soon to draw any conclusions.

    That might be a wise answer for the entire field, says Princeton's Lemischka. There are good evolutionary reasons for suppressing cell plasticity in the body. As yet, very little is known about how to change the rules while averting the dangers of running wild—a worry that applies to potential therapies derived from ES cells as well as adult cells.

    So is plasticity biology's “cold fusion”? No, scientists say. Even some skeptics believe something is going on in these experiments, even if they don't know exactly what. “I think [therapies with transplanted stem cells] will eventually work,” says Grompe. But “we've raised a lot of false hopes for quick fixes, and that's not going to happen.” He and others say a closer comparison might be with gene therapy—greatly hyped 20 years ago but still without much to show for itself. James Thomson of the University of Wisconsin, Madison, who first isolated human ES cells back in 1998, agrees. “I'm not looking forward to the backlash 3 years from now when people say, ‘What happened to stem cells?’” he says. What can scientists do about it? Says Thomson: “We need to educate the public that science takes a long time.”


    Russia Can Save Kyoto, If It Can Do the Math

    1. Paul Webster*
    1. Paul Webster is a writer in Moscow.

    Russia's ratification of the Kyoto treaty might put the pact over the top. But some take a chilly view of the reliability of its greenhouse gas emission numbers

    MOSCOW—The U.S. withdrawal from international negotiations over carbon emissions last year dealt a blow to the Kyoto Protocol that many thought might be fatal. A year on, however, Russia has emerged as an unlikely savior.

    To come into force, the treaty must be ratified by enough industrialized nations to account for 55% of carbon emissions in 1990, Kyoto's baseline year. The U.S. withdrawal put its leading 36% share off limits, making participation by the other major players even more important. Russia—which accounts for 17% of 1990 emissions—holds second place. Its government deliberated for more than a year before President Vladimir Putin declared in April, “We'll do it.” A final review of the protocol is due for completion by midsummer, with ratification expected in the fall.

    What led Russia to become an environmental champion? Its economy has traditionally relied on smokestack industries and burning fossil fuels, and until recently climate change was seen benignly as an antidote for shoveling snow. But the treaty gives the cash-strapped Russian government a financial incentive to think green. Russia expects its carbon emissions to be down by 20% from 1990 levels when Kyoto comes into force in 2008, the result of an economic downturn that has shuttered factories and shrunk agriculture. The regrowth of forests has sequestered more carbon.

    As a result, Russia will win huge amounts of pollution credits under Kyoto's emissions-trading system. “Russia will have a near-monopoly on emissions credits,” says economist Richard Baron, an expert in the trading system at the Organisation for Economic Co-operation and Development (OECD) in Paris. Those credits can be sold to countries, in particular Europe and Japan, whose emissions have increased since 1990. The windfall could earn Russia tens of billions of dollars.

    A dark cloud hangs over Russia's greenhouse bonanza, however, in the form of doubts about the accuracy of its emissions inventories. In 4 years' time, those inventories will be open to international scrutiny by the other 83 Kyoto-signatory nations. If Russia can't prove its reduction in emissions since 1990, its partners are unlikely to let it cash in. “If they can't show they have an appropriate inventory, there's no middle ground,” says Baron. William Chandler, a specialist on Russian climate change policy at Pacific Northwest National Laboratory's office in Washington, D.C., agrees: “The situation is urgent.”

    In 1997 a panel of scientists convened by the United Nations to review data compiled by the Russian Federal Service for Hydrometrology and Environmental Monitoring (Roshydromet) revealed glaring methodological problems and striking data gaps. The U.N. review team labeled Roshydromet's approach “insufficient” and highlighted its poor presentation of data and its lack of uncertainty levels and information on data-collection methods. Methane emission figures, the U.N. team said, were “highly unreliable since they were based upon hypothetical assumptions.” Estimates for emissions from the oil and coal industries and the carbon consumption of Russian forests and peatlands were similarly criticized.

    Three years later, a second U.N. review team evaluating a second set of data from Roshydromet reported some progress but pointed to many lingering problems. One is a complete absence of figures from important industries including pulp and paper, asphalt, glass, iron and steel, and nonferrous metal production. The team concluded that the new data were “of average or low quality.” According to review team member Raisa Mäkipää of the Finnish Forest Research Institute, “We were not satisfied.”

    The current government review prior to ratification is based partly on a third set of data from Roshydromet that have not yet been made public. But Alexei Kokorin, a campaigner with the Worldwide Fund for Nature in Moscow who says he has seen the latest Roshydromet data, believes that it still “doesn't conform to international norms.”

    Climatologist Yuri Izrael, Roshydromet's principal greenhouse gas inventory investigator, agrees that the data quality in the first two inventories was “variable” and that “we still need better information.” He insists, however, that Russian greenhouse gas data quality has improved steadily since Roshydromet began work in 1994. “Our latest inventory is much better than the previous inventories were,” he says.

    On the slide?

    Despite official figures, natural gas flaring seems to have increased in these satellite images of Siberia in 1992 (top) and 2002.


    One of the biggest obstacles to a more accurate accounting of greenhouse gas emissions is the Russian petroleum industry—now the world's largest producer. Kokorin says that the industry is “very shy” about its emissions data and that the latest Roshydromet inventory doesn't include them. Russian oil companies say such tallies are provided in confidence to the government. A recent World Bank report highlighted one sore spot: the widespread industry practice of burning off byproduct gases in giant torchlike flares.

    According to the World Bank, Russian government estimates based on industry data indicated that 2.6 billion cubic meters of gas were flared in 2000. But an alternative assessment commissioned later by the World Bank in collaboration with the Russian government estimated 10.25 billion cubic meters of flaring. According to Bent Svensson, head of the World Bank's international flaring project, obtaining reliable Russian data for the petroleum sector has been “very difficult.”

    At the other end of the carbon chain, the contribution of Russia's forests in reducing emissions is also very uncertain. Finland's Mäkipää, who helped review Roshydromet's 2000 inventory, found the official inventories unreliable after a more thorough study of Russian forest data. The data depend on highly uncertain estimates of tree age, she says, and are simply not usable for Kyoto requirements: “The quality of the data don't allow objective evaluation of the size of the forest carbon sink.”

    A lone bright spot in Russia's emission inventories is the data produced by Russia's mammoth electrical utility, United Energy System of Russia (UES), which produces an estimated 25% of all Russian carbon emissions. According to Ludmilla Khoudogarova of the Russian Academy of Sciences' Energy Research Institute, UES produced a reasonable account of emissions from its 370 power plants for Roshydromet in 1995, and that account has improved with each review. “It's possible groups could buy [emission] credits directly from Russian companies like UES that can actually prove their numbers,” says OECD's Baron.

    Apart from the opacity of Russian industry, the basic competence of Russian climate change researchers is also causing concern. A 1999 review of the research by Nina Poussenkova of the Russian Academy of Sciences' Institute of World Economy and International Relations revealed plenty of well-trained scientists. But their isolation from the mainstream, combined with a Soviet-era fear of challenging official information, makes them uneasy in the highly internationalized and politicized climate change field. Lack of funding is also a problem. The U.N. team that reviewed Roshydromet's data in 2000 declared that Russia's financial commitment to climate change research was “marginal.”

    As Russian officials sharpen their pencils for a final review of the Kyoto accord, Russian climate change researchers hope the government will boost its investment in climate change research to meet the protocol's scientific requirements. In the absence of better data, Pacific Northwest's Chandler says that Russia is ill equipped to participate in an international system built first on trust, then verification. “Without accurate data, the system crashes,” Chandler warns. “It just won't work.”


    A Generation Gap in Brain Activity

    1. Laura Helmuth

    As the field of cognitive neuroscience matures, researchers are starting to see how the brain's behavior changes with advancing years

    As people get older, some things just don't work the way they used to. Some of these changes seem straightforward: A once cast-iron stomach begins to balk at greasy food, or knees complain after too many trips down the ski slopes. But what happens to the aging brain has been more mysterious.

    By some measures brain function appears to decline as we age. Behavioral studies have shown, for example, that older people are slower on easy tasks and less accurate on difficult ones; their memories are leaky; they're easily distracted in tests of attention. But some of these findings might simply be a function of what researchers are able to study in the lab. “Older adults don't show impairments in everyday life,” says Denise Park of the University of Michigan (UM), Ann Arbor, indicating what she calls a “disconnect” with the lab results.

    Hoping to see how the brain changes as it ages, researchers have increasingly been using imaging techniques for the past 5 years or so. Although they have uncovered some striking patterns—as shown by presentations at the inaugural meeting of a conference* dedicated to the cognitive neuroscience of aging—they also have added to the mystery. Some researchers have observed that certain brain areas physically shrink over time, whereas others have found that the patterns of neural activity in 60- or 70-year-olds often bear little resemblance to those in the 20-something subjects who populate most brain functional imaging studies. “We see one set of activations in young people, but older subjects are quite strikingly different,” says Park.

    Exactly how these changes relate to cognitive failings in old age remains unclear, however. Indeed, a major debate at the meeting centered on whether the shifts in brain activity are good news or bad. Older people might exercise extra brain areas to compensate for a decline in normal function, some suggest, whereas others view the change as a symptom or even a cause of age-related mental lapses. As John Gabrieli of Stanford University points out, “so many things are going on at once” as the brain ages that the challenge is to figure out “which changes in the brain correspond to which changes in the mind.”

    Answers to such questions might help researchers with another crucial goal, says UM's Patricia Reuter-Lorenz: figuring out which mental skills are hard-wired into specific brain regions and which can be accomplished by more than one set of regions. The latter might be more amenable to yet-undiscovered cognitive or pharmacological interventions that allow people to compensate for sputtering brain circuits.

    The march of time

    Death comes for some brain neurons before others. Naftali Raz of Wayne State University in Detroit has depicted the “neuroanatomy of aging” by imaging specific brain regions in healthy people of various ages. These cross-sectional studies indicate that some areas are stable over time, others undergo a seemingly inexorable decline, and some reach a turning point and only then start to dwindle.

    One of the better preserved parts of an older brain is the occipital cortex, the region at the back that largely handles visual information. The frontal lobes are less fortunate. Raz found that one section, the so-called dorsolateral prefrontal cortex, shrinks by about 5% per decade between the ages of 20 and 80. The hippocampus, a part of the brain crucial for memory, holds its own until middle age. But after age 45 or so, it loses about 7% of its volume per decade.

    Comparisons between different people of different ages, no matter how well matched they are on measures of health or education, leave open the possibility that factors other than age might account for some of the apparent changes. But Raz says they don't appear to. At the meeting, he presented new data from a longitudinal study that tracked the same subjects' brains for 5 years, confirming the pattern seen in the cross-sectional studies.

    Gabrieli points out that it's tricky to “relate specific biological changes to specific cognitive changes,” but he says the patterns seen by Raz and others might reflect what he identifies as two major changes that shape the aging brain. The first is a gradual, lifelong decline in circuits that include the frontal lobes. Thanks to this effect of age, over time people are slower to process complicated visual scenes, get tripped up on complex reasoning tasks, and have a lower capacity for so-called working memory, or keeping thoughts in mind for later use. As behavioral tests on older subjects have shown, this slowdown hits pretty much everyone and is part of normal aging.

    The second effect, related more directly to memory, hits some people harder than others. It centers on the hippocampus and surrounding tissue called the entorhinal cortex. Gabrieli and colleagues have followed a group of healthy old people since the early 1990s. Some have very good memories; others are more forgetful, although none have been diagnosed with Alzheimer's disease. Those with good memories have entorhinal cortices that look pretty hearty, whereas in those with the worst memories, the region is relatively small—a possible early sign of Alzheimer's, Gabrieli says.

    A whole-brainer

    Neuroimaging studies have uncovered another major shift that occurs as people age—one some researchers see as hopeful. Neurobiologists have long known that the left and right sides of the brain have specialized functions. When young people exercise their so-called verbal working memory—say, by keeping a list of words in mind for a few minutes—the left frontal lobe lights up. In contrast, when they juggle spatial information, such as recalling where a dot appeared on a computer screen, the right frontal lobe is in charge.

    But older people, it appears, are more broad-minded: They use both frontal lobes when performing either type of working memory task. “We saw this in 1997 but didn't put too much importance in it,” says Roberto Cabeza of Duke University in Durham, North Carolina. But as study after study began uncovering the same double activity, “it started looking like a phenomenon.” The “long-standing view” in neuroscience, Reuter-Lorenz says, is that certain parts of the brain perform certain functions, but studies showing widespread activation in older people make it clear that this “may have to be revised.”

    People are still trying to figure out what the phenomenon means. One question is whether the difference is “neurogenic”—due to age-related changes in neural connections—or “psychogenic”—a side effect of older people using different mental strategies to solve the same problem. For instance, an older person wary of relying on spatial skills to remember a location might come up with a verbal description of the spot, thus calling the left hemisphere into play.

    Cabeza says that the evidence points to a neurogenic source in most cases: The furious firing in the front of older brains shows up whether people are remembering pairs of words, recognizing faces, or preparing to match up locations in space. It's unlikely that the strategy would work well in such different tests, he maintains.

    However it arises, loss of specialized activity in the brains of older subjects could be an unfortunate development. Having specialized brain regions is generally thought to be a good thing—a division of labor that, for example, lets the left hemisphere build sophisticated language abilities while the right concentrates on navigation.

    When a younger person is working a verbal puzzle, Randy Buckner of Washington University in St. Louis and others have shown, there's some activity in the right hemisphere at first, but it's quickly squelched. This efficient silencing is lost in older people, Buckner suggests, through a breakdown in communication between the hemispheres. The two sides of the brain are “just not recruited well,” he says—a sign of underlying pathology that creeps in with age.

    Others look on the bright side. Reuter-Lorenz, for one, sees extra frontal activation as a “correlate of graceful aging.” The elderly subjects in her experiments are just as accurate in working memory tests as young college students. And in some cases, the fastest old subjects are the ones with the greatest activity in the “extra” hemisphere. Older people might be “recruiting additional brain areas in a compensatory way,” she says, and brain imaging studies are capturing what is simply a healthy response to what she calls the “decline in efficiency of circuitry typically used in younger brains.”

    Cabeza tested this notion directly. He classified his older subjects as either high-functioning or low-functioning according to a battery of standardized tests. Then he observed their brains under positron emission tomography (PET) scans as they recalled pairs of words. In young subjects, the left side of the brain lit up, as expected, during this verbal task. Old subjects with poor memories likewise relied upon the left hemisphere. But old subjects who performed as well on memory tests as young people used both the right and left hemispheres, he reported at the meeting—suggesting that extra activation is indeed beneficial.

    Measurable skills.

    With age, people perform worse in spatial, memory, and problem-solving tests—but their vocabularies grow.


    Potentially compensatory changes aren't limited to the frontal cortex, as shown by Cheryl Grady of the Rotman Research Institute at the University of Toronto. When young people try to recognize pictures of faces, a constellation of brain regions lights up. But Grady and her colleagues found that in older people matching faces, accuracy was correlated with activity in the amygdala—a region that normally processes emotions. Older subjects might pay more attention to facial expressions to aid their memories, Grady says, possibly in a case of psychogenic compensation.

    But even Cabeza, Reuter-Lorenz, and others who suggest that extra activity helps an older brain cope admit that what they see as compensation might come at a cost. The frontal lobes do most of the brain's glamorous work—the problem solving, working memory, and other tasks that fall under what psychologists call “executive control.” If what's easy for a 20-year-old requires a lot of effort when one is 70, Reuter-Lorenz asks, “what happens when the going gets rough?” Other studies have shown that even young subjects sometimes use both hemispheres if they're really concentrating; perhaps older subjects tax the frontal lobes' processing power even during simple tasks, she suggests, and aren't able to access it in more trying times.

    It's also not clear when the differences in brain activation kick in—whether 40-year-olds' frontal lobes act more like those of their children or their parents. “We know nothing about middle-aged adults,” says Park of the dearth of functional imaging studies that include people from a range of ages.


    Pinning down where and when certain abilities falter with age might suggest ways to counteract the changes. Finding out what happens when extra areas of the frontal lobe are recruited in older people is “something that's on a lot of people's minds,” says Reuter-Lorenz. Some abilities might be hard-wired, performed by strictly defined circuits whose signals can't be rerouted. But others might be amenable to compensation from more spry parts of the brain.

    Despite some grim findings of cognitive loss described at the meeting, Park points out that people have plenty of areas of expertise that are preserved or even grow over time. For example, vocabulary tests are one of the few lab measures that show a reliable advantage for older subjects—which should come as some comfort to those who have been trounced by a great-aunt in a game of Scrabble. But researchers don't have standardized tests for other advanced skills—knowledge of banking, say, or how to navigate a city's streets. “People develop a large mental scaffolding for expertise,” says Park, “and it's relatively easy to hang new information on a scaffold created over time.”

    Researchers have even less of a handle on how to measure intangible qualities, such as wisdom. “We all hypothesize that as you get older, you get wiser,” says Park. But it's awfully tough to find a quantitative measure, much less one that can be assessed while the aged but wise recline in a PET scanner.

    • *Symposium on Neuroscience, Aging, and Cognition, San Francisco, 12–13 April.


    Physicists Prepare to Catch Cosmic Bullets

    1. Robert Irion

    Researchers can't agree where the most energetic particles in the universe come from, or even how many of them strike Earth. An observatory under construction by physicists in 19 nations might change that

    Just like any other place on Earth, the Pampa Amarilla in western Argentina is daily shot through with billions of bits of matter, cascading from the sky at just shy of the speed of light. What's different about this ancient lakebed is that soon physicists will be there ready to catch these intense sprays. In a few years, 3000 square kilometers of the desert—an area roughly the size of Rhode Island—will be scattered with 1600 detectors forming one giant observatory. The researchers who are building it, and planning a twin instrument in Utah, hope it will end decades of confusion about the origins of our highest energy visitors from outer space.

    The visitors in question are particles called ultrahigh-energy cosmic rays (UHECRs). The fiercest of these travelers slam into Earth's atmosphere packing more than 100 exa-electron volts (100 EeV, or 1020 eV) of energy. That makes the world's most powerful particle accelerator, the 1012 eV Tevatron at Fermi National Accelerator Laboratory (Fermilab) in Batavia, Illinois, look like a pea shooter. Each of these tiny subatomic particles—thought to be protons or occasionally heavier atomic nuclei such as helium and carbon—can pack as much energy as a brick dropped onto your foot or a fastball from a professional baseball pitcher.

    “It's very hard to understand how to produce these particles with what we think we know about conventional physics,” says Nobel laureate James Cronin of the University of Chicago. Indeed, no one knows for sure where UHECRs come from. Theories abound, from the nearby descendants of quasars to ultramagnetic neutron stars to the annihilation of clumps of superheavy dark matter. But these guesses rest on the slightest of foundations: perhaps a dozen particles above 100 EeV and a few dozen at somewhat lower energies. “It's like the early work on gamma ray bursts,” comments astrophysicist Robert Streitmatter of NASA's Goddard Space Flight Center in Greenbelt, Maryland. “The number of papers is many multiples of the number of events.”

    That might soon change with the construction of the $54 million Pierre Auger Observatory near Malargue, Argentina, named after the French physicist who in 1938 first detected air showers from cosmic rays with detectors high in the Alps. A test array of about 30 detectors has already captured some incoming UHECRs; the full observatory should be running within 3 years. Funding permitting, Auger physicists will build an identical facility in the desert of Millard County, Utah, a few years later, enabling them to watch the sky over both hemispheres.

    Cronin, the project's spokesperson, says the utter mystery of UHECRs puts the Auger team on the precipice of physics, as if perched on the snow-capped Andes above the array. “We're doing something different with our careers; it's a little off the wall, a little risky,” he says. “Particle physicists will ask, ‘What theory are you checking? What particle are you looking for?’ I don't have the slightest idea.”

    Beyond the cutoff

    The cosmic ray quandary is rooted in a calculation called the GZK cutoff. In 1966, American physicist Kenneth Greisen and Soviet physicists Georgi Zatsepin and Vadim Kuz'min calculated that particles above 40 EeV face draining journeys through space. Beyond that threshold, the microwaves that permeate space—the remnant heat from the big bang—appear like gamma rays to the relativistic particles. The cosmic rays collide with the faux gamma rays, losing roughly one-fifth of their energy in each encounter. The physicists figured that over a few hundred million light-years, any particle traveling faster than the GZK cutoff will have been battered down below it. So any UHECRs detected on Earth must come from nearby, usually no farther than 100 million light-years.

    Desert sentinels.

    The Pierre Auger Observatory near the Andes will spy cosmic rays with water tanks (top) and fluorescence telescopes (bottom).


    In light of this cosmic energy limit, physicists expected few or no events of 100 EeV or higher. Although quasars in the distant universe probably spit out such bullets, our neighborhood seemed serene. Modest arrays of particle detectors did catch a few eye-popping events (Science, 19 May 2000, p. 1147). But the rates were so low—barely one UHECR per square kilometer of ground per century—that researchers weren't sure whether they were flukes. “It took the community some time to accept that the ultrahigh-energy events were real,” says particle physicist Glennys Farrar of New York University (NYU) in New York City.

    Firmer evidence has come from two teams using different methods to spot cosmic rays. One approach uses the world's largest network of ground detectors: the Akeno Giant Air Shower Array (AGASA) near Kofu, Japan. When a cosmic ray crashes into Earth's atmosphere, it unleashes a shower of particles—muons, electrons, gamma ray photons, and others—in an expanding cone that flashes to the ground. A single UHECR can trigger an air shower involving more than 100 billion particles. AGASA spots a fraction of them with 111 detectors spread out over 100 square kilometers. A special plastic in the detectors scintillates when some of the particles zip through. With this information, the team reconstructs each event's energy using simulations of shower behavior (Science, 14 August 1991, p. 891). Since 1990, AGASA physicists have seen about 60 events past the GZK cutoff—and 10 events with energies above 100 EeV.

    In contrast, experiments at the U.S. Army's Dugway Proving Grounds in Utah look skyward. An instrument known as Fly's Eye and its successor, High-Resolution Fly's Eye (HiRes), use segmented mirrors to monitor the air for flares of ultraviolet light—the sign of nitrogen atoms fluorescing at the disk-shaped leading edge of the UHECR air showers. The technique works only on clear, moonless nights. However, HiRes physicists at the University of Utah in Salt Lake City and elsewhere think the technique yields a reliable, direct measure of a cosmic ray's energy. Fly's Eye and HiRes have seen only two or three events above 100 EeV in more than a decade, although the best detectors have operated for just a few years.

    The gulf between AGASA and HiRes has puzzled physicists. Debates about it have spiced up recent cosmic ray conferences, including a session at an April meeting* in Albuquerque. Akeno Observatory director Masahiro Teshima said that although some adjustments in energy calculations might occur as the AGASA team refines its analysis, they won't be drastic. “We cannot change the energy scale by a factor of 2 or 3,” he said at the meeting. “I'm very sure of the existence of these super-GZK particles.”

    However, the latest HiRes results, now in preparation for publication, strengthen the Utah team's conviction that AGASA physicists have systematically endowed their events with too much energy. “We see the GZK cutoff in action,” says physicist Gordon Thomson of Rutgers University in New Brunswick, New Jersey. “Our data take a nosedive at that energy.” The team also sees signs of a “pileup” of events near the 40-EeV energy level, says physicist Pierre Sokolsky of the University of Utah. That's what one would expect for particles that start out as UHECRs at great distances from Earth and then lose energy along the way. A few sources closer to our galaxy could account for the small number of extremely energetic events detected by HiRes, he notes.

    The Auger Observatory might resolve the discrepancy. Physicists have combined both methods into the world's first “hybrid” cosmic ray observatory. The 1600 enclosed tanks, each containing 10,000 liters of purified water and spaced 1.5 kilometers apart, will catch air showers with energies above 1 EeV. Meanwhile, 24 fluorescence telescopes will overlook the entire array to watch for UV flashes. When the sky is clear and totally dark—about 10% of the time—the physicists will get combined data for the showers, helping them analyze each technique's systematic errors.

    Engineering tests in the Argentinean pampa have already revealed one unforeseen technical challenge. “We can't leave anything that invites the resident cows to scratch themselves,” says project manager Paul Mantsch of Fermilab. The physicists have had to hide all cables and make sure the tanks have no sharp edges to minimize cattle-catalyzed repairs.

    A zoo of sources

    Once the observatory is complete, Auger physicists hope to see thousands of events above 10 EeV, some of which undoubtedly will transcend the GZK cutoff. Time will tell whether there are enough to refine theories on the possible origins of UHECRs. “Right now, I don't know of any plausible sources,” says Chicago's Cronin. “That's what makes it exciting.”

    Ideas for how to spawn fantastically energetic cosmic rays within 100 million light-years of Earth fall into two categories. The first, dubbed “bottom-up,” maintains that the particles don't start out that fast but are boosted by vast natural accelerators. Supermassive black holes in the cores of nearby galaxies could do the trick. At the Albuquerque meeting, a team led by physicist Diego Torres of Princeton University reported suggestive associations between the directions of the UHECRs detected by AGASA and four very bright nearby elliptical galaxies—possibly the remnants of quasars active billions of years ago. Black holes at their cores might spin rapidly enough to power a raging electromagnetic field of perhaps 100 billion billion volts, shooting off cosmic rays like sparks.

    However, NYU's Farrar and physicist Tsvi Piran of Hebrew University in Jerusalem suggest that relying on the incoming direction of UHECRs might be misleading. It's possible, they say, that all detected UHECRs come from the most active galactic core in the nearby universe: Centaurus A, a galaxy in the southern sky 11 million light-years from Earth. Farrar says that the paths of the particles could be bent by intergalactic magnetic fields, making them appear to come from all over the sky. This would require the magnetic fields to be stronger than now suspected.

    Astrophysicist Jonathan Arons of the University of California, Berkeley, thinks we need only look inside normal galaxies, such as our Milky Way. In work he will soon submit for publication, Arons points to magnetars, unusual highly magnetized neutron stars forged by certain supernovas. A magnetar spinning thousands of times a second at birth could whip up plasmas that accelerate particles to 1000 EeV (a zetta-electron volt) or more. “This could be an interesting signature of some of the craziest supernovas you'll ever run into,” Arons says. Astrophysicists Pasquale Blasi of the Arcetri Astrophysical Observatory in Florence, Italy, and Angela Olinto of the University of Chicago have also proposed this idea.

    In contrast, some theorists prefer “top-down” ways of producing UHECRs, without the need for an insanely powerful accelerator. For example, the cocoon of dark matter enshrouding our galaxy might contain clumps of “superheavy” dark matter that popped into existence in the early universe. Over the millennia such clumps, dubbed “WIMPzillas” by physicist Edward Kolb of Fermilab and colleagues, would occasionally collide and annihilate each other, spewing out ferocious cosmic rays (Science, 19 February 1999, p. 1095). Others have proposed similar scenarios involving the decays of magnetic monopoles, cosmic strings, and other massive relics from the big bang.

    “The theorists get more creative as the evidence gets more compelling,” says Auger physicist James Beatty of Pennsylvania State University, University Park. “But all of the mechanisms have the problem that, in one way or another, the very existence of these particles is far-fetched.”

    If the Auger Observatory doesn't see enough UHECRs to unravel this knot of hypotheses, its successors might take to orbit. Both NASA and the European Space Agency (ESA) are studying proposals to scan the atmosphere for the fluorescence flashes of UHECR impacts from space. The ESA proposal, called the Extreme Universe Space Observatory, would consist of a single downward-pointing telescope on the international space station and might fly as soon as 2008. NASA is looking a few years farther down the road toward a two-satellite mission called Orbiting Wide-angle Light-collectors.

    Whatever the outcome of experiments under construction or in the works, most of the bizarre notions about UHECRs will likely crash and burn. The teams wouldn't have it any other way. “Particle physics is so cut and dried with the Standard Model,” says Sokolsky. “Here, it's just wild.”

    • *Joint meeting of the American Physical Society and the American Astronomical Society's High-Energy Astrophysics Division, 20–23 April.

  16. Sorting Out Chromosome Errors

    1. Jon Cohen

    Researchers are struggling to understand—and possibly overcome—aneuploidy, the most common cause of miscarriage

    Humans are not particularly good at making babies. We have a flaw that becomes more pronounced as we age, and the limitation cannot be helped by candlelight and violins, Viagra, or, for the most part, fertility clinics. More often than in any other species, embryos we conceive have an abnormal number of chromosomes, a condition called aneuploidy. Most of the time, aneuploidy leads to miscarriage, but it is also responsible for Down syndrome and other mental and physical problems. “Aneuploidy is the most important problem in human reproduction,” says Terry Hassold, a cytogeneticist at Case Western Reserve University in Cleveland, Ohio, who studies the many odd chromosomal configurations that humans create. “It's unquestionably the most common genetic problem our species has, and we don't have a clue why it happens.”

    Researchers might not have discovered why we conceive so many aneuploid offspring, but by studying embryos, eggs, and sperm in humans and other species, they have made solid progress in determining how, where, and when the process of distributing chromosomes to progeny goes awry. The advent of in vitro fertilization (IVF), which has high failure rates that appear strongly tied to aneuploidy, has also pushed the field forward. So, too, has the discovery that some chromosomes are more prone to aneuploidy than others. “There's been an extraordinary spurt in terms of interest and new knowledge about aneuploidy,” says Hassold, who has been in the field for more than 25 years.

    Nowhere is the spurt more tantalizing than in studies of the molecular machinery that drives meiosis, the cell division process that leaves eggs and sperm each holding half the total number of chromosomes—and the main source of aneuploidy (see p. 2181). “Until very recently, we didn't know what to look for,” says Dorothy Warburton, a cytogeneticist at Columbia University in New York City and an authority on miscarriage. “As we begin to recognize the molecular players, there'll be obvious things to do.” This applies both to basic researchers who are attempting to understand how meiosis stumbles, as well as fertility specialists.

    Three's a crowd

    The first estimates of the frequency of aneuploidy came in a landmark 1975 study. French researchers Joëlle and André Boué, along with Philippe Lazar, analyzed the chromosomes in nearly 1500 first-trimester miscarriages. They found chromosomal abnormalities in a startling 61% of the samples. Of these, aneuploidies—usually three copies of the same chromosome or a single copy of the female sex chromosome—accounted for more than two-thirds of the abnormalities. Subsequent large studies showed slightly lower rates, leading to the now widely accepted view that chromosomal abnormalities explain half of all miscarriages, with aneuploidies accounting for roughly 60% of the problem. (Other relatively common abnormalities include three or more complete sets of chromosomes, called polyploidy, and “translocations,” in which unmatched chromosomes combine with each other.)


    The older the oocyte, the more likely its chromosomes (red) will get stuck during meiosis, possibly due to faulty spindles (green)


    All but a small percentage of aneuploidies end in miscarriage. The only trisomies that aren't lethal—those involving chromosome 13, 18, or 21 (Down syndrome), or sex chromosome combo XXY, XYY, or XXX—make up the single largest genetic cause of mental retardation and developmental disabilities. Warburton points out that chromosomes 13, 18, and 21 carry the fewest genes; trisomies of larger chromosomes are apparently so disruptive that they always cause miscarriage. Only one monosomy, X0, isn't lethal; it causes physical but not cognitive problems.

    Aneuploidy occurs far more often in humans than in other species, says pathologist Kurt Benirschke, former director of the Center for Reproduction of Endangered Species at the San Diego Zoo. “But it's very rarely studied in other species,” cautions Benirschke, now a professor emeritus at the University of California, San Diego. Aneuploidy in mice, the most intensively investigated mammal other than humans, occurs in no more than 2% of fertilized eggs.

    In part because humans have such high rates of aneuploidy, miscarriage is staggeringly common. Allen Wilcox and colleagues at the National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, North Carolina, stunned many people in 1988 when they reported in The New England Journal of Medicine what is still one of the most-cited estimates of human miscarriage rates. They asked 221 women who were attempting to become pregnant to collect urine samples each day. The researchers assessed some 30,000 samples for human chorionic gonadotropin, a hormone that spikes when an embryo implants in the uterus. They found that 31% of embryos miscarry after implantation begins.

    The urine test does not account for other lost fertilizations that never make it to implantation. Many studies have attempted to peek through the window between conception and the week or so before implantation by assessing the chromosomes of embryos fertilized through IVF. Abnormalities run as high as 70%, with many embryos carrying a “mosaic” of both aneuploid and normal cells. Wilcox and others caution that IVF data do not necessarily reflect the general population: Most people seek IVF because they are having fertility problems; the embryos analyzed are often those left unused; and the technique itself might alter chromosomes. Still, says Wilcox, “IVF has added something to our complete ignorance” about the frequency of aneuploidy in the earliest stages of development.

    Scrambled eggs

    In the intensive hunt for the causes of aneuploidy, only one clear-cut risk factor has emerged: maternal age. But as fly geneticist R. Scott Hawley stresses, even that association is often misunderstood. “It's not how old the woman is,” says Hawley, who studies aneuploidy at the Stowers Institute for Medical Research in Kansas City, Missouri. “It's how close she is to menopause.”

    Females make all of their eggs, or oocytes, when they are fetuses, and they are born with between 1 million and 2 million. Women go through menopause not when they reach a specific age, but when they have about 1000 oocytes left, most of the others having died through “atresia,” a little-understood process of programmed cell death. The closer a woman is to menopause, the more frequently embryos formed from her eggs will have aneuploid chromosomes.

    Many theories have attempted to explain why aging human oocytes are prone to aneuploidy. They share one underlying perspective: It's linked to how the eggs are made. Oocytes in a developing female fetus begin with two copies of each chromosome. Meiosis—a two-stage process of cell division referred to in shorthand as MI and MII—reduces the 46 chromosomes to 23. But, for some reason, shortly after MI begins, it arrests, effectively throwing the oocytes into a deep freeze. These oocytes will not “thaw” and reenter meiosis until they vie to become the one egg that is typically ovulated each month during a woman's reproductive years.

    Split decisions.

    When meiosis works properly (left), pairs of homologous chromosomes divide evenly into four eggs. Errors in meiosis (right) lead to “aneuploid” eggs that have an extra or missing chromosome.


    Arrest is a generalized feature of female meiosis in a range of species, from mammals to mollusks, says cytogeneticist Patricia Hunt of Case Western, although the evolutionary reason is unclear. But human eggs, with our comparatively long life-spans, face extra perils, because they have to wait as long as 50 years to complete their journey through MI and MII. “It strikes me as dangerous to do,” says Hunt. As decades pass, oocytes have an increasingly difficult time separating their chromosomes into two even groups. Studies by Case Western's Hassold and others have shown that 90% of all chromosomal abnormalities have a maternal origin, and the vast majority of those trace back to an MI error in which chromosomes do not properly separate, or “disjoin.”

    There's more than one road to aneuploidy, but the field has long sizzled with debate about the mechanisms behind the most common pathways. In the once-popular “production line” theory, posited by University of Cambridge geneticists S. A. Henderson and Robert G. Edwards (the famed IVF pioneer) in 1968, the oocytes first produced by the fetus are more fit and are the first to be ovulated. As a woman approaches menopause, this theory holds, she has more and more “bad” eggs. The theory has since been disproven, but it helped lead to today's favored theory.

    When MI begins, the maternally and paternally inherited copies of each chromosome connect to each other at places called chiasmata. This allows homologous chromosomes to, in essence, combine in their own version of sex and swap similar pieces of DNA. Henderson and Edwards found that mouse oocytes had fewer chiasmata as they aged, and the chiasmata they did have were more often located near the ends of the chromosomes. As a result, these chromosomes exchanged less DNA, a process known as recombination. Reduced or absent recombination thus served as a surrogate marker for the underlying problem: Poorly placed or absent chiasmata somehow gummed up disjunction.

    Many experiments have verified Henderson and Edwards's suggestion that homologous chromosomes with few or unfortunately located chiasmata are prone to nondisjunction. But the team's other suggestion, which ties the maternal age effect to chiasmata becoming creakier over time, hasn't held up.

    In 1996, two groups came up with a new theory to explain the discrepancy. Fly geneticist Hawley, then at the University of California, Davis, Kara Koehler, and co-workers studied nondisjunction in the X chromosome of Drosophila melanogaster. Neil Lamb and Stephanie Sherman of Emory University in Atlanta, Georgia, teamed up with other researchers, including Hassold, to examine nondisjunction in humans with Down syndrome. Both teams arrived at what's now known as the “two-hit” theory.

    In hit number one, diminished recombination—caused by a lack of chiasma or their mislocation—creates “susceptible” chromosomes. “If you take a pair of chromosomes and it doesn't have an exchange, it is in bad shape,” explains Hassold. But here's the catch: Recombination occurs in the fetal oocyte before MI arrest sets in, so a 40-year-old woman has the same percentage of oocytes with susceptible chromosomes as she did when she was 20.

    What does change with time, however, is the ability of the molecular players in meiosis to carry out their assigned roles. This is hit number two. Younger eggs, in other words, have a strong, healthy cast of stagehands, allowing these oocytes to disjoin even funky chromosomes. But as eggs age, parts of the cast—the spindle, for example, which helps pull chromosomes apart —begin to stumble, making nondisjunction of susceptible chromosomes increasingly common.

    Divided minds

    The two-hit model has won over many researchers, but some still have their doubts. Columbia's Warburton says she thinks the model is “a pretty good attempt” to explain how aberrant chiasmata and the maternal age effect fit together. “But it doesn't seem to fit all chromosomes,” she cautions. Chromosome 18 in particular challenges the two-hit hypothesis. Many researchers have shown that trisomy 18 most often originates in MII, and in those cases, there's no evidence of reduced recombination in MI, indicating that a different mechanism must be responsible.

    A model proposed by Roslyn Angell, who recently retired from the University of Edinburgh, U.K., purports to accommodate the findings in all chromosomes. Angell examined more than 200 oocytes rejected for IVF—clinicians routinely remove more eggs than needed and then select those they think are of the highest quality—and discovered a unique mechanism for aneuploidy.

    Chromosomes are typically depicted with an X-shape because they consist of two sister “chromatids” that are Siamese twins of a sort. After fertilization, MII normally severs this bond to form single chromatids, with one set turning into the female's contribution to the embryo and the other being cast off as trash. In Angell's studies, aneuploidy—without exception—resulted from sister chromatids prematurely separating in MI rather than MII, which ultimately can leave an egg shortchanged or overstocked with that chromosome. This precocious separation, Angell found, also occurs more frequently in older women, which leads her to suggest that the bonds between sisters become weaker over time. In her studies, this is particularly true for chromosome 16, the most common trisomy in humans.

    One too many.

    People with Edward's syndrome, or trisomy 18, are born with distinctively clenched fingers.


    Although some researchers questioned whether Angell's results were an IVF artifact, Case Western's Hunt and colleagues found evidence that premature separation occurs in a more natural setting, too. They studied 117 oocytes obtained during tubal ligations and routine gynecological surgeries and confirmed that premature separation occurs. But they doubt Angell's assertion that it explains all nondisjunction, because they also found whole chromosomes—not just chromatids—that did not properly separate.

    A few theories about why humans suffer such high rates of aneuploidy build on much simpler notions of biology. One suggests that the longer the interval between sperm meeting ovulated egg, the more likely are abnormal embryos. Experiments have shown this is clearly the case in frogs, rodents, and other species, and it has led some to suggest that humans are so prone to aneuploidy because we do not have a mechanism, such as estrus, that optimizes the timing of fertilization.

    There's some evidence that mistimed meetings lead to aneuploidy in humans. In the NIEHS pregnancy study, the researchers found that women who became pregnant from intercourse that had occurred in the 5 days before they ovulated—and thus had viable sperm immediately ready to greet the egg—had significantly fewer miscarriages. A paper in the 11 May issue of The Lancet, however, finds no such association in a study of nearly 1000 women using natural family planning.

    Columbia's Warburton has long sought evidence to support what she calls the “limited oocyte pool” hypothesis. Unlike the production-line idea, this scenario contends that it's not a question of the first eggs made in the fetus being the healthiest and the first ones to ovulate. Rather, it holds that aneuploidy occurs more frequently as women age because, as their stock of oocytes becomes depleted with time, fewer oocytes compete to become the one that is ovulated. This leads to more substandard oocytes making their way into the fallopian tubes. Warburton herself has reservations about the hypothesis. “As I've thought about it more, it's [become] unclear to me whether the pool is the cause of the problem or a sign of the problem,” she says.

    Spindle doctors?

    Case Western's Hassold, who used to do genetic counseling with couples, says he'd love to figure out some way to help clean up messes made by meiosis. “My Holy Grail is to deliver some sort of aneuploidy-reduction pill,” says Hassold. “The difficulty is there are a number of routes to aneuploidy.”

    Molecular studies might reveal the best places to intervene. The spindle, for example, appeared to deteriorate with age in both Hunt's study of oocytes and another that looked directly at non-IVF eggs. Using mutant mice, Hunt and colleagues report in the May issue of Human Reproduction that the spindle itself often appears abnormal when nondisjunction occurs. They suggest that aberrant hormone levels, often found in women as they approach menopause, might disturb the final maturation of oocytes, which in turn could affect the production of motor proteins that move chromosomes along the spindle. Recent studies have identified a bevy of other proteins that control many steps of meiosis, from the pairing of homologous chromosomes to the repair of mismatched DNA during recombination to the “cohesins” that glue sister chromatids together. Theoretically, treatments directed at one of these proteins might tune up meiotic engines and prevent them from stalling as frequently as they do.

    A few aneuploidy interventions have already entered the clinic, although the results to date remain sketchy. One is called ooplasmic transfer, which builds on work that fingers defective mitochondria in the cytoplasm of eggs (the ooplasm) as a key suspect in aneuploidy. The theory suggests that as women age, these mitochondria become compromised and cannot produce as much adenosine triphosphate; that deficit prevents proper disjunction. To steer around this potential problem, Jason Barritt, Jacques Cohen, and co-workers at the Institute for Reproductive Medicine and Science of Saint Barnabas in Livingston, New Jersey, pioneered a technique to transfer ooplasm from a young woman's egg into the egg of an older woman.

    In July 2001, the U.S. Food and Drug Administration decided that this procedure was a form of gene therapy and temporarily halted its use while reviewing the technique for approval. More than two dozen babies have been born as a result of this controversial procedure, which has been strongly criticized because it transfers the donor's mitochondrial DNA to the baby, creating a child with three genetic parents.

    A less controversial but still unproven method of preventing aneuploidy that has made great headway in the past few years is preimplantation genetic diagnosis (PGD). In this IVF technique, embryos are screened for chromosomal abnormalities before they are implanted. The idea is that by reducing the number of aneuploid embryos, the IVF success rate should increase.

    But geneticist Wendy Robinson, who studies aneuploidy at the University of British Columbia in Vancouver, Canada, cautions that PGD has several limitations. Given that IVF embryos often contain mosaic combinations of normal and aneuploid cells, PGD, which samples only one embryonic cell and typically only looks at a handful of chromosomes, might miss a lot of abnormalities, says Robinson. On the flip side, she notes, PGD might detect a mosaic cell that was destined to become the placenta, not the fetus. “I'd worry that you may be throwing away perfectly good embryos,” she says.

    Then again, Robinson, who works closely with a clinic in Vancouver that specializes in treating miscarriages, strongly supports the push by researchers to try to intervene. “When I was younger, I may have said ‘It's nature's way,’” says Robinson. But, for career reasons, she did not have a child until she was 36. “It really has changed my views.” And Robinson is confident that researchers will find ways to prevent many aneuploidies. “Eventually,” she predicts, “we'll have more women having babies at age 40 than now.”

  17. Quirks of Fetal Environment Felt Decades Later

    1. Jennifer Couzin

    Normal babies who might have encountered adversity in the womb grow up to have a higher incidence of chronic diseases. Researchers are starting to figure out why

    A fertilized egg has hoop after hoop to jump through during its upcoming 9 months of gestation. “We pass more biological milestones before we are born than we'll ever pass at any other point in our lives,” says Peter Nathanielsz, director of the laboratory for pregnancy and newborn research at Cornell University in Ithaca, New York. Scientists have long believed that these milestones are molded by the environment an embryo, and later a fetus, encounters in the womb. Without enough folate, a fetus can't build and seal a backbone, for instance; a deluge of alcohol can interfere with delicate nervous system wiring; and certain medications taken by the mother might inhibit limb growth in the fetus she carries.

    But some effects of gestational molding might lay dormant for decades before rearing their heads. A series of epidemiological studies began pointing to this conclusion in the late 1980s. Unexpectedly, adults who had been born with normal but low birth weights were found to have a higher risk of a constellation of adult diseases, including heart disease and type II diabetes. It was a startling idea. For years researchers have known that low birth weight, often associated with prematurity, is linked to a range of problems at birth, such as respiratory infections. But the new studies argued that low but normal birth weight—even in full-term, healthy babies—is a marker for susceptibility to diseases that show up 50 years later. And the list of adult ailments in which low birth weight has been implicated continues to grow: A May report in The Journal of the American Medical Association linked a woman's birth weight to her risk of developing gestational diabetes.

    At first, researchers were skeptical. “There has been a kind of seeing what you want to see” in the epidemiological data, says Nigel Paneth, a pediatrician and perinatal epidemiologist at Michigan State University, East Lansing. Still, the theory that fetal environment influences adult disease susceptibility has been gaining legitimacy, particularly as it moves into the molecular biology arena. Indeed, some of the early skeptics are now pressing hard in animal studies to find out what, precisely, is behind the link between low birth weight and susceptibility to adult diseases.

    At best, birth weight is a crude measure of fetal growth, and most believe that differences in birth weight, which rarely span more than 2 kilograms in full-term infants, are simply a rough marker for conditions encountered in the womb. A growing number of researchers are examining variables such as maternal stress, placental development, and embryo implantation that might underlie the perplexing find. Poorly formed kidneys, for example, might falter at regulating blood pressure. But for the most part, the mechanisms by which the fetal experience contributes to adult disease remain enigmatic.

    Few had even considered such a delayed effect when British epidemiologist David Barker of the University of Southampton gave biologists a jolt in the late 1980s. After poring over birth and death records of thousands of people born in Hertfordshire, U.K., during the first third of the 20th century, he determined that babies weighing less at birth were likelier to die of heart disease. His statistical analysis of birth and death certificates revealed that 2.3-kg babies had double the rate of death from coronary heart disease of 4.5-kg babies. He later added hypertension, stroke, and type II diabetes to the list. Barker hypothesized that maternal malnutrition was largely responsible.

    “I was skeptical,” recalls Janet Rich-Edwards, an epidemiologist at Harvard Medical School in Boston, about her first encounters with the so-called Barker hypothesis. She wondered about all the data Barker's analysis couldn't capture because his subjects weren't available for interviews: socioeconomic status, family history, smoking habits. But Rich-Edwards had her own extraordinary, living cohort on her hands: 121,700 nurses who make up the Nurses' Health Study, a group that continues to supply scientists with valuable medical data. In 1992, the team added a question about birth weight to the routine questionnaire the volunteers filled out.

    “I was sure I was going to be able to adjust away this association between birth weight and later disease” by taking into account other factors, says Rich-Edwards. “In fact I couldn't. … My own data convinced me.” Her group found that a woman who weighed between 2.3 and 2.5 kg at birth—low, but within the normal range—had a 23% higher risk of heart disease and an 80% higher risk of type II diabetes than someone who weighed between 3.2 and 3.9 kg. These epidemiologists have also found that babies weighing about 2.5 kg at birth had about half the chance of developing breast cancer as adults, compared with those in the 4-kg range— suggesting that there's no straightforward relationship between low birth weight and susceptibility to disease.

    The risks of maternal malnourishment were reinforced by a study of Dutch adults conceived or born during late 1944 and early 1945, when the Nazi army halted food transport to occupied areas of the Netherlands. Food consumption plunged from 1800 calories a day to as little as 400. Tessa Roseboom, now an epidemiologist at the University of Amsterdam, and her colleagues found that babies conceived while their mothers were severely malnourished tended to share the same problems in adulthood as the people in Barker's study. Many of her volunteers didn't have low birth weights, however, suggesting that a troubled environment before birth might leave no easily measurable trace behind.

    Taking shape.

    Conditions in the womb appear to have an impact on risk of disease in adulthood.


    Food and stress

    To uncover the molecular actions that can go awry early on and prompt disease decades later, researchers are reaching beyond the risk factors first identified by the fetal origins field, such as the baby's weight or length. Simon Langley-Evans, a nutritionist at the University of Nottingham, U.K., experimented with feeding pregnant rats a low-protein diet. Normally their diet would be upped to 12% protein during pregnancy, but Langley-Evans gave them 9%—what they would consume if they weren't pregnant. As seen in previous studies, the animals' offspring grew up to have high blood pressure.

    But Langley-Evans had a hunch that low protein wasn't directly responsible for the hypertension. Rather, he hypothesized, it was blocking a critical enzyme in the placenta that keeps stress hormones in the mother's blood from reaching the fetus. To test this, he gave pregnant rats a drug that stopped them from producing stress hormones. The offspring of these rats, fed low-protein diets, then had normal blood pressure—suggesting that the hormones, not the diet, were behind the hypertension. Furthermore, when pregnant rats on high-protein diets were given a drug that inhibited the placental enzyme, their offspring had high blood pressure, just as if their mothers had ingested too little protein. Says Langley-Evans: “My experience is any manipulation of the diet has pretty much the same effect”-inhibiting the enzyme, which goes by the unwieldy name of 11β-hydroxysteroid dehydrogenase (type 2), and allowing more stress hormones into the fetus's bloodstream.

    Even well-fed pregnant animals can bathe a fetus with excess stress hormones if they are under significant stress. Marelyn Wintour, a fetal physiologist at the University of Melbourne, Australia, found that injecting the stress hormone cortisol into pregnant sheep gives their offspring high blood pressure for years. She presented her research at the Federation of American Societies for Experimental Biology conference in April.

    Endocrinologist Jonathan Seckl of Western General Hospital in Edinburgh, U.K., believes that excess levels of stress hormones in the fetus “reset” a major mediator of stress in the body, the hypothalamic-pituitary-adrenal (HPA) axis, making it hypersensitive to even banal events. In other words, the body churns out glucose, cortisol, and other stress-related substances when they wouldn't normally be needed—a result now observed in rats, mice, sheep, and low-birth-weight humans. Elevated glucose levels could help explain the high rates of type II diabetes found in those whose mothers were under stress before birth, such researchers claim.

    Stress hormones like cortisol are also known to be powerful regulators of gene expression. Researchers including Harvard pediatric nephrologist Julie Ingelfinger say these hormones might well turn on and off genes critical to the fetal development of organs such as the kidney, further contributing to later disease.

    Why don't these effects show up until well after birth? Varying theories seek to answer that question. Cornell's Nathanielsz suggests that the animals are born fighting against, say, high blood pressure, and at some point their defense mechanisms collapse. Langley-Evans says it's possible that the animals are born susceptible to certain diseases, and other factors that contribute to them, such as poor diet, can push those more at risk over the edge.


    Pigs lacking adequate protein during gestation are born with fewer nephrons (black dots).


    From the outside in

    One puzzle researchers face is that low-birth-weight babies aren't all born to anxious mothers subsisting on bread and water. Many perfectly healthy women give birth to infants whose growth is somehow retarded in the womb, a reminder that it's not only the mother's condition that matters, but what reaches her fetus through the placenta.

    Remarkably little is known about placental development and defects. In humans, a fertilized egg has about a week after fertilization to implant itself in the walls of the uterus. Implantation reshapes uterine arteries so they can better deliver nutrients and oxygen. As the fetus grows, the placenta becomes larger and thinner—a process called remodeling—to sustain its charge.

    Although drastic defects in the placenta often lead to miscarriage, more subtle problems seem to cramp development of fetal organs, particularly the kidneys and heart. “If the placenta is poorly formed and does not remodel properly, this puts cardiovascular stress on the fetus,” says Kent Thornburg, director of the heart research center at Oregon Health & Science University (OHSU) in Portland. Thornburg's group is currently testing a hypothesis that improper placental remodeling in the latter half of pregnancy contributes to heart disease in adult sheep.


    Stressed-out ewes give birth to lambs with high blood pressure.


    In another OHSU lab, nephrologist Susan Bagby is using pigs to explore how stunted kidney development might, along with an overactive HPA axis, contribute to high blood pressure later on. Because kidneys control the body's balance of salt and water, they are a crucial moderator of blood pressure. Furthermore, the functional units of the kidney, called nephrons, all form before birth in humans. “The number of nephrons that you have is very important in determining the capacity of the kidney to deal with the world,” says Bagby. Langley-Evans has found that low-protein diets during pregnancy cut the number of nephrons in rat offspring by a third. Researchers are only beginning to consider whether some kidney failure in adult humans might have its roots in the womb.

    Even more mysterious than placental development is what comes before it: implantation. Richard Schultz, a developmental and reproductive biologist at the University of Pennsylvania in Philadelphia, has found that variations in artificial embryo culture can dramatically alter gene expression.

    The true start, of course, comes not with implantation but with the genes endowed during the combination of egg and sperm. Fetal origins research, which began with both feet firmly planted in the environmental camp, has struggled with how big a role genes might play. Birth size, for example, is probably partly genetic. Researchers suspect that the kind of “smallness” that puts babies at risk of later disease is not genetic. At-risk babies, the theory goes, were destined to be larger but suffered retarded growth in the womb. They can be distinguished from genetically small infants, many in the field say, by their proportionally large heads. A popular theory holds that some form of stress before birth prompted these fetuses to restrict their body's growth in order to protect their brain.

    Ultimately, most agree that genetic and environmental influences are braided together. There might be genetic variations in the fetus or mother that, say, prevent the placenta from properly remodeling or make it more likely to admit excess cortisol.

    Sculpting destiny

    If the 9 months spent in the womb help shape susceptibility to disease, what, if anything, can be done to reverse or even prevent ill effects? Given how little is known about critical periods in pregnancy when a given stressor might produce a given defect, the likelihood of preventing problems before they start remains remote.

    Reversing these problems could prove slightly more feasible. One possibility might be halting a common phenomenon among babies born smaller than intended, called catch-up growth. These infants born at, say, the 20th percentile for growth hit the 80th percentile by the time they're school age. Some theorize that this occurs because a baby conditioned in the womb to anticipate fewer nutrients gains more from each gram of food it consumes. Studies in humans and animals suggest that catch-up growth makes adult diseases associated with low birth weight much likelier. Bagby is experimenting in her pigs to see whether inhibiting catch-up growth—which, in humans, might be easier said than done—will preserve kidney function and normal blood pressure in adults.

    In another approach, treating newborn rats between 2 and 4 weeks after birth with drugs to counteract high blood pressure, and then withdrawing the treatment, has also been shown to permanently reverse the effects of low protein prior to birth, says Langley-Evans.

    Still, researchers say they're a long way from addressing the implications of troubled fetal environments in the clinic, especially because low birth weight remains the only simple measure of susceptibility to later disease. Rebecca Simmons, a neonatologist at the University of Pennsylvania and Children's Hospital of Philadelphia, routinely treats low-birth-weight babies. But at a loss to quantify risks, she rarely volunteers information on the likelihood of later disease with parents. “The reason we're not talking about it with the parents now,” she says, “is that we don't know what to do.”

  18. Cells Exchanged During Pregnancy Live On

    1. Marcia Barinaga

    Microchimerism, viewed at first as an oddity, has been linked to autoimmune diseases and complications of pregnancy

    A mother's love is enduring. But most mothers would be surprised to discover that there's a similarly enduring physical bond: Cells from a fetus can live on in the mother's body for decades after pregnancy, a situation called microchimerism. Likewise, a mother's cells can also survive for many years in her child.

    When this phenomenon was first reported in the mid-1990s, scientists scoffed at the notion that these cells could persist for so long, tolerated by their host's immune system. “Everyone said it can't be true,” says rheumatologist Michael Lockshin, director of the Barbara Volcker Center for Women and Rheumatic Disease at the Hospital for Special Surgery in New York City. “But now everyone who looks finds it.”

    In some cases, the cells might be benign guests: self-perpetuating lines of stem cells that can reproduce and even give rise to other types of cells, all without harming their host. But a growing body of research, still preliminary, suggests that the cells might also be at the root of some autoimmune diseases and other conditions.

    Indeed, microchimerism might help explain one of the puzzles about autoimmune diseases: why many of them strike more women than men. No one knows how many women carry foreign cells around from past pregnancies, but several studies have shown that women with certain autoimmune diseases are more likely to harbor such cells than healthy women. “When you see that this is a real phenomenon, it gives you a different perspective,” says pediatric hematologist William Reed of the Children's Hospital Research Institute in Oakland, California. “You begin to ask yourself whether a disease might have a pathogenesis that you've never considered before.”

    And it's not only the long-lived cells that might be making mischief. Reproductive biologists have known for some time that fetal cells course through the bloodstream of pregnant women, but in the past 4 years researchers have discovered that this temporary invasion might be implicated in two common complications of pregnancy.

    Inner turmoil

    Fetal microchimerism was uncovered quite by chance. In 1992, medical geneticist Diana Bianchi, then at Children's Hospital in Boston, was trying to develop a method for prenatal diagnosis that relied on isolating fetal cells from the blood of pregnant women. Her team was separating out cells that carried a protein known as CD34—a marker for so-called hematopoietic stem cells that give rise to cells of the immune system—based on a hunch that CD34 would be a good marker for fetal cells.

    Blood from 13 of the pregnant women they studied contained CD34-positive cells with a Y chromosome, indicating that the fetuses from which the cells came were male. But amniocentesis showed that only nine of those women were carrying male fetuses. “We were mystified,” says Bianchi, who is now at Tufts-New England Medical Center in Boston. They checked to see whether any of the four women with unexplained male cells had other children who were male, and two of them did. The other two had previously terminated pregnancies in which the sex of the fetus was not known. “That is when the hypothesis began to take shape,” says Bianchi.

    Under mom's skin.

    A cell with a green-stained Y chromosome, presumably from a son, was found in a skin biopsy from a woman with systematic sclerosis.


    To test the idea that fetal cells from a past pregnancy can linger, Bianchi and her colleagues examined the blood of mothers who were not pregnant. They chose eight mothers of boys, because testing for the presence of a Y chromosome could easily distinguish the sons' cells from their mothers'. Six of the women, including one whose youngest son was 27 years old, had male cells still circulating in their blood. The idea was so surprising that it met resistance. “I lost count of how many times this paper was rejected,” Bianchi told the audience at a meeting in April.* The study was finally published in the Proceedings of the National Academy of Sciences in January 1996.

    Meanwhile, on the other side of the country, Lee Nelson, an immunologist and rheumatologist at the Fred Hutchinson Cancer Research Center in Seattle, had formulated a theory that fetal cells lingering in the mother might be at the root of autoimmune disease. Nelson studies autoimmune diseases such as scleroderma, a debilitating condition characterized by inflammation of the skin, which is often called systemic sclerosis when it advances to involve internal organs. The symptoms resemble graft-versus-host disease (GVHD), a complication that sometimes arises in bone-marrow-transplant recipients, when white blood cells derived from the donated bone marrow attack the recipient's tissues. Because scleroderma, like many autoimmune diseases, is more common in women than in men and often arises after a woman's childbearing years, Nelson wondered whether it might be caused by an immune reaction set off by pregnancy.

    In 1994, before seeing Bianchi's work, Nelson heard from a colleague that researchers at CellPro, a Seattle biotech company, had found fetal cells in a woman years after pregnancy. Although that work was never published, Nelson contacted Jeff Hall, the CellPro scientist who had made the observation, and Hall told her about Bianchi's work. Nelson wrote a paper outlining her hypothesis that fetal cells might trigger autoimmune disease, which was published in Arthritis & Rheumatism in February 1996.

    Nelson contacted Bianchi, and the two began collaborating to search for fetal cells in the blood of female scleroderma patients. They tested 17 patients and 23 healthy women, all of whom had given birth to at least one son. They found male (presumably fetal) cells in most of the patients and in some of the healthy controls. But overall, the scleroderma patients had 30 times as many fetal cells in their blood as the healthy women did, an average of seven male cells per 10 milliliters of blood. The foreign cells they identified were cells from the fetal immune system, including antibody-producing B cells and T cells—the type of cell that is responsible for the cellular immune response that contributes to scleroderma—as well as natural killer cells and monocytes. In short, circulating in the women's blood were cells that were tuned to protect the fetus against foreign invaders and that could conceivably recognize the women's own tissues as foreign and attack them.

    The researchers also found evidence that, just as in GVHD, the compatibility between the foreign cells and their host seems to play a role. The histocompatibility, or HLA, genes encode proteins that help the immune system identify and kill cells that have foreign proteins on their surface, such as virus-infected cells. Each HLA gene comes in up to 100 different forms, and immune cells will also kill cells that have forms of the HLA proteins that are different from their own. This is why HLA genes must be carefully matched between bone marrow donors and recipients.

    Each person has two copies of each HLA gene, one from each parent, so a mother has one copy in common with her child. The child's other copy, which comes from the father, is usually different. In women with scleroderma, however, Nelson found that for one of the HLA genes, known as DRB1, both copies of the gene carried by the fetal cells matched their mother's genes. The fetus either had the same two genes that the mother did, or the fetus had two identical copies that matched one of the mother's genes. In either case, the mother's immune system would not recognize that fetal HLA gene as foreign.

    But the so-called HLA compatibility for the DRB1 gene alone can't explain why the mother's immune system doesn't kill the fetal cells, Nelson says. The other HLA genes are unlikely to be matched between fetus and mother, and those mismatches should cause the mother's immune system to destroy the fetal cells. “Why these cells persist in the face of all these mismatched HLAs is a very interesting biological question,” she says.

    The fetal cells do persist, though, and their HLA compatibility with the mother for the DRB1 gene is associated with an increased risk of scleroderma. Nelson's team calculated that a woman who has given birth to a child whose DRB1 genes each match one or both of hers has a ninefold greater risk than average of developing scleroderma.

    If these fetal cells are somehow mounting an attack on the mother's tissues, one would expect to find fetal cells at the site of inflammation. In 1998, Carol Artlett, Sergio Jimenez, and their colleagues at Thomas Jefferson University Hospital in Philadelphia found this to be the case: They identified male cells in the skin lesions of 11 out of 19 women with scleroderma.

    Meanwhile, other researchers looked for fetal cells in patients with other autoimmune diseases. Two groups, Bianchi's at Tufts and that of Michael Klintschar at Martin Luther University in Halle-Wittenberg, Germany, found increased numbers of fetal cells associated with Hashimoto's thyroiditis, an autoimmune disease that reduces the patient's production of thyroid hormone and strikes more women than men.


    A cell with a green-stained Y chromosome in a mother's liver biopsy suggests that fetal cells endure for decades.


    With child, with cells

    Although researchers were surprised to find fetal cells persisting in the mother for years after birth, it has long been known that fetal cells enter the mother's blood during pregnancy. Now researchers suspect that these cells are at work in at least two diseases of pregnancy—one an autoimmune disease, the other probably not.

    Dermatologist Selim Aractingi of Tenon Hospital in Paris studies skin conditions in pregnant women, including polymorphic eruption of pregnancy (PEP), which resembles a bad case of hives. In 1998, Aractingi and his colleagues reported finding male cells in the skin lesions of five out of 10 women with PEP who were carrying male fetuses. The presence of the male cells seemed specifically associated with PEP, because the researchers found no male cells in comparable skin samples routinely removed during caesarian sections from 13 women who were delivering boys but did not have the disease.

    That same year, Wolfgang Holzgreve, an obstetrician and geneticist at the University of Basel, Switzerland, was working—as Bianchi had been in Boston—on developing a way to isolate fetal cells from maternal blood for prenatal diagnosis. In the course of that study, his team made an unexpected discovery: Women with a serious complication of pregnancy called preeclampsia had manyfold more circulating fetal cells than healthy pregnant women had. “In normal pregnancy, the level of fetal cells is about one in 1 million cells in maternal circulation,” Holzgreve says. “In preeclampsia, it could be one in 1000 or more.”

    Preeclampsia, which causes dangerously high blood pressure, impaired kidney function, and edema, usually occurs in the third trimester of pregnancy and often forces an immediate delivery of the child to save the mother's life. The health and survival of babies born this way would be improved if physicians could prepare them in advance for a premature birth—for example, by administering treatments to accelerate lung maturation. Holzgreve wondered if the high numbers of fetal cells could be used as a predictor of preeclampsia. Together with his co-worker Sinuhe Hahn, he chose a group of women who, because of placental abnormalities detected by ultrasound, were thought to be at increased risk of preeclampsia. At the 20th week of pregnancy, the researchers drew blood from the women and analyzed it for fetal DNA. “There was a strong correlation between the level of fetal DNA and the likelihood of developing preeclampsia,” Holzgreve says.

    Holzgreve's team also found a parallel between the amount of fetal DNA in the mother's blood and the severity of the disease. Researchers have long suspected that in preeclampsia, some toxin in the blood damages endothelial cells lining organs such as the kidneys. Now Holzgreve suspects that the fetal cells or free fetal DNA are that toxin. Preliminary studies with endothelial cell cultures suggest that fetal cells and DNA are toxic to endothelial tissue, he says: “It could be a very direct effect of the [fetal] material on the maternal tissue.”

    Inheritance tax.

    A boy with juvenile dermatomyositis hosts a blue-stained white blood cell (right) with two X chromosomes (pink) and no Y chromosomes (green).


    Two-way traffic

    Mothers might bear the brunt of this newly discovered cellular invasion, but it turns out that men and women who have not borne children are not exempt. Cells of close relations can colonize their kin in two other ways: Twins share cells in the womb and can harbor these cells into adulthood; and a mother's cells can stick around in her child's bloodstream for years.

    Maternal cells have been linked to at least one autoimmune disease. Two research teams, one led by Artlett of Thomas Jefferson University and the other by pediatric rheumatologist Ann Reed at the Mayo Clinic in Rochester, Minnesota, independently found maternal cells in the inflamed muscle tissue of children and young adults with autoimmune conditions that attack the muscles. Reed's group studied children with juvenile dermatomyositis, and Artlett's group looked at boys with either dermatomyositis or a related condition called polymyositis. The researchers found maternal cells in the bloodstream and tissues of nearly all of the boys they examined who had the autoimmune conditions, compared with 20% or fewer of those who did not. And when maternal cells were present in healthy boys, they were generally in lower abundance.

    Like Nelson's group, Reed's team also found a striking association between disease and certain HLA relations. In this case, it wasn't a simple matter of the son's and mother's HLA genes matching. Rather, 85% to 90% of the patients had a particular version of an HLA gene known as DQ. The gene doesn't always cause trouble, but it might somehow enable maternal cells to persist in children. What's more, Reed's team found that the children without dermatomyositis who had maternal cells in their blood carried the same form of the DQ gene as did the boys with the disease. “The gene seemed to influence somehow the persistence of these cells in the children,” says Reed.

    That might be the key to explaining another nagging puzzle in autoimmunity research, Reed suggests. For some reason, people with certain HLA types are more likely to suffer from some autoimmune diseases. “Maybe it is not the HLA gene per se causing autoimmunity,” she posits. “Maybe it is allowing chimerism to occur and then that is triggering the disease.” Her current hypothesis for dermatomyositis, she says, is that there is a mutual tolerance between the patient and his mother's cells, perhaps enabled by the patient's DQ gene. Then some second event causes the tolerance on the part of the mother's cells to break down, and the mother's cells attack the patient's tissues.

    Both teams found evidence that stem cells from the mother have set up housekeeping in the patients' bone marrow—a sort of mini-bone marrow transplant. Maternal cells in the patients include T cells and B cells, which could conceivably survive for years, but the researchers also found maternal-derived neutrophils. Neutrophils generally live for only a day, says Reed, so they must have been produced by resident stem cells.

    Blood brothers

    Although it is natural for the blood of mothers and their children to mix during pregnancy, blood transfusions mix the blood of total strangers. Microchimerism has popped up in some transfusion patients as well.

    After most blood transfusions, any white blood cells in the donor blood are rapidly cleared by the recipient's immune system, says transfusion medicine specialist Michael Busch of the University of California (UC), San Francisco. But in patients who have received massive transfusions of 10 to 30 units, it is common for donor cells to persist for years. What's more, says Busch, who made the finding with colleagues at BloodSource in Sacramento, California, and the UC Davis Medical Center, the degree of microchimerism is very high. “One [percent] to 3% of all circulating white cells in these patients are of donor origin,” says Busch. “It is not one in 100,000 or one in a million like in scleroderma.” And the cells that persist all come from just one of the many donors whose blood the patient received. The researchers don't know yet what causes microchimerism in these patients or whether it will lead to autoimmune or other disease.

    Indeed, those who study microchimerism in all its forms agree that the discoveries so far have raised far more questions than answers. For example, it is possible that much of the population harbors foreign cells without suffering ill effects. In others the cells might be triggered in some unknown way to do damage. Or the cells in those cases could be innocent bystanders, not culpable in the disease process. “Do we really know that these cells are involved in disease pathogenesis?” Nelson muses. “No.” William Reed of Children's Hospital in Oakland, who worked with Busch on the transfusion study, agrees. “This is really just at the observation stage,” he says, “with people standing around saying, ‘Gee isn't this neat, what does it mean?’”

    But that might soon change. The most popular model for how the chimeric cells might cause autoimmune disease is through a reaction akin to GVHD. Until recently, there had been no evidence that chimeric cells taken from their hosts react to the patient's tissues. But in February, a team at the University of Florence, Italy, reported in Arthritis & Rheumatism that T cells derived from male offspring in the blood and skin of women with scleroderma could be cultured in the lab and shown to react against the patient's tissue. “This is the most exciting paper that has come out in the last year,” says Thomas Jefferson's Artlett. “To me it says these cells are definitively involved.” Ann Reed's team at the Mayo Clinic has similar results; her group isolated persistent maternal cells from dermatomyositis patients and showed that they react against the patient's tissues.

    Even if foreign cells are shown to respond to the patients' tissues, however, most researchers agree that they can't be carrying out the entire attack themselves; there just aren't enough of them. “Scleroderma looks a lot like graft-versus-host disease,” says William Reed, “but the levels of cells you find in those patients is nothing like what you have in graft versus host. Something is missing.”

    “My premise is that they aren't doing the bulk of the destruction,” says Ann Reed; “I think they are the initiator.” Once the foreign cells have started an inflammatory reaction, she suggests, the patient's own immune cells are attracted to the scene, where they do much of the damage. Ann Reed is among those who believe that these intruding cells might play an important role in a number of autoimmune diseases. “People said, ‘Prove it. Prove they are there, prove that they mean something.’ We are slowly doing that. But it takes time.”

    Lest microchimerism get a particularly nasty reputation, Nelson points out that the majority of people who harbor foreign cells—whether from their children, twin, mother, or blood donor—are healthy. “This is likely to be a broad-based biological phenomenon,” she says. “And the best guess, since it is common, is that it may have beneficial roles, it may have neutral roles, and just in selected situations such as a particular lineup of HLA genes across generations, it can become bad.”

    In her talk at the recent meeting, Bianchi of Tufts mentioned two bizarre cases that suggest that microchimeric cells can build tissues as well as attack them. One subject in Bianchi's thyroid study was a 48-year-old mother who had a goiter removed. To her surprise upon examining the removed goiter tissue, Bianchi discovered that one whole section of the woman's thyroid was predominantly male, presumably from her son. Bianchi cites another case, of a woman with hepatitis C who had a liver biopsy. “Part of her liver was entirely male,” Bianchi says, “and it was surrounded by female tissue.”

    Bianchi suggests that, in cases like that of the woman with hepatitis C, circulating fetal stem cells might help repair damaged or diseased tissue. In some cases, rather than cause the disease, she suggests, “maybe the cells are responding to the disease. Wouldn't it be amazing if one of the benefits of being pregnant is that you get, as a reward, a second population of stem cells?” If so, that would be just one more way that mothers and children continue to take care of each other.

    • *The Society for Women's Health Research third annual conference on Sex and Gene Expression, San Jose, California, 4–7 April.

  19. Research on Contraception Still in the Doldrums

    1. Constance Holden

    A billion young people are heading toward their reproductive years, but few new birth control methods are on the horizon

    While scientists are beavering away at improving human reproduction, commensurate efforts are lacking on how to curb the process. By 2020, about 1.2 billion people, or 16% of the world's population, will be entering their childbearing years. “We are about to have the biggest proportion of young people the world has ever seen; reproductive health services are about to be inundated by a tidal wave of teenagers,” says population expert Felicia Stewart of the University of California, San Francisco (UCSF). “Frankly, I think we're not ready at all.” Some 90% of those entering reproductive age will be in the developing world, where there's a particularly pressing need for new forms of fertility control that are cheap, safe, reliable, convenient, reversible, and culturally acceptable.

    This should be “a major time for investment” in new forms of contraception, says Stewart, who was formerly in charge of population affairs at the Department of Health and Human Services. But contraception research, which had its heyday in the 1950s and 1960s, hasn't produced a major breakthrough since the introduction of the birth control pill. And there are still only two choices for men: condoms and vasectomy.

    Only a handful of companies are engaged in research on new contraceptive methods. One is Schering in Berlin; another is Organon in West Orange, New Jersey, which has just launched a hormone-releasing vaginal ring (NuvaRing), approved in November. But very few others are striving for new breakthroughs. Big pharmaceutical companies left the field in droves in the 1970s, says Carl Djerassi of Stanford University, the father of the birth control pill. Now, he says, “of the 20 largest pharmas in the world, only two have any commitment” to new contraceptives: Wyeth, and Ortho, a branch of Johnson & Johnson. “The only work most are doing is minor modifications” of existing products, he says.

    Few options.

    A Gambian health care worker discusses contraceptive devices, demand for which is expected to grow.


    Most companies have been driven away by the same forces at work 2 decades ago: liability worries, tough government regulations in the United States and other countries, and concerns about profitability, a big problem for products where the greatest demand is in poor countries. That leaves governments, international agencies, and private foundations to pick up the tab—with the U.S. government being the number one provider.

    Funding has been stagnant for decades. Tellingly, there are no up-to-date figures on global expenditures for contraceptive R&D, and no one has attempted a statistical roundup since the mid-1990s. As a result, there are no more current figures than those in a 1996 report by the Institute of Medicine,* which reported that, in terms of constant dollars, worldwide funding peaked in 1972. And a new report from Johns Hopkins University relates that donors would have to quadruple their efforts to fill the same proportion of contraceptive needs in 2015 as they do now.

    Nonetheless, a trickle of products continues to flow into the market, such as the vaginal ring and a new skin patch for women. And a couple of promising new approaches are coming down the pike, both carrying benefits beyond fertility control.


    The AIDS epidemic has spurred new initiatives to develop spermicides that also act as microbicides against sexually transmitted diseases. The U.S. National Institute of Child Health and Human Development (NICHD) is currently running a seven-center clinical trial on BufferGel, a vaginal microbicide that is used with a diaphragm. Although compounds that kill germs almost always attack sperm, too, “it's hard to find something that's both that doesn't irritate the vaginal lining,” says Diana Blithe of NICHD's Center for Population Studies, which is spending about $16.2 million a year on the project. Ironically, though, the nearby National Institute of Allergy and Infectious Diseases (NIAID) is spending far more—$44 million—on developing a microbicide that kills germs but does not disable sperm.

    Clinical trials on BufferGel show why companies shy away from such research. It costs several million dollars a year to test its efficacy as a contraceptive, says Blithe. And, she notes, separate tests of its efficacy as a microbicide—being conducted at NIAID—are particularly difficult. Researchers have to target a population at high risk for sexually transmitted diseases, and to satisfy ethics requirements, couples must be advised to use condoms in addition to the gel. Huge numbers of subjects are needed because the only useful data come from women whose partners failed to don condoms. Blithe says that although diaphragms don't enjoy great popularity anywhere, the hope is that women might reconsider the method if the gel gains a reputation as an effective dual-purpose product.

    Oh, babies.

    The newly fertile population is expected to jump in the next 20 years.


    Twofer for men

    Spearheaded by the Population Council in New York City, work is also picking up on a hormone-based male contraceptive. One obstacle to the decades-old vision of a long-acting male contraceptive, says James Catterall, director of research in reproductive physiology at the Population Council, is that some kinds of hormonal intervention can stimulate prostate growth and potentially contribute to cancer.

    But sugar-coating this pill might make a difference. Scientists at the Population Council and at Schering in Berlin, the company that has licensed the product, are working on a patch or implant that can suppress sperm production while at the same time supplying desirable hormone supplements. It would shut down testosterone—and therefore sperm—production by stimulating the immune system to create antibodies to a hormone called GnRH.

    Supplementary hormone is needed to replace the missing testosterone. So the key to the product is a potent synthetic androgen, called MENT, developed by the Population Council, that does not stimulate the prostate but does keep muscle mass and libido at desirable levels. In addition to providing contraception, says Catterall, the therapy could furnish androgen supplements for aging men. Such a product might override some males' resistance to contraception and feed into the growing interest in male hormone replacement therapy—an “explosive area,” according to Robert Spirtas, chief of contraceptive research at NICHD.

    Unrealized promise

    But contraceptives for the 21st century are yet to materialize. Stanford's Djerassi says there are a number of technologies—such as a reversible vasectomy or a contraceptive vaccine—that have been scientifically obtainable for more than a decade but that are at the moment dead in the water. Hopes for vaccines, for example, have been pretty thoroughly dampened due to worries about reversibility and side effects. And the fruits of recent advances in biology “haven't even yet begun to be addressed in contraceptive research,” says UCSF's Stewart. Few researchers are working on interventions that can block a specific phase of sperm or egg development, for instance, without affecting the whole body as do hormone-based contraceptives.

    Donald Patrick McDonnell, a molecular pharmacologist at Duke University Medical Center in Durham, North Carolina, says that “probably one of the major advances in recent years” has been the development of selective estrogen receptor modulators (SERMs), such as tamoxifen. They have contributed to advances in hormone replacement therapy—where market demand is strong—but, he says, researchers have been slow to explore them for contraception. But SERM-like compounds, says McDonnell, could contribute to a generation of contraceptives that are both highly specific—acting either just on the hypothalamic-pituitary-adrenal axis or the uterus—and dual-purpose as well, providing hormone replacements that help prevent cancer or osteoporosis.

    But Djerassi, for one, is not optimistic about a renaissance any time soon in contraceptive research. He says the Pill never could have been developed in the current climate. He did his pioneering work before the post-thalidomide tightening of drug regulations and before the arrival of the “litigation explosion,” whose beginning was marked by a suit over an intrauterine device, the Dalkon Shield, which bankrupted A. H. Robins and cast a temporary pall over such devices.

    Clinical trials, always expensive, are now even more so. In a typical trial, according to Regine Sitruk-Ware of the Population Council, researchers must follow 10,000 cycles including 200 women for 2 years. That means starting with 1200 women, given the dropout rate. In Europe regulations require twice that number of cycles.

    Instead, companies prefer to tinker with existing products, such as pills with lower doses of hormones. Most companies are doing well with the Pill, so “if they develop something new, they're essentially competing with themselves,” explains Stewart. And something new that lasts a long time is far less profitable than a pill that must be taken daily. “It's hard to find a [corporate] partner interested in a 12-month ring”—one suitable for poor women who don't visit clinics often, says Sitruk-Ware.

    To reengage the private sector in contraceptive research, says Stewart, “we've got to change the rules of the game”—that is, offer financial incentives as well as measures to protect companies from liability. Allan Rosenfield, dean of Columbia University's School of Public Health, says rules to encourage public-private partnerships would help. If the government would offer a buffer against liability, as it has done with vaccines such as those for childhood diseases, “that would be a major step forward,” he says. But don't hold your breath, he adds: “There's no way this government would ever do that for contraceptives.”

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