# News this Week

Science  20 Oct 2000:
Vol. 290, Issue 5491, pp. 418
1. BIOETHICS

# Helsinki's New Clinical Rules: Fewer Placebos, More Disclosure

1. Martin Enserink

After 3 years of intense debate, medical researchers and ethicists have agreed on international standards that would dramatically tighten the rules for clinical research and put new limitations on the risks to which patients may be exposed. Meeting in Edinburgh, U.K., on 7 October, the general assembly of the World Medical Association (WMA) gave a clear victory to patient-advocacy groups seeking a change in the way experimentation on humans is conducted. The WMA voted to approve a revised version of the 1964 Declaration of Helsinki, the cornerstone of clinical research ethics, that reduces ambiguity in existing guidelines and could force changes in the design of future drug trials.

Already, critics are warning that the newly proposed restrictions on the use of placebos, or dummy treatments, are at odds with common practice and clash with policies of the U.S. Food and Drug Administration (FDA). Indeed, researchers and drug companies may soon find themselves in an ethical conundrum: In order to test a drug for approval on the U.S. market, they would have to use research protocols that the declaration specifically rejects. Journal editors would be affected as well: Those wishing to abide by the Helsinki rules would have to turn away articles based on methods that are widely used today.

The controversy centers on the use of placebos to get quick and clear-cut results in clinical trials. According to the new declaration, placebos may be used only when there are no other therapies available for comparison with a test procedure. If there's an appropriate drug already on the market, then a trial should compare any new treatment to the existing product, the declaration says. That way, patients volunteering to participate in a trial wouldn't run the risk of getting a fake treatment and being worse off as a consequence of participating. “Our main objective is to protect our patients,” says WMA secretary-general Delon Human.

Patient groups are jubilant. “It's a major improvement,” says Peter Lurie of Public Citizen, a group in Washington, D.C., that lobbied heavily to influence the outcome of the revision process. But FDA officials Robert Temple and Susan Ellenberg, who pleaded in favor of placebo-controlled trials in a paper in last month's Annals of Internal Medicine, disagree strongly. The declaration's paragraph on placebos “doesn't make sense to me,” says Temple.

Placebo use has led to major controversies over the past few years—for instance, in trials in Africa and Asia to find out whether short-term use of an antiviral drug could prevent the transmission of HIV from a mother to her newborn baby. Public Citizen decried those trials, arguing that by using a placebo, the researchers allowed some babies to become infected, although they had the means to prevent it. But researchers argued that the trials had to be designed this way to get credible information. Besides, they said, their approach was ethical, because the mothers wouldn't have received the drugs if they hadn't been included in the trial (Science, 27 February 1998, p. 1299).

Researchers and activists have also faulted the use of placebo control groups in testing drugs for non-life-threatening ailments in the Western world, such as depression, chronic pain, and arthritis. Stopping treatment for the sake of a trial, they argue, causes unnecessary suffering and, in the case of mental illness, may cause patients to become violent to themselves or others. But Temple argues that comparing new drugs to a placebo is often the only way to establish efficacy, especially in treating diseases like depression, where success rates vary widely from trial to trial (Science, 21 April, p. 416).

The Declaration of Helsinki—prompted originally by the gruesome medical experiments of the Nazi era—contained a phrase suggesting that placebo use should be restricted, but the wording wasn't explicit. To end the ambiguity, expert groups, including the American Medical Association, pushed for a revision in 1997. Several early drafts drew strong criticism from opponents of placebo-controlled studies, like Lurie. They rejected one draft, for example, from a panel chaired by prominent Yale bioethicist Robert Levine. Levine's group proposed relaxing the Helsinki rules considerably in 1999 to allow the use of placebos if they don't cause death or disability. This version also implied that research subjects in the developing world should get the best care normally available to them. But Levine's attempt proved “totally unacceptable” to the medical community, says WMA's Human. “It was very useful,” says Human, “because it defined exactly what we didn't want.”

The final version, drawn up by a four-member committee that included Human, does exactly the opposite, stating that new drugs should be tested against the best current treatments—period. This standard would rule out the controversial placebo-controlled perinatal HIV trials, says Human. He also suggests that the use of placebo control groups in the developed world should end. “We're very pleased,” says Harvard University epidemiologist Karin Michels, who, along with her colleague Kenneth Rothman, has long argued against the widespread use of placebos in clinical trials. “We really see this as a big success.”

The FDA says it's studying the revised document and doesn't have an official reaction yet. But Temple, associate director for medical policy in the agency's Center for Drug Evaluation and Research, says it will be hard to live by the letter of the declaration and carry out trials that meet the FDA's demands for scientific rigor. “The answer is not clear,” he says. Human agrees: Adhering to FDA guidelines could mean violating the new Helsinki document. But the FDA isn't always right, Human suggests: “Bob Temple and the FDA are one voice. But we are a global organization, and this was a consultation from one side of the world to the other.”

The new document contains several other provisions aimed at strengthening the patient's position. For instance, it asks researchers to divulge to participants how the trial is funded and whether they have any conflicts of interest. Such disclosures are rarely required now.

Another surprising provision, some say, is that the new document asks that all study results, “negative as well as positive, should be published or publicly available.” “That is really wonderful news,” says Kay Dickersin, who directs the Center for Clinical Trials and Evidence-Based Healthcare at Brown University in Providence, Rhode Island. Currently, trials showing that a drug has no efficacy are often buried, says Dickersin. Researchers don't get around to writing them up, journal editors don't want to waste space on them, and pharmaceutical companies don't want to publicize their failures. The result of this so-called “publication bias” is often an unrealistically rosy picture of a drug's efficacy, which the new declaration may help prevent, says Dickersin.

It isn't clear that any of these new principles will be widely accepted, however, because the declaration doesn't have the power of law. “But you have to start somewhere,” says Human. “This is an ethical document. What we hope is that it will be adopted in many national regulations and legislation.”

2. ARCHAEOLOGY

# Paintings in Italian Cave May Be Oldest Yet

1. Michael Balter

Traces of what could be the world's oldest known cave paintings have been found in northern Italy. Stone slabs bearing images of an animal and a half-human, half-beast figure were uncovered during excavations by an Italian team at the Fumane Cave northwest of Verona. The slabs, painted with red ochre, had apparently fallen from the cave roof and become embedded in floor sediments previously dated to between 32,000 and 36,500 years ago. That would make the images at least as ancient as some found in the Grotte Chauvet in southern France—the current record holder at 32,000 years—and possibly even older (Science, 12 February 1999, p. 920). More important, cave art experts say, the new paintings bolster other evidence that humans engaged in sophisticated symbolic expression much earlier than once thought.

“This is an extremely exciting discovery,” says archaeologist Randall White of New York University. Cave art expert Michel Lorblanchet of the University of Toulouse in France agrees. “The Grotte Chauvet has shown that we already had a very elaborated art” by 32,000 years ago. With Fumane, Lorblanchet says, “we now have confirmation.”

Moreover, White and Lorblanchet say that there is little reason to doubt that the paintings are as old as the Italian team claims. “The [radiocarbon] dating is even better” than at Chauvet, Lorblanchet says. At Chauvet, very small samples of charcoal drawings were taken directly from the cave walls—a tricky technique that is prone to error. Although the inorganic red ochre paintings at Fumane cannot be dated by similar techniques, radiocarbon dates from plant and animal remains buried in the cave floor sediments where the art was found “are practically sure,” Lorblanchet says.

Fumane Cave, which has been under excavation since 1988, had already revealed rich evidence of occupation by early humans, including stone tools. The painted slabs were discovered last year but kept a closely guarded secret until this week. Paleontologist Alberto Broglio of the University of Ferrara, who co-directs the dig with geologist Mauro Cremaschi at the University of Milan, told Science that the paintings were covered with a thin layer of calcite that made them difficult to see. This summer an Italian art restorer removed much of the calcite. Although the team has not yet figured out what the images on three of the slabs represent, the other two appear to depict some sort of four-legged beast and an 18-centimeter-tall human figure with the head of an animal—which Broglio says is similar to images often seen in more recent caves and called “sorcerers” by cave art experts.

Lorblanchet says that the finding of a sorcerer at Fumane “does not surprise me,” because a similar motif—a strange hybrid of rhinoceros and human—has also been found at Chauvet. Another spectacular example, a statuette of a human with a lion's head dated to at least 30,000 years ago, was uncovered in southern Germany in 1939. With the discovery at Fumane, White says, “we now have this image in three different places during this early time period.”

This concurrence has cave art experts really excited. “It is one thing to represent a horse, but another thing to represent something that is a figment of the collective imagination, something that doesn't exist in reality,” says White. “People had ideas about the world that were abstractions, which we can only describe as religious. We are looking at a widespread belief system that is very ancient.”

3. ECOLOGY

# Plant Invader May Use Chemical Weapons

1. Mari N. Jensen*
1. Mari N. Jensen is a science writer in Tuscon, Arizona.

In Montana and other parts of the northwestern United States, an imported purple-flowered plant called knapweed grows so thickly that it looks as if ranchers are cultivating it as a crop. Yet in the Caucasus foothills of the Republic of Georgia where knapweed is native, this plant is so uncommon that plant ecologist Ragan Callaway of the University of Montana in Missoula had to enlist the help of local botanists just to find any at all.

Ecologists would love to know what explains the rollicking success of invaders such as knapweed, which in the United States is an aggressive, thistlelike weed that cows don't eat and ranchers and government agencies battle using herbicides. A traditional answer is that all the invaders' natural predators and pathogens have been left behind. On page 521, however, Callaway and colleague Erik Aschehoug offer a novel explanation for the success of invasive plants. By comparing how one species of knapweed, Centaurea diffusa, behaves with its natural neighbors and with foreign plant species that evolved separately, they found that the invader gains an edge in its adopted home not only by ditching its herbivores but also by wielding weaponry: chemicals exuded from its roots that hamper its new neighbors' growth.

The work is “important” because it could help predict which organisms will be successful invaders, says Kevin Rice, a plant population biologist at the University of California, Davis. “We haven't considered [such underground interactions] in the past.” The finding that some plants use subsurface chemical warfare on foreign soil also suggests, says Callaway, that a popular strategy of fighting exotics—siccing insects from back home on the plants—may be even less effective than experts think.

To explore knapweed's underground maneuvers, Callaway and Aschehoug, now a plant ecologist with The Nature Conservancy in Ventura, California, began by pitting C. diffusa, or diffuse knapweed, plants against either American or Eurasian grasses in pots. The researchers found that the American grasses produced 85% less leaf and root mass when they were planted with knapweed, whereas knapweed's growth was unaffected. A more complex picture emerged when Eurasian grasses were planted with knapweed: The grasses' biomass dropped by 50%, and the knapweed's growth declined as well.

Callaway and Aschehoug suspected that knapweed's roots exude organic chemicals that stopped the Montana plants from absorbing nutrients. To test that idea, they added to their knapweed-grass pairs some activated carbon, which sucks up organic molecules. They found, Callaway says, that the addition “changed the balance of competition. The American grasses did better, and the Eurasian grasses did worse.” Confirmation that knapweed interferes with the American grasses' ability to take up nutrients came when the team then injected the soil with a radioactive form of one potentially limiting nutrient—phosphorus—and tracked how much the various plant pairs snagged.

In the absence of the carbon, knapweed more than halved the amount of phosphorus captured by the American grasses; adding the carbon helped these grasses a bit. In contrast, the Eurasian grasses not only took up just as much phosphorus when grown with knapweed, but also appeared to pack weapons of their own: Adding carbon in the presence of knapweed drastically reduced the amount of phosphorus the Eurasian species took up, suggesting they had been pumping out chemicals that helped them compete with knapweed.

“The big deal is that Centaurea is interacting really, really differently with its long-term neighbors than with its new neighbors,” says Callaway. His next step will be teasing out whether knapweed's as-yet-unidentified root chemicals affect its competitors directly or have an indirect effect by changing how soil microorganisms interact with plants.

4. EDUCATION

1. Jon Cohen

Cambridge and Oxford universities compete in everything from chess to cricket, but for nearly a century Oxford has had the field to itself with its Rhodes Scholars program for attracting non-British students. Now Cambridge, thanks to a new $210 million trust announced on 11 October by the Bill & Melinda Gates Foundation, is launching a new high-visibility scholars' program of its own, which each year will fund at least 225 students from outside the United Kingdom. The university will select Gates Cambridge Scholars based on merit, not need, focusing on academic ability and leadership potential. The program will support students from any country; Rhodes Scholars, in contrast, must come from one of 19 jurisdictions. The scholars, who will receive about$40,000 a year in support, will live together in what will be called the Gates House. “We are hoping that the young people we select will be motivated to use their education to put something back into society for the benefit of a much wider community,” explained Bill Gates Sr., CEO of the foundation and father of the Microsoft co-founder, in a prepared statement. The Gates Foundation currently has roughly $21 billion in assets, making it the largest philanthropy in the world. Gates Cambridge Scholars will be able to pursue either a graduate degree or a second bachelor's degree, a particular attraction for students who have attended undistinguished schools in poorer countries. Although the program will not evaluate a student's financial situation, “the large bulk of the scholarships will go to people who wouldn't be here otherwise,” predicts Anne Lonsdale, pro-vice chancellor for international relations at Cambridge. Cambridge already has scholarship funds set up for overseas students, but the new gift dramatically changes the amount of available resources. “Instead of having to worry about every penny that goes into scholarships, suddenly we have all this money,” Lonsdale says. “We're deeply happy.” 5. ARCHAEOLOGY # Arizona to Take High Road to Preservation? 1. Mark Muro* 1. Mark Muro writes from Tucson, Arizona. TUCSON, ARIZONA—Building freeways is big business in this rapidly growing state. So it's news when state transportation officials agree to weigh a proposal to set aside a scientifically valuable parcel of a federal highway project for future research rather than excavating it and selling it to the highest bidder. Should it proceed, Arizona could set a new standard of stewardship for government agencies that determine the fate of ancient relics. The renovation of Interstate 19 at its intersection with I-10, a major north-south corridor in the state, almost inevitably means uncovering the remains of previous civilizations. And antiquities laws require extensive investigation of any major site. These efforts, which often include surplus lands that are intended for sale, can be a boon to science: Eight major digs involving I-10 over the past 5 years have yielded important findings about the origins of prehistoric village life in the Southwest. However, even a careful excavation leaves one fewer site for the next generation of researchers. That dilemma has led to a novel plan for a small parcel near a$60 million, three-level interchange barely 2 kilometers from downtown Tucson. Under the plan, one of the nation's most prolific road-building agencies would retain and actively manage a plot of unneeded land for the sole purpose of preserving its archaeological resources. “This is a big deal,” comments Jim Walker, the southwest regional director of the Archaeological Conservancy, a nonprofit group based in Albuquerque, New Mexico, that acquires and preserves key archaeological sites. “Highway rights-of-way are hot zones for archaeology, and we need to preserve much more raw data in the ground for more advanced research. This sets an important precedent for preserving archaeology rather than doing a one-time excavation before the bulldozers come in.”

The preserve plan, which has been tentatively embraced by the highway department, entails key portions of the so-called Julian Wash site, one of the largest and longest occupied Hohokam culture village sites in the Tucson area. About half the 22-hectare site has already been destroyed by urban development and road building, and next year construction begins on six lanes of new freeway that will slice through other sections of the site.

The state has retained Desert Archaeology Inc. (DAI) of Tucson to conduct extensive “data recovery” on the site in compliance with the National Historic Preservation Act. These excavations will likely reveal scores of pit houses and other features reflecting the village's continuous occupation from 500 B.C. to A.D. 1150. That period encompasses the arrival of agriculture and pottery in southern Arizona and the rise of the Hohokam, one of the main prehistoric cultures of the Southwest. “Julian Wash is important because it was a very large village and very long-lived,” says Henry Wallace, the DAI project manager overseeing the dig. “This excavation will give us important data on key questions about site organization, early economies, and cultural change over time.”

The current dig, however, does not address two other pieces of the site, totaling 4 hectares, that will be opened up when the Arizona Department of Transportation (ADOT) shifts existing lanes of traffic a couple hundred meters to the west. Such land would ordinarily be sold off when the interchange project is completed. However, researchers believe crucial sections of the prehistoric village lie intact beneath the present roadbed. It is this area that DAI wants ADOT to set aside as a fenced preserve, at least until another entity can assume control, and perhaps indefinitely.

The idea has taken some getting used to. “This has never been done before,” says Bettina Rosenberg, the department's historic preservation coordinator. “And we're not in the preservation business.” Indeed, while other state transportation agencies may retain archaeological features along their rights-of-way, none seems to carry them on otherwise salable property, and none seems to protect them with so formal an arrangement as ADOT is contemplating. Still, the department has warmed to the preserve idea in part because of the challenge of selling a relic-filled parcel that requires perhaps $2 million in archaeological excavation—by ADOT or the developer—before it can be built upon. Such a sale might also be very time-consuming. “We're seeing this as good for archaeology, and it will save us the cost of doing more data recovery,” says Rosenberg. She and DAI scientists note that the preserve would leave the cost of future excavations to others. Archaeologists, for their part, are ecstatic about the stewardship. William Lipe, an anthropologist at Washington State University, Pullman, stresses that the “finite” number of archaeological sites makes it imperative that “you put some sites in the bank for future research.” And Stephen Lekson, a curator at the University Museum of the University of Colorado, Boulder, suggests that the speed of technological advances—ranging from carbon dating in the 1940s to archaeomagnetic dating in the 1970s to the present era's use of ground-penetrating radar—vastly improves data collection. “We need to keep sites around, because we keep getting better at analyzing them,” he argues. “Let's leave some raw data for the archaeologists of the future.” No one is more pleased than Bill Doelle, DAI's president. Although DAI stands to lose several million dollars in potential fees for excavating the preserve site, Doelle says he's willing to pay the price to conserve archaeological sites. “This business has to be about more than just digging for dollars. We have an ethical duty to the future of archaeology, and to Native Americans with ancestral ties to these villages, to leave some archaeology in the ground.” 6. RESEARCH FUNDING # Windfall for French Biomedical Agency 1. Michael Balter PARIS—Researchers at France's giant biomedical research agency, INSERM, are rejoicing over a 16% hike in the organization's research budget for 2001. The windfall, announced by INSERM director-general Claude Griscelli last week, is the biggest such increase since 1983. It will give the organization's 260 laboratories an extra$13 million over the current research budget of about $83 million. In addition, 100 new research posts will be created, bringing the total number of scientists to nearly 4000. The new money represents “a significant sum,” says neuroscientist Marc Peschanski, director of the INSERM Laboratory of Neuroplasticity and Therapeutics near Paris. “It will really mean something” to the labs. Geneticist Judith Melki, director of the Molecular Neurogenetics Laboratory in the Paris suburb of Evry, adds that the influx of new money will help boost the “rather modest” support that INSERM labs have received in recent years. Griscelli told Science that such a big increase was “entirely unexpected.” Indeed, other public research organizations were awarded smaller amounts—the basic research agency CNRS, for example, will receive a 9% research boost. Griscelli says that one reason the government smiled so brightly on INSERM may be that the agency has been willing to shape its research agenda according to priorities laid down by the research ministry, which wants to see life sciences research pay off in new therapies and products (Science, 8 September, p. 1667). Whereas CNRS researchers have strongly resisted what many see as government meddling in research directions, INSERM has largely accepted the government's guiding hand. Thus the new money will be spent in a number of priority areas, including gene therapy, vaccines, psychiatric research, and epidemiology. Griscelli insists that basic science will continue to receive strong support at INSERM: “I do not want to prioritize by diminishing funds for fundamental research.” 7. PHYSIOLOGY NOBEL # Celebrating the Synapse 1. Michael Balter Arvid Carlsson first started thinking that he might win a Nobel Prize nearly 40 years ago. Since then, he says, “I've been up and down about it many times.” Carlsson need not have fretted. His pivotal discovery—that dopamine is a key neurotransmitter in the brain—not only led to treatments for Parkinson's disease and schizophrenia, but also sparked a revolution in neuroscience that has helped keep the field on a constant high ever since. Last week, the Nobel Assembly recognized those achievements, awarding the Nobel Prize in physiology or medicine to Carlsson of the University of Gothenburg in Sweden and to two other pioneers in the study of nerve cell communications: Paul Greengard of Rockefeller University in New York City, who figured out how dopamine and other neurotransmitters trigger their target neurons when they bind at the synapse, the junction between two nerve cells; and Eric Kandel of New York's Columbia University, who built on these insights to demystify some aspects of learning and memory. “This prize is really a celebration of the synapse,” says neuroscientist Corey Goodman of the University of California, Berkeley. He and others applaud the Nobel committee's decision to honor nearly a half-century of neuroscience research. “These people are all towering figures” in the field, says neurobiologist Charles Stevens of the Salk Institute for Biological Studies in La Jolla, California. The story of this year's prize began in the 1950s, when Carlsson, now 77, overturned conventional wisdom by proving that dopamine—once thought to be merely a precursor in the synthesis of the neurotransmitter norepinephrine—is an important nervous system messenger in its own right. In one key experiment, he and his colleagues gave rabbits a drug that depletes norepinephrine in the brain, putting the animals into a temporary stupor. Carlsson found that the rabbits could be roused with injections of L-dopa, which the brain converts to dopamine. According to the then-prevailing view, the dopamine should have been converted to norepinephrine. But he found only dopamine in the animals' brains—indicating that it was responsible for the rabbits' recovery. Carlsson and others later discovered that Parkinson's disease, which causes rigidity and tremors, results from degeneration of dopamine-producing neurons in a brain region involved in movement control. That finding led to the use of L-dopa as a therapy for Parkinson's patients. Further studies on the connections between neurotransmitter levels and mental states spawned a wealth of drugs, including Prozac, that fight psychosis and depression. In the 1960s, Greengard, 74, took Carlsson's insights several steps further by exploring how dopamine, norepinephrine, and serotonin trigger responses in their target neurons. Back then, Greengard says, most neuroscientists believed that nerve transmission was purely electrical, propagated by the flow of charged ions across nerve cell membranes. But he showed that this is only half of the story. In most neurons, Greengard found, the neurotransmitters exert their effects biochemically, by triggering a so-called second messenger inside the target cells. This in turn activates an enzyme that adds phosphate groups to cellular proteins, thus setting off a chain of events that alter nerve cell properties—for example, heightening or damping sensitivity to electrical stimulation. To date, Greengard and his colleagues have identified more than 100 brain proteins phosphorylated as a result of neurotransmitter activity, including one that serves as a kind of master control switch for dopamine. The link between phosphorylation and nerve cell signaling inspired the research of Kandel, 70, into how the brain learns and remembers. Kandel, who started his career as a psychiatrist, began dabbling in learning and memory research as a postdoc at the National Institutes of Health in the late 1950s. But although his work there recording electrical impulses from the hippocampuses of cats resulted in some “very nice papers,” Kandel says, he wanted a system that was easier to work with. So he went to Paris to study with Ladislav Tauc, an expert on the sea slug Aplysia, famous for its giant neurons. “With Aplysia, you can just record from those cells until the cows come home,” Kandel says. Over the following years, Kandel demonstrated that the responses of Aplysia's nerve cells to various stimuli—such as touching the animal's tail or giving it an electrical shock—were amplified according to the strength and duration of the stimuli. These heightened responses could last for days or weeks, thus demonstrating a form of learning and memory. In general, weak stimuli gave rise to short-term memory, and stronger stimuli to long-term memory. Kandel, sometimes in collaboration with Greengard, went on to show that short-term memory is created by means of phosphate addition to proteins that make up the pores in the cell membrane that calcium and other ions involved in nerve transmission flow through. Long-term memory, he found, forms when stronger stimuli trigger the synthesis of new proteins that change the shape of the synapse and its sensitivity to further stimuli. This finding helped solved a long-standing puzzle: why protein synthesis is necessary for long-term memory but not for short-term memory. It was also the culmination of decades of work that began with Carlsson's pioneering discoveries in the 1950s. When asked how winning the Nobel Prize might change their lives, all three recipients told Science they hoped the impact would be minimal. “I will try very hard not to let it affect my life,” says Kandel. “I already like my life.” 8. PHYSICS NOBEL # Achievements Etched in Silicon 1. Charles Seife Silicon, rather than gold, might be an appropriate material for this year's Nobel Prize in physics. For it was with silicon that the three recipients—Jack Kilby of Texas Instruments in Dallas; Herbert Kroemer of the University of California, Santa Barbara; and Zhores Alferov of the A. F. Ioffe Physico-Technical Institute in St. Petersburg, Russia—made their crowning achievements. Computers, cell phones, and CD players rely on technology they made possible. Silicon technology has come a long way. In late 1947, scientists at Bell Laboratories invented the transistor, ushering in the computer age. John Bardeen, Walter Brattain, and Wil-liam Shockley won the 1956 Nobel Prize for the invention, but their transistor was not ideal. Although it was much smaller and more reliable than the vacuum tubes it replaced, manufacturers still had to solder thousands of transistors and other components to a circuit board to construct even the most rudimentary computers. “By that time people could visualize electronic equipment that couldn't be built—it was too expensive, too bulky, and too unreliable,” Kilby recalls. In 1958, Kilby was stuck in the laboratory; just hired by Texas Instruments, he hadn't earned the vacation time to get away for the summer. But Kilby made good use of the extra time in the lab: He came up with a radical solution to the assembly problem. Instead of taking lots of individual transistors and soldering them together, Kilby put all the components of a circuit on a single wafer of semiconducting crystal such as germanium or silicon. He did this by taking a wafer of germanium, covering bits of it with black wax, and then exposing the wafer to acid, so that areas that weren't protected by the wax were etched away. In this way, he carved transistors, resistors, condensers, and other elements on the same wafer and out of the same materials—all layered and wired on one crystal. This “integrated circuit” avoided the labor problems, space constraints, and quality-control issues that plagued circuit boards assembled from individual transistors. Kilby and a competitor, the late Robert Noyce of Intel, are credited with inventing the integrated circuit that put the computer revolution in high gear. Kroemer wins his Nobel Prize for refining the basic transistor that made it faster and more efficient. The key to this invention lies in modifying the flow of negatively charged electrons and positively charged “holes”—spaces vacated by electrons—through semiconductors. Though electrons carry the charge, the holes act as if they were particles that carry a positive charge. In an ordinary transistor, when electrons flow in one direction, holes swim upstream in the opposite direction. Unfortunately, the bigger the reverse flow of holes, the less amplification the transistor gives—the less powerful it is. The so-called heterojunction bipolar transistor solves this problem by using layers of two complementary semiconductors (such as gallium arsenide with aluminum gallium arsenide), whereas traditional transistors used just one (such as silicon). Electrons can cross from one semiconductor layer to the other easily, but holes cannot (or vice versa, depending on the configuration). By doing this, “you prevent holes from flowing in the reverse directions—they run into a potential barrier,” explains Jim Merz, a physicist and vice president at Notre Dame University in Indiana. “This was Kroemer's idea; he realized that it had huge implications.” Better yet, Kroemer realized—as did Alferov—that these heterogeneous semiconductors could be turned into lasers. By arranging materials in the proper fashion, it's possible to create a trap for electrons and holes—a region where they can flow in but can't flow out. When an electron and a hole meet inside this trap, they recombine, releasing light. This light, in turn, incites more trapped electrons and holes to recombine. It's just like a traditional laser, but it can be made out of semiconductors. Kroemer and Alferov's brainstorm led to the development of radio satellites, base stations for mobile phones, fiber optic cables, and CD players, notes semiconductor laser researcher Al Cho of Lucent Technologies' Bell Laboratories in Murray Hill, New Jersey. “I think they certainly are pioneers.” 9. CHEMISTRY NOBEL # Getting a Charge Out of Plastics 1. Robert F. Service* 1. With reporting by Dennis Normile in Tokyo. Polymers generally make good insulators: Witness the plastics shrouding the wires in your home. But this year's Nobel Prize in chemistry was awarded to a trio of researchers—Alan Heeger of the University of California, Santa Barbara; Alan MacDiarmid of the University of Pennsylvania in Philadelphia; and Hideki Shirakawa of the University of Tsukuba in Japan—for discovering that plastics can be made electrically conductive. The discovery paved the way for revolutionary applications such as full-color displays for cellular phones and plastic electronics for computerized merchandise, as well as still-futuristic hopes of computing with molecules and creating cheap, large-area solar cells. The initial discovery of electrically conductive plastics stemmed from a wonderful bit of serendipity. In an experiment in the early 1970s, one of Shirakawa's students accidentally added excess catalyst to a brewing batch of plastic called polyacetylene. The result was a shiny silvery film. Shirakawa told MacDiarmid of his discovery during a visit to Kyoto University in 1975. “When MacDiarmid saw it, he was very surprised,” says Shirakawa. It turned out MacDiarmid and Heeger —who was also at the University of Pennsylvania at the time—had been experimenting with metallic-looking films from polymers made from inorganic building blocks. They were trying to learn more about the changes that take place as materials change from insulators to metals. The inorganic polymers were scientifically interesting because they showed hints of this change, but they couldn't be modified easily like organic polymers, says Heeger. So when MacDiarmid returned to Pennsylvania and told Heeger of Shirakawa's work, “I said, ‘This is what we're looking for,’” recalls Heeger. MacDiarmid invited Shirakawa to visit the University of Pennsylvania, and the researchers quickly set about modifying the polymers and testing the results. In one case, they used an iodine vapor to oxidize the film, a treatment they knew could change the film's optical properties. But that was the least of the changes: The conductivity shot up by 10 million times. Polyacetylene, like other conducting polymers discovered since, is a chainlike molecule with alternating double and single bonds. When excess charges are added to the molecule—as happens during oxidation—these charges can then hop along the alternating bonds with relative ease. That discovery created a field that has been hopping ever since. Despite widespread agreement among chemists that the trio selected by the Nobel committee are worthy recipients, the selection is “rather controversial,” says Stephen Forrest, a materials scientist at Princeton University. Many applications of plastic electronics, it turns out, are based on more recently discovered relatives of the metallic polymers that Heeger, MacDiarmid, and Shirakawa originally experimented with. These behave like silicon and other semiconductors. The Nobel committee chose not to honor the discoverers of these materials. Forrest and most others call that understandable. “Now the research has more emphasis on semiconducting polymers,” says Zhenan Bao, a chemist at Lucent Technologies' Bell Laboratories in Murray Hill, New Jersey. “But it's all based on [Heeger, MacDiarmid, and Shirakawa's] early concepts.” Such concerns haven't clouded the moment for the Nobel recipients. “It's been a wonderful week,” says Heeger, who adds that the prize came as a “complete surprise.” MacDiarmid says that when he was told the news by a friend who saw it on the World Wide Web, he didn't believe it. “I thought it must be a hoax. But then I immediately got calls from reporters in France and Germany and thought maybe this is real.” The reaction has been particularly enthusiastic in Japan, where Shirakawa's selection is the first chemistry Nobel Prize awarded to a Japanese researcher in 19 years. It was front-page news the following morning, and throughout the week newspapers ran large photos, such as shots of Shirakawa receiving bouquets of flowers from students at Tsukuba University. And it prompted the editors at the Mainichi Shimbun, one of Japan's largest daily newspapers, to say they hoped the award would serve as a catalyst to “update the nation's dilapidated and cramped research facilities.” 10. ECONOMICS NOBEL # Dealing With Biases and Discrete Choices 1. Charles Seife To a biologist, “micro” means bacterium-sized. To an economist, it means people-sized. And this year's Bank of Sweden Prize in Economic Sciences, given in honor of Alfred Nobel, goes to two researchers who gave the field of microeconomics—the study of individuals' economic behavior—new tools to help draw conclusions from imperfect data. As any scientist knows, statistical investigations are prone to error; inadvertent biases in choosing the sample or systematic errors can doom a project. The situation is even dicier for economists who take statistical samples of complex, semirational objects like human beings. James Heckman of the University of Chicago wins half of this year's prize for coming up with ways to deal with selection biases. He developed two methods that formalize the handling of such biases, and then he used them to analyze things such as how wages affect the behavior of married women in the labor market. Burton Singer, a demographer at Princeton University, says he believes Heckman's best achievements were not in the mathematical methods, but in what he was able to do with them. For instance, Heckman analyzed whether African Americans in Southern states like South Carolina were being helped more by education improvements or by the civil rights movement. “Government legislation had a more profound impact than schools per se,” as did activism in the African-American community, says Singer, who notes that this surprising conclusion “had almost been ignored by the economic community.” Heckman also thinks his most valuable work is applied rather than theoretical. “Economics is a field where you're solving real problems,” said Heckman by telephone from Brazil, where he and his students are studying education and economics. “Being able to tackle real problems has always been an attraction for me.” Daniel McFadden of the University of California, Berkeley, tackled a different conundrum: how to quantify discrete choices rather than continuous ones. “Before McFadden did his work, economists were concerned with buying amounts—how many oranges a consumer buys, et cetera,” says Charles Manski, an economist at Northwestern University in Evanston, Illinois. “But many important choices are discrete: Do you go to college or not? Do you buy an auto or not?” McFadden had two insights that allowed him to tackle discrete problems. First, he came up with a way of comparing apples and oranges—or buses and cars, as the case may be. “For instance, if you choose to commute to work, you can go by bus, by rapid transit, or by auto,” says Manski. “If you think of an auto as a bundle of characteristics—values for travel time, cost, and comfort—and a bus as a different bundle, you can compare them.” Now that all these different choices were directly comparable, he could model how consumers behave when given those choices. This is where McFadden's second insight comes in: He turned the discrete choices into more continuous, tractable functions by looking at them in terms of probabilities. For instance, a consumer might have, say, a 20% chance of taking a car to work, a 40% chance of taking a bus, and a 40% chance of taking rapid transit. This method, inspired by similar approaches used by psychologists, turned discrete problems into continuous ones—and it led to his helping design the Bay Area Rapid Transit system. Marketers, sociologists, political scientists, and others are indebted to McFadden's methods, says Steve Lerman of the Massachusetts Institute of Technology: “The number of applications, in both marketing and economic analysis, must be in the thousands—maybe even higher.” 11. MALARIA # A Renewed Assault on an Old and Deadly Foe 1. Eliot Marshall Lots of money and new scientific tools have invigorated the fight against malaria. But the disease is unyielding, and the current weapons are losing their effectiveness After languishing for decades in the scientific backwaters, malaria research is suddenly being swept into the mainstream. International finance and aid organizations have declared malaria a global health priority, and money is beginning to pour in. Researchers who have been doggedly pursuing an intractable foe with limited resources now have the means to follow new leads—just as some promising avenues are opening up. And funding agencies are finally beginning to plug a critical gap: a lack of research and medical capacity in developing countries where malaria exacts its deadly toll. It's quite a turnaround. Despite malaria's horrific handiwork—it kills more people each year than any other infectious disease except AIDS and tuberculosis (TB)—it has been hard to muster support for a disease that “doesn't happen here.” True, malaria has long received funding from a few deep-pocketed organizations, like the U.S. Army and Navy, which in many places lost more soldiers and sailors to parasites than to gunfire. The U.S. Centers for Disease Control and Prevention (CDC) in Atlanta also had a powerful mandate to be involved: Fighting malaria is precisely what CDC was created for in 1946. But basic research funding grew slowly: When the AIDS budget at the U.S. National Institutes of Health (NIH) broke the$1 billion mark in 1991, for example, NIH's investment in malaria was just over $10 million. Now, signs of change are everywhere. The World Bank is pledging$300 million to $500 million in interest-free loans to fight the disease, especially in Africa. The World Health Organization (WHO) has set an ambitious goal of cutting malaria deaths in half by 2010 (see sidebar on p. 430). In major speeches, President Clinton has thrown his support behind efforts to develop a malaria vaccine. Indeed, the finance chiefs of the world's wealthiest nations, the G-8, have promised billions of dollars to fight malaria, AIDS, and TB. The Bill & Melinda Gates Foundation is donating$115 million for antimalaria research, education, drugs, and vaccines. And at the National Institute of Allergy and Infectious Diseases (NIAID), still the largest single contributor to malaria research, the budget is expected to rise above $52 million in 2001. “There is a momentum we didn't have before,” says NIAID director Anthony Fauci. “All of a sudden things are happening that were unimaginable a few years ago.” What changed? No single force, but a confluence of events has opened pocketbooks. As Western economies have boomed, charities have become flush with stock earnings, for instance, and the U.S. Congress has promised NIH a 5-year budget doubling by 2003—all of which makes it easier to deliver on humanitarian urges. Self-interest plays a role as well, as politicians come to view countries wracked by poverty, disease, and war as a threat to national security. Says Fauci: “The important policy-makers in government are now talking about making this [alleviating the disease burden in poor countries] part of our foreign policy.” ## Rising toll Whatever the logic, the malaria research community welcomes the support. And the need has never been greater. More than 400 million people, WHO estimates, fall ill with malaria each year, and between 1 million and 3 million die—mostly children younger than 5 years, and most of them in Africa. Public health experts believe the toll has actually been increasing in recent years, although numbers are impossible to verify. But no one disputes that the drug-resistant strains of the parasite are spreading, rendering cheap and effective drugs ineffective. Scientifically, too, the time is ripe for a fresh attack. New resources are coming on line, such as data from the malaria genome project, which is deciphering the genetic code of the most deadly parasite, Plasmodium falciparum (see p. 439). Tom Wellems, a molecular biologist at NIAID, is ecstatic. The genome is a “tremendous” tool for zeroing in on how the parasite wreaks its havoc, he says, and for finding new drug targets. Equally important, says Fauci, young people are entering the field again. These scientists will need all the new tools they can get. In the 1950s, WHO optimistically targeted malaria for eradication. After all, the United States and other Western countries had wiped out the disease, and Latin American countries were making progress. But today—with billions of people at risk of malaria in a swath that extends from Central and South America through Africa to South Asia—thoughts of eradication have disappeared. Indeed, even control seems like a distant dream. Not only is the disease notoriously difficult to fight, given the cunning strategies employed by the parasites that cause it, P. falciparum and P. vivax. But malaria is also exacerbated by poverty, malnutrition, inadequate housing, and ill-funded public health systems. Pesticides and other barriers against mosquitoes help. The use of pesticide-soaked bed nets, for instance, has produced “a substantial reduction in child mortality” in western Kenya, where children may be exposed to 300 infective mosquito bites a year, says Richard Steketee, chief of CDC's malaria branch. “Few other interventions” have produced such credible evidence of effectiveness, he adds. A malaria vaccine would be the ultimate weapon; most experts believe the disease cannot be controlled without one. But despite encouraging advances, a vaccine isn't likely soon (see p. 434). Antimalarial drugs are the last certain defense, and even they are failing as resistant strains of parasites spread across the globe (see map). First established in Asia, resistant strains have now spread into Africa. Chloroquine, the mainstay, is no longer useful in most of these endemic areas. Even later generation drugs like mefloquine and sulfadoxine-pyrimethamine can no longer be relied upon in much of Southeast Asia and are beginning to fail in Africa, as parasites acquire resistance to multiple drugs. A handful of new drugs are under development, but they are years from being approved (Science, 17 March, p. 1956). Alarmed, WHO in 1993 called for a renewed global effort. ## A turning point WHO's plea didn't produce an immediate response. But in 1996, Britain's Wellcome Trust, the largest biomedical charity in the world, echoed the appeal with its own audit of the world's investment in malaria research. Worldwide, the trust said, just$84 million was being spent annually on malaria research. But the economic cost of the disease in Africa alone was probably more than $2 billion a year. The meager research investment, it continued, “is very low compared with other disease areas … and appears to be declining further.” Since then, the annual investment has climbed to more than$100 million.

At about the same time, public health leaders began to focus on sub-Saharan Africa. After AIDS, malaria was still the most threatening disease on the continent. Harold Varmus, then director of NIH, argued that his agency should be doing more to address global health issues—and that malaria was one where you get a big bang for the buck. Maxime Schwartz, director of the Pasteur Institute in Paris, was equally committed but had no comparable budget to contribute. Varmus, Schwartz, and representatives of Britain's Medical Research Council and the Wellcome Trust met on the NIH campus in Bethesda, Maryland, in 1995 and 1996 to chart a new strategy for malaria. A key goal, they agreed, should be to build technical infrastructure in Africa and encourage Africans to take the lead in research.

They also laid the groundwork for an unprecedented meeting of African scientists in Dakar, Senegal, in January 1997. “Maxime insisted that the meeting should be in Dakar,” says Louis Miller, a leader of NIAID's malaria effort, to ensure participation of African scientists. It worked. Some 50 African scientists from 22 countries, many of whom had not met before, attended along with 75 outside scientists. This “extraordinary” gathering, as Varmus called it, produced bold declarations—most notably, a push for a new multilateral fund to support African research. The various funding agencies also planned to enlist the help of reluctant pharmaceutical companies, concerned about a lack of profit, in developing badly needed new drugs. Varmus said that funding would be worked out at a session in The Hague in July 1997.

At The Hague, the Americans “were met with some skepticism” when they sought commitments from people who could write big checks, says one U.S. participant who asked to remain anonymous. “Some of the Europeans greeted our enthusiasm … as our trying to take over.” The joint research fund never materialized. Nor did pharmaceutical company executives offer to bankroll drug development projects. But the two meetings did create a new outfit: the Multilateral Initiative on Malaria (MIM), “a loose confederation” supporting African research (Science, 18 July 1997, p. 309).

## New promises

With funding from NIH, WHO, and the Rockefeller Foundation, among others, MIM has awarded 23 grants, ranging in value from $92,000 to$250,000 per year, to collaborations that include African principal investigators at 18 sites in Africa (see p. 431). As part of MIM, NIAID is also helping to ensure that African scientists have adequate resources. Malaria researchers “are not short on concepts,” explains Fauci; “what they are short on is … materials.” In 1998 the agency began building a repository of research reagents to be distributed for free. It provides antibodies, cell libraries, and DNA clones with oligonucleotide primers to “all legitimate malaria researchers,” says project manager Yimin Wu. So far, 85% of its users are still American or European. But Wu plans to promote the repository to African scientists with training sessions. NIAID also plans to extend Internet connections to Africa. NIH's National Library of Medicine (NLM) has already established electronic beachheads at seven African research sites, setting up satellite dishes, receiving stations, and small local networks. “We've tried to do that in a big way in Bamako, Mali,” says Fauci. “It's like having an NIH lab in the middle of the jungle.” The NLM group, along with the Africa Program of the American Association for the Advancement of Science (Science's publisher), is urging 11 biomedical publishers to subsidize free online access to their journals for African scientists during a 3-year trial.

Enthusiasm breeds yet more enthusiasm. Almost weekly, it seems, organizations are pledging more funds to fight malaria and other infectious diseases. But now comes the hard part: transforming that enthusiasm and basic science into treatments that can be used in rural villages. In some ways, Miller concedes, “we're worse off than we were in the 1950s,” because effective pesticides like DDT are less available and the cheap antimalarial drugs have lost their potency. But with malaria now high on the political agenda and researchers armed with new tools, it may not be so crazy to think again about bringing malaria under control.

12. MALARIA

# Can WHO Roll Back Malaria?

1. Michael Balter

GENEVA—Ask malaria experts around the globe to rate the World Health Organization's (WHO's) performance in the fight against malaria, and you'll probably get an earful. Yet if you ask the same experts whether WHO is the right organization to lead a renewed onslaught against the disease, you are likely to get an unequivocal “yes.” “We have to criticize WHO” for its past performance, says tropical medicine researcher Nicholas White of Mahidol University in Bangkok, Thailand. But if malaria is to be brought under control, he says, WHO's technical know-how and moral authority will be crucial. “We have to enthusiastically support them.”

WHO Director-General Gro Harlem Brundtland needs that support. In October 1998, just 3 months after she took office, Brundtland announced Roll Back Malaria (RBM), a multiagency crusade that aims to cut malaria mortality in half over the next 10 years. Brundtland might just be the one to pull it off, say numerous public health experts. But “it will take an absolutely stupendous effort of leadership, coordination, and investment,” cautions Kevin Marsh, coordinator of a collaborative research program in Kenya run by the Kenya Medical Research Institute (KEMRI), Britain's Wellcome Trust, and Oxford University. Although some researchers question whether the goal is realistic, most agree that RBM has already achieved an important political end: putting malaria higher on the agenda of political leaders, especially in Africa.

Malaria was high on the agenda once: In the 1950s and 1960s, WHO spearheaded an effort to eradicate the disease. But by several accounts, the organization began to fumble in the late 1960s, when it became clear that eradication efforts had failed in most parts of the world. “This failure seems to have knocked all the stuffing out of [WHO staff],” says Marsh. WHO drastically scaled back its technical staff in afflicted countries, largely leaving local health workers to treat the sick. “Malaria control programs collapsed,” says Brian Greenwood, a malaria researcher at the London School of Hygiene and Tropical Medicine. “Since they couldn't eradicate malaria, they eradicated the [malaria researchers].” In creating RBM, Brundtland has made malaria again one of WHO's top priorities. RBM focuses on four goals: rapid treatment of children with life-threatening malaria; treatment of pregnant women infected with the malaria parasite; increased use of insecticide-impregnated bed nets; and emergency control of malaria in areas afflicted with warfare or natural disasters.

WHO will carry out these tasks in tandem with the three other “founding partners” of RBM: the United Nations Development Program, UNICEF, and the World Bank. In addition, more than 50 other organizations, ranging from the Nigerian health ministry to nongovernmental organizations such as Médecins Sans Frontières, are participating in this loose coalition. While some partners are donating personnel, others are contributing cash. The World Bank, for example, has pledged between $300 million and$500 million in interest-free loans to African countries for malaria prevention and control.

Even with the renewed energy and extra resources, cutting malaria deaths in half by 2010 will be a formidable challenge. For instance, RBM last April convened a malaria summit in Abuja, Nigeria, where nearly two dozen African heads of state pledged to take concrete steps to combat the disease. But if this “Abuja declaration” is to have any effect, it must be translated into real action on the ground, says entomologist John Vulule, acting director of the KEMRI field station in Kisian, Kenya. “This may be an uphill task given the levels of poverty in sub-Saharan Africa,” Vulule adds.

Already, some experts complain that the RBM campaign has been slow to reach areas most affected by malaria. In India, says Neeru Singh, a deputy director of the Indian Council of Medical Research, “there is no RBM activity in the Madhya Pradesh district,” a hard-to-reach region where serious malaria outbreaks occur each year. RBM officials counter that their job is not to micromanage what goes on in each country but to help foster political commitment and technical support. And they also deflect concerns that by participating in a broad-based coalition, WHO is diluting its own leadership role. “WHO is trying to facilitate a partnership but not control it,” says David Heymann, director of the agency's division of emerging and communicable diseases. Clearly, in its renewed war on malaria, WHO will need all the allies it can muster.

13. AFRICAN RESEARCH

# Against All Odds, Victories From the Front Lines

1. Gretchen Vogel

In one of the world's poorest countries, Malian and American researchers attack malaria in the lab and in the clinic

BANDIAGARA, MALI—The rains, when they come, come hard in this remote town a few hundred kilometers south of the Sahara desert. Toward the end of a sweltering afternoon, dark clouds form in the south and east, the glaring sunlight dims, gusts of wind raise choking clouds of dust, and the first large drops smack into the ground. Soon the din of rain on metal roofs drowns out any attempt at conversation. The soil that 6 months of the year receives little or no rain attempts to soak it in, but within minutes dusty streets turn to streams and then to rivers. Gardens and fields turn to swamps and then ponds—even lakes—as the rain pours down in sheets. Then, almost as quickly as it dimmed, the sky brightens, the thunder fades into the distance, and the steady flow of water off the roofs slows to a trickle. The downpour's effects remain, however: Roads become quagmires as heavy trucks churn the mud.

With the rains come the insects. The flies and beetles, which manage to thrive even in the dry season, are suddenly joined by flying ants, damselflies, a profusion of moths, and, of course, mosquitoes. And with mosquitoes comes malaria. The local word for malaria translates literally as “sickness of the green season.”

Researchers, too, arrive with the rains. Each year, scientists from Africa and the rich countries of the North come to this community of 12,000, which lies 9 hours from Mali's capital, Bamako, looking for answers to some basic questions about malaria. Bandiagara is best known as a staging point for tourists who visit the villages of the Dogon ethnic group that cling to the famous cliff that shares the town's name—the Bandiagara escarpment. It is a place where research traditions meet ancient cultures.

The researchers who come to study the sickness of the green season are documenting malaria transmission in the community and monitoring the patterns and mechanisms of chloroquine-resistant parasites. They are also probing the complex question of natural immunity—in essence, how people can coexist with the parasite without showing severe symptoms of the disease. And they are looking for clues to one of the most puzzling questions of all: why the parasite kills more than 1 million people each year. Malaria exacts much of its horrific toll on children who develop deadly anemia or a virulent cerebral form of the disease. They come down with a sudden fever, suffer wrenching seizures, and slip into a coma. Without treatment, half of them die. No one knows why some develop cerebral malaria while their friends and siblings, who also carry the deadly Plasmodium falciparum parasite in their blood, do not.

Basic questions about malaria persist in part because the Plasmodium parasite is a complex foe. But other, equally complicated diseases such as diabetes are well understood, notes Terrie Taylor of Michigan State University College of Osteopathic Medicine in East Lansing, who studies cerebral malaria in Malawi. Much of the problem, she says, has been a lack of research interest in a disease that affects primarily the developing world. “Malaria remains a scourge because almost no one in an endemic area has looked at it systematically,” Taylor says. To make a significant contribution, “you don't have to be brilliant, you just have to be there.”

## Being there

That realization has become something of a rallying cry for international organizations funding malaria research. Agencies including the World Health Organization (WHO), the Wellcome Trust, the Bill & Melinda Gates Foundation, and the U.S. National Institutes of Health (NIH) have ramped up funding for malaria research (see p. 428), and all have said that building research capacity in Africa, where 90% of the world's malaria cases occur, is a top priority.

But the logistical hurdles are daunting. Malaria hits hardest in the world's least developed countries, where it kills the poorest of the poor. In places such as Malawi, Kenya, and Thailand, local doctors are stretched to the limit and trained scientists are scarce. Researchers must often build and equip their research space from scratch—providing everything from bedsheets to power generators. And they have to achieve a delicate balance between meeting the ethical standards of U.S. and European research sponsors and respecting the cultural mores of local communities.

Researchers in this West African nation —one of more than a dozen African countries where malaria research is under way—are proving that these obstacles are surmountable. In Mali, most people live on less than $1 per day, and life expectancy is 47 years. But at the Malaria Research and Training Center (MRTC) in Bamako, “we are showing by example that world-class science can be done in a poor country,” says pharmacist and microbiology Ph.D. student Abdoulaye Djimde. The center, funded mainly by NIH, the U.S. Agency for International Development (USAID), WHO, and the European Union, has two goals: researching the multiple facets of malaria and training young scientists. And, despite unreliable Internet connections, crowded labs, and scarce supplies, MRTC is succeeding at both, says Tore Godal, executive secretary of the United Nations Global Alliance for Vaccines and Immunization. ## On the edge Although conditions in Bamako can be trying, the city is strikingly modern compared to much of Mali. Most of the MRTC scientists conduct their main research in remote towns like Bandiagara, where electricity comes only from generators and the food supply is determined by recent harvests. Most buildings in Bandiagara are constructed with wattle and daub, a mixture of straw and mud. Their top edges, carefully sculpted during the dry season, begin to wash away with the first few rains. Pigs roam the streets with goats, sheep, and chickens; donkey carts are far more common than cars. Children and women must draw water from wells scattered throughout town, then carry it home on their heads. But the remote setting does not hinder the team of Malian and American researchers who have been monitoring malaria here since 1997. Led by epidemiologist and physician Ogobara Doumbo, co-director of MRTC, and Christopher Plowe, an infectious-disease expert at the University of Maryland School of Medicine in Baltimore, the Bandiagara Malaria Project is laying the groundwork for future trials of a hoped-for vaccine. Backed by NIH and the University of Maryland, the team also includes two American physicians, a pharmacist from Niger, and eight Malian doctors and pharmacists. The researchers' presence has already had a measurable effect. Before they arrived, malaria killed scores of children every year. But in the past few years, malaria deaths have been rare; indeed, last year only one child from an outlying village succumbed. When a child develops a high fever or slips into a coma, parents now know to bring the child to the research team's clinic, which provides treatment without charge. “When we first met the team [members] … I thought they were [with] another of the many programs that do nothing,” says a community elder through an interpreter. “But 3 years later I know that's not true. There are more small children alive today thanks to their presence here.” Building that kind of trust is essential. Before the researchers can begin their work each rainy season, they must pay their respects to Bandiagara's leaders. Team representatives present the local leaders with traditional gifts of kola nuts (bitter, caffeine-containing nuts that are a traditional sign of respect) wrapped in banana leaves and request their permission to begin research. In rural Mali, as in much of West Africa, decisions are made by a council of elders on behalf of the whole community. “You can't do anything if you don't have consent from the community leaders,” says Djimde, who grew up in a small village not far from Bandiagara. “They want to know who you are, whether your words are trustworthy.” The leaders, in turn, explain the projects to the rest of the community. “To win their trust is the most difficult part. And once you win their trust, you have to keep it. Every year, you have to go through the whole process again,” says Djimde. At first, the leaders were suspicious. Many were disturbed that, by collecting so much blood, the researchers might harm the children. And some of the local doctors and traditional healers were afraid the researchers might undermine their business. But Doumbo, who also grew up nearby, was able to allay their fears (see sidebar). After securing the leaders' blessings, the team recruits families to participate based on a random geographic distribution. By now, says Boureïma Ouologuem, one of the team's two local translators and “guides,” most parents jump at the chance. For the trouble of bringing their children into the medical center once a week, which often interrupts field work, the families are compensated with a sack of millet or rice at the end of the study season. The children also receive regular checkups and free medication for common ailments such as intestinal parasites, persistent coughs, and of course malaria. One of the team doctors, Ando Guindo, claims he can tell a distinct difference between the children in the study and those who aren't enrolled: The study children are, in general, both bigger and healthier. In Malian society the consent of the elders implies consent from the whole community. The written consent documents required by ethics boards and funding agencies in the United States mean little in this setting, where two-thirds of the population is illiterate. But the research team, working in two cultures, takes both types of consent seriously. Ouologuem and Akouni Dougnon carefully explain to parents the aims of the study, the requirements for participants, and the possible risks—in French at first, but often switching to a local language: Peuhl, Bambara, or one of the dozens of Dogon dialects. Those languages have no words for foreign concepts like “research,” much less “case-control study,” but the Malian members of the team have devised ways to explain the work. Most parents apply a thumbprint to the signature line of the several-page consent document, written in English and French. ## Notable successes At the health center, the children line up to register for this season's study. In a cinder-block building with simple ceiling fans, no running water, and screenless windows, two team members record each child's family, location of home, and birth date—often noted simply as 15 June of a given year, as many birthdays go unrecorded. Four team doctors chart the children's weight, height, and temperature, then check for any signs of anemia or other illness. Other team members draw blood. Several drops go onto a glass slide to be checked for malaria parasites; some blood is checked for iron and hemoglobin levels; and some is analyzed for immune system cells, antibodies, and other proteins. By comparing samples from children who develop cerebral malaria or severe anemia with controls who are infected but show no symptoms, the team hopes to discover more about what triggers the deadly symptoms. A drop of blood from each participant also goes on a slip of filter paper for later DNA analysis to check the genetic traits of both parasite and host. The team has already produced notable results. This month in Blood, NIH parasitologist Tom Wellems, Doumbo, Plowe, and colleagues report that a genetic trait common in the Dogon ethnic group—two-thirds of the population in Bandiagara—seems to protect children against severe malaria. The trait, a mutation in one of the genes that codes for the hemoglobin protein, seems to be as effective in protecting carriers as the sickle cell trait is in other populations. The team has also been able to document the presence of a newly identified chloroquine-resistance gene in the local population, lending strong support to the lab-based results Wellems's team reported in the 18 October issue of Molecular Cell. ## Growing connections Until now, the project's main link to the outside world has been a satellite phone. When its batteries are correctly charged, the connection isn't bad, although the$3 per minute fees add up quickly. Now NIH is installing equipment in Bamako and the MRTC's field sites that will bring high-quality Internet and phone connections to researchers in their villages. It may also provide MRTC with a U.S. phone number—so that a call to or from NIH in Bethesda, Maryland, will be local.

The MRTC laboratories in Bamako were already an oasis of technology compared to the field sites. And over the past year, lab space at the facility has doubled, thanks to funds from NIH and USAID. The new labs are equipped with state-of-the-art freezers, fume hoods, polymerase chain reaction (PCR) machines, and computers, as well as a section that complies with U.S. Food and Drug Administration clinical laboratory standards, says entomologist Robert Gwadz of NIH, who has been instrumental in developing the facility.

Access to such technology will be vital to the careers of the younger scientists at MRTC, such as Djimde, who earned his pharmacy degree in Bamako and expects to receive his Ph.D. in microbiology from the University of Maryland School of Medicine in early 2001. Djimde will return to Mali full time next summer to set up his lab in the new addition. He will join two other recent Ph.D.s as a full-time faculty member at the medical school in Bamako. Both have won prestigious reentry grants from WHO that support their research for 2 years, and Djimde hopes to win one as well. Still, “they need to come to a place where they don't have to worry that purchasing a PCR machine would be their entire budget for a year,” says Richard Sakai, an entomologist who has been the U.S. National Institute of Allergy and Infectious Diseases' scientist-in-residence in Bamako for nearly 10 years. The returning researchers are first-rate, he says. “We need to give them sufficient funds and sufficient challenges to keep them interested. The challenge for us is to make those conditions available to them.”

14. AFRICAN RESEARCH

# Traditional and Modern Medicine Merge to Save Lives

1. Gretchen Vogel

BANDIAGARA, MALI—Before the Bandiagara Research Project began, cerebral malaria was not associated with the “the disease of the green season,” the local term for malaria. Seizures and coma were considered symptoms of wabu, caused by a bird that cries out at the same time a child cries and steals a child's spirit. Parents did not consult the local doctor but rather the traditional healers.

The respect seems to flow both ways. At a meeting to discuss the current research project at the beginning of the rainy season, one healer told the team, “Before the study started, many children suffered, and we lost many 2- and 3-year-olds. But two hands can wash each other. I hope the collaboration will continue forever.”

15. VACCINES

# Searching for a Parasite's Weak Spot

1. Gary Taubes

Using a host of new technologies, vaccine developers are trying to target the parasite at every stage of its complex life cycle

“Optimism” is not a word usually associated with the pursuit of a malaria vaccine. “Frustration” is more appropriate. Over the years, researchers confronting the extraordinarily complex parasite have suffered a string of disappointments interspersed with some high-profile setbacks, as promising candidate vaccines have failed to perform up to expectations. The scientific obstacles are enormous: Compared to a virus, with its dozen or so genes and relatively monomaniacal approach to evading the human immune system, the malaria parasite has 14 chromosomes, perhaps 7000 genes, and a four-stage life cycle as it passes from humans to mosquitoes and back again. It is almost demonically efficient at evading the human immune response. Yet, something akin to legitimate optimism has been creeping into the field.

A recent clinical trial in The Gambia demonstrated that a vaccine made from a single malaria recombinant protein can protect humans against the parasite—albeit for only a few months. Dozens of new vaccines are in the works, employing a host of technologies that promise to attack the parasite at every vulnerable point of its multistage life. And after years of subsistence funding, money is pouring into malaria vaccine research (see p. 428). The Bill & Melinda Gates Foundation plans to disburse $50 million through its new Malaria Vaccine Initiative, for instance, and at the U.S. National Institutes of Health (NIH) funding for overall malaria research has increased fivefold in the past decade and should exceed$50 million for 2001.

Researchers now predict that within 5 or 10 years they will have a successful vaccine that will actually save lives. But in malaria vaccine research, the concept of “success” comes with caveats. Few researchers expect that the first or even second generation of malaria vaccines will work like a polio or tetanus vaccine and protect the majority of those inoculated for a decade or longer. For example, the military, which has long taken a lead role in malaria vaccine development, is aiming for a vaccine that is 95% effective for 6 months, says Grey Heppner, chief of immunology at the Walter Reed Army Institute of Research in Silver Spring, Maryland. That would be enough to protect troops deployed for short periods in an endemic area.

Protecting the vast populations that live in malarial areas presents a much greater challenge. Ultimately, researchers hope to devise a vaccine that prevents death—which means targeting children under 5, who constitute 90% of malaria fatalities. The goal is to protect these children from the worst of the parasite's onslaught, although not necessarily from the disease itself. Then, the logic goes, the children will acquire their own natural immunity and, like adults in these regions, experience only mild symptoms from repeated infections. But that will likely require a vaccine that induces multiple immune responses against multiple stages of the parasite, says Steve Hoffman, head of the vaccine development program at the Naval Medical Research Center in Silver Spring, Maryland. “Nobody has ever made a vaccine like that before.” Moreover, a truly global vaccine will have to handle not only Plasmodium falciparum—the most virulent parasite, against which most current research is directed—but P. vivax as well.

## A worthy foe

Vaccines against viruses work by stimulating the immune system with either an attenuated version of the virus or a piece of the virus. That primes the immune system to recognize the real thing when it comes along and eliminate it quickly from the bloodstream. “Those viruses or bacteria for which we do have vaccines look the same the whole time they are inside of you,” notes Hoffman. This is not so for malaria, with its four different stages (see sidebar).

The cycle begins when an infected mosquito injects threadlike sporozoites into the bloodstream. The sporozoites travel to the liver, where they burrow into cells and multiply into tens of thousands of tiny round merozoites. Bursting out of the liver cells, the merozoites invade red blood cells; there, they multiply madly and cause the primary symptoms of the disease. Finally, some of the parasites enter a sexual stage and become gametocytes, which are taken back up into the mosquito, where they reproduce and the cycle begins anew.

These stages have evolved in part to avoid the two primary defenses of the human immune system: antibodies, which seek out and destroy invaders in body fluids, and killer, or cytotoxic, T cells, which attack infected cells. The sporozoites, for instance, don't remain in the bloodstream long enough to be hunted down by antibodies, and the merozoites don't stay in the liver long enough for killer T cells to mobilize and attack the infected cells. And once the merozoites move into red blood cells, they are safe from both arms of the immune system. Meanwhile, the parasite multiplies furiously at every step, so that even if an immune response is 99% efficient at wiping out the parasite at any one stage, there are likely to be enough parasites left to multiply and cause disease.

Over the half-century that researchers have struggled to create a malaria vaccine, they have based their belief that it can be done on two models. One is naturally acquired immunity: the fact that people in endemic areas who survive to adulthood no longer die of malaria and often don't show symptoms when they do get infected. When blood from these adults is transfused into children with high parasite loads in their bloodstream, the antibodies in the transfused blood dramatically reduce the children's load. The second is known as the attenuated sporozoite model. Since the 1940s, researchers have known that the sporozoites in an infected mosquito can be debilitated by zapping the mosquito with radiation. If enough of these attenuated sporozoites are then introduced into a human, they can protect against malarial challenge for up to 9 months. “The problem,” says New York University malaria vaccine researcher Elizabeth Nardin, “is that you have to expose the individual to the bites of hundreds or thousands of irradiated infected mosquitoes.” The technique, needless to say, does not represent a viable vaccine strategy, but it has led researchers to study the attenuated parasites to find what in the sporozoite triggers the antibody response.

The initial breakthrough came in 1979, when vaccinologists Ruth and Victor Nussenzweig, a husband-and-wife team at New York University (NYU), identified the primary antibody target on the attenuated sporozoites. It is a protein that constitutes the bulk of the parasite's surface coating, now known as the circumsporozoite protein (CSP). Once John Dame and colleagues at NIH cloned the CSP gene in 1984, the race was on for a viable vaccine. One team from the Army, the Navy, and SmithKline Beecham Biologicals (SB Bio) of Belgium put the CSP gene into bacteria and generated a recombinant protein that they injected first into mice and then, in 1987, into six human volunteers. The Nussenzweigs and collaborators at NYU, the University of Maryland, and Hoffmann-La Roche synthesized a portion of the CSP protein and used that synthetic peptide as their vaccine, first successfully in mice and then in three human volunteers. “We both immunized at the same time and both got more or less the same results,” says Hoffman, who was then a young member of the Army-Navy-SB Bio team. “We each got one individual protected. So we proved the principle, but the response was not as good as anyone wanted it to be, not nearly good enough for the field.”

Among the volunteers who developed malaria were Hoffman and Rip Ballou, another young researcher on the Army-Navy-SB Bio team. The chilling experience —contracting the disease when they thought they were protected—taught them not only how nasty malaria is, they later said, but also just how difficult it is to beat. “That vaccine worked by making antibodies that prevent the sporozoite from getting into the liver,” says Hoffman—a strategy that made a lot of sense. The problem was that “if just one of those sporozoites gets through, you have 30,000 merozoites 1 week later. Then you lose the battle, because the immune response you generate to fight the sporozoite doesn't do anything to those 30,000 merozoites. It doesn't even see them.”

Since then, the two groups have been working methodically to boost the antibody response generated by their vaccines. The Army-Navy-SB Bio team, for instance, tested two dozen vaccines between 1986 and 1996, when their perseverance paid off. They inoculated seven subjects with a modified version of the CSP vaccine—known as RTS, S—and six were protected against a malaria challenge 3 weeks later. This was followed with an RTS, S field trial in 306 adult volunteers, organized by the British Medical Research Council in The Gambia. The results “were unprecedented,” says Joe Cohen of SB Bio. In the first 2 months, the vaccinated group experienced 71% fewer clinical episodes of malaria than a control group. Over the next few years, the researchers plan to continually boost the immune response generated by the vaccine while testing the current version in progressively younger volunteers. Eventually, if the vaccine remains safe and effective, they hope to test it in children between ages 1 and 5. “We feel there is good chance this vaccine will behave as well if not better in children,” says Cohen. “Of course we won't know until we actually evaluate it.”

## Strength in numbers

Despite the success so far of RTS, few researchers believe that it—or, for that matter, any vaccine made from a single malaria antigen—will be sufficient to control malaria in the field. That will require deploying not just one weapon but the full armamentarium. Since that realization has sunk in, the intense competition of the early days has evolved into a sprawling collaboration, as researchers explore the potential of various antigens—some 40 are now under study—from the different stages of the parasite's life cycle. They hope to elicit both antibodies and killer T cells and to attack the parasite wherever it makes itself visible.

One promising strategy is to ambush the parasite at another weak point: when it leaves the liver, but before it invades the red blood cells, where it becomes effectively unreachable. This could be done by targeting the surface proteins of the merozoite. A successful antibody attack against these proteins might prevent the merozoite from penetrating the red blood cells, says Heppner of Walter Reed, which in turn should prevent the cyclic amplification that leads to the clinical disease. “If we can find a vaccine that can block invasion of merozoites into red cells,” he says, “then we [should be able to] completely suppress the infection or suppress it to a trivial level.” Numerous research teams are trying to do just that.

In an effort to prevent transmission of the disease, NIH scientists have embarked on a novel strategy to create a vaccine that would muster antibodies against the reproductive stages of the parasite that appear only when it's in the mosquito. In its sexual stage, the parasite normally lurks in red blood cells until it detects a drop in temperature that suggests it is now safely in a mosquito's gut. Then it leaves its hiding place, reproduces, and starts the process of infecting the mosquito. Using a Trojan horse approach, Louis Miller and his colleagues are trying to create antibodies that can be smuggled into the mosquito's gut with the mosquito's blood meal. When the parasites break out of the blood cells, the antibody will be there to take them on. “Once the antibodies are taken up along with the parasite,” says Miller, “they will block the fertilization of the male and female sexual stages and, if that doesn't work, block the zygote produced from burrowing its way through the stomach wall and infecting the mosquito. This mosquito then can't transmit the parasites back to other people.” Miller and his colleagues think such a transmission-blocking vaccine could be used in combination with a more traditional vaccine and preventive methods such as impregnated bed nets to help eliminate malaria from an endemic region—provided that transmission rates in that area are not tremendously high. So far, the NIH team has demonstrated that the transmission-blocking vaccine works in mice and rabbits; it hopes to begin human trials by next fall.

## Mix and match

In numerous labs, researchers are now looking for the right mixture of antigens, or a “gemische,” in the words of Gina Rabinovich, director of the Gates Foundation's Malaria Vaccine Initiative, that could constitute the ultimate vaccine. “You [would] have some liver-stage antigens, some blood-stage antigens, eventually maybe a transmission-blocking component, and maybe something specifically against Plasmodium vivax, for a world where you have transmission of both [P. vivax and P. falciparum]. This would be the combination to end all combinations, although it makes all the issues in the creation or production of any other vaccine look simple.”

This is where a new technology known as DNA vaccines might make a big difference. These are stretches of “naked” DNA containing genes for viral proteins, which are expressed when the DNA is taken up by muscle and other cells in the body. DNA vaccines have a few enormous advantages and one potential—and equally enormous—disadvantage. The latter is that nobody knows whether DNA vaccines can induce a sufficient immune response to protect a human against any disease, let alone malaria. As for the advantages, DNA vaccines are relatively easy to make compared to a synthetic or recombinant protein (Science, 5 December 1997, p. 1711). Moreover, DNA vaccines, unlike a recombinant or synthetic peptide vaccine, call into action the killer T cells, which destroy infected cells. And that would provide researchers with a long-sought weapon: a way to target infected liver cells, which so far have evaded attack.

Adrian Hill and colleagues at Oxford University have created a DNA vaccine with genes coding for antigenic parts of six different malaria proteins, while Hoffman's team has cocktails with the genes from five, eight, and even 15 different antigens in the same vaccine. In animal studies, these cocktails have raised both antibody and killer T cell responses to attack the malarial parasite in the bloodstream as well as in liver cells. Both teams are also betting on a technique known as a prime boost, in which a DNA vaccine is used to ready the immune system and then a second vaccine—either a recombinant protein or a recombinant virus—is used to enhance the immune response. On 18 September, Hill and colleagues began a field trial in The Gambia of their six-part DNA vaccine, followed by an attenuated version of the vaccinia virus that eliminated smallpox, modified to express the same malarial components as their DNA vaccine.

As science advances, the lingering pessimism in the field arises more from the financial constraints of developing a malaria vaccine. According to a recent NIH report, it takes more than a quarter of a billion dollars and a dozen years to take a vaccine from research through licensing in industry. Much of that money is spent on large-scale clinical trials, which are still a distant dream of malaria vaccine researchers.

“Within 5 years there will be a number of vaccines out there being tested for efficacy,” predicts NIH's Miller. “Within 10 years, some will be ready for a company to try to get them licensed.” The ultimate challenge, says Miller, may be convincing a company to take a chance on a vaccine for a market that is enormous but is also hopelessly impoverished.

16. VACCINES

# Malaria Parasite Outwits the Immune System

1. Gary Taubes

Given a few million years of evolutionary practice, the malaria parasite has become extraordinarily proficient at one task: evading the immune system of its human hosts. This it does in four distinct life stages.

Taking Plasmodium falciparum, the parasite that causes the most virulent strain of malaria, as an example, the cycle in humans begins when an infected Anopheles mosquito partakes of a blood meal and in the process dribbles her saliva, which contains thousands of threadlike sporozoites, into the bloodstream. Within 5 to 10 minutes, far too short a time for the immune system to muster an efficient antibody response, the sporozoites home in on the liver and infect liver cells. There, they are safe from antibodies but susceptible to killer T cells—if they stick around the 10 to 12 days necessary for these defending forces to mobilize. They don't. Instead, the sporozoites begin multiplying furiously. Each sporozoite forms a schizont, which contains some 30,000 round, compact merozoites. In less than a week, the schizonts rupture, killing the liver cells in the process and spilling millions of merozoites into the bloodstream. The merozoites quickly infect red blood cells—the one cell type in the body where they are safe from both antibody and killer T cell defenses.

In the red blood cells, these merozoites can go one of two ways. They can participate in a repeated process of amplification, fueled by the hemoglobin in the blood cell. Each merozoite forms another schizont, this time with 20 new merozoites in it. After 48 hours, the schizonts rupture and the cycle continues. The result is a 20-fold amplification of the parasite burden in the blood cells every 2 days.

Those merozoites that don't form schizonts in the red blood cells can develop into a sexual stage—becoming males and females, known as gametocytes—and reinfect mosquitoes. These are taken up into the mosquito's gut when it takes a blood meal from an infected person. In the gut, the female produces an oocyst—a cross between an egg sack and a cyst—out of which emerge new sporozoites. Over the next 2 weeks, the sporozoites infect the gut, the bloodstream, and fi-nally the saliva glands of the mosquito, at which point she is prepped to reinfect humans.

People don't experience symptoms until the parasite invades the red blood cells. Inside the red blood cells, the parasite extrudes knobs onto the surface of the blood cells that cause them to stick to the lining of blood vessels and capillaries. The knobs, yet another evolutionary response to the human defense mechanisms, keep the infected blood cells from passing through the spleen, where they would be purged. By decreasing the rate of blood flow, they also result in one of the primary symptoms of malaria: severe anemia. Malaria's characteristic fever and chills arise when the schizonts rupture the red blood cells and in the process release not only merozoites but also a “malaria toxin.” Moreover, as the parasites amplify in the bloodstream they can eventually infect two out of every three red blood cells. Explains Steve Hoffman, director of the malaria vaccine program at the Naval Medical Research Center in Silver Spring, Maryland: “Now you have, say, 60% fewer red blood cells and 60% less oxygen-carrying capacity, and you have severe anemia right then and there.”

17. DRUGS

# Reinventing an Ancient Cure for Malaria

1. Eliot Marshall

As drug resistance renders cheap antimalarials ineffective, a promising candidate has emerged from an overlooked source

Nowhere on Earth is malaria more threatening than in northwestern Thailand. Here, the deadliest form of the parasite, Plasmodium falciparum, has been toughened by decades of exposure to antimalarial drugs—conditions that promote the survival of drug-resistant strains. In this caldron, says researcher Nicholas White of Bangkok's Mahidol University, a “nightmare scenario” is brewing: Local parasites are becoming resistant to every cheap drug that works. The old standby, chloroquine, “is gone, just about everywhere,” agrees Pierro Olliaro of the World Health Organization (WHO), and resistance to newer drugs is emerging.

Flush with new funds and aiming at targets now being provided by genome sequencers, researchers are trying to concoct the next generation of antimalarials (see p. 439 and Science, 17 March, p. 1956). But those drugs are a decade away, while the need today is urgent. The situation would be truly desperate, White says, if it weren't for the arrival in the 1990s of a new type of antimalarial from Asia: artemisinins. These drugs haven't yet been approved for clinical use in Western countries, although they have been used as herbal remedies in China for 2000 years. White, whose team is funded by Britain's Wellcome Trust and WHO, is championing one member of this family, a water-soluble form called artesunate. He thinks it may be the most potent new weapon against malaria in decades. It could also be a lifesaver for children in remote villages.

Abundant clinical data show that artesunate knocks down the number of parasites in the blood faster than any other drug does, according to Steve Hoffman of the U.S. Naval Medical Research Center in Silver Spring, Maryland. If given early, it can stop an infection from progressing to a deadly coma. Yet it must be used with care, says White, partly because of concerns about neurotoxic effects but mainly to avoid promoting drug resistance. The past approach, using malaria drugs one at a time and replacing them as they toppled “like dominoes,” only encouraged resistance. So White argues that artesunate must be deployed in combination with other drugs to hit the parasite with a complex challenge.

Artemisinins were unknown to Westerners until about 20 years ago, says Steven Meshnick, an epidemiologist at the University of Michigan, Ann Arbor. But 2 millennia earlier, Chinese herbalists noted that fevers could be treated with a tea based on the flowering plant qinghao (Artemisia annua). An ether extract of qinghao, qinghao-su, gained scientific prestige in China during the 1960s, when a search for organic drugs revealed that it was effective against the mouse form of malaria. Chinese researchers developed new drugs and tested them on thousands of patients, publishing medical reports in English in the early 1980s.

One of the first Westerners to pounce was Dan Klayman, a U.S. Army malaria researcher, now deceased. Unable to obtain samples of qinghao from China, Klayman eventually found some Artemisia annua, a weed called sweet wormwood, growing near his lab in Washington, D.C., and cultivated it. (Klayman's review of the Chinese clinical work and his lab's work on qinghao's chemistry appeared in Science, 31 May 1985, p. 1049.) In the 1980s, both Army- and WHO-sponsored researchers began testing an oil-based version for intramuscular injection to treat severe malaria. It was a stable formula, but it was sometimes poorly absorbed, and in animal studies it injured the brainstem at high doses. White thinks it was a mistake not to move quickly to other formulations.

Meanwhile, Chinese research in the 1970s and 1980s suggested that the water-based artesunate formulation given by tablet or needle worked well. China now manufactures artesunate, and White estimates that more than 1 million people have used it safely in Asia. What's more, says White, recent trials in Thailand suggest that a combination of artesunate and mefloquine is rapidly absorbed and immediately bioavailable and is seemingly “a better drug.”

But artesunate got trapped in a regulatory cul-de-sac. Although Chinese researchers had published safety and efficacy data, Western authorities looked askance at their research methods. WHO, for instance, declined to launch major clinical trials of artesunate in the 1980s, after some of its advisers expressed concerns about neurotoxic effects seen in animal studies. White suspects the biggest roadblock was that artesunate “came from the wrong place.” It didn't have the right regulatory “credentials … which people seem to regard almost as religious edicts.”

In 1994, as mefloquine resistance spread through Thailand, WHO decided to support trials of artesunate with mefloquine as the last line of defense. White and co-authors reported in the 22 July issue of The Lancet that this oral therapy yielded efficacy of “nearly 100%.” Today, WHO, the Wellcome Trust, and others are backing advanced clinical trials in Africa as well, promoting a rectal suppository formulation, or “rectocap.” The goal, says a WHO official, is to have a product that can be distributed to remote areas and administered in an emergency, giving a family time to get a child to the clinic before deep coma sets in. The advantage of this low-tech version, says WHO, is that it would be cheap, easy to store, not dependent on needles, and usable even in a child who is vomiting. Another advantage, says White, is that artesunate, especially in combination therapy, is less likely than older drugs to promote resistance, because it is rapidly eliminated from the body. “There is no sign of resistance to date,” says White, “although you should never be complacent about malaria.”

Given the promising data from Thailand, White and WHO officials are eager to deploy artesunate more widely. Because no drug company has taken the initiative, WHO plans to submit a drug application to the U.S. Food and Drug Administration (FDA) to obtain a license to develop the rectocap in collaboration with a European manufacturer. WHO doesn't need FDA's approval for research, but having it would make it easier to manufacture the drug later. An FDA official observes that artesunate looks “very, very promising,” while cautioning that its potential toxicity must be studied further.

But White is impatient. “We need new combination drugs in the villages—yesterday,” says White, who thinks they might have been able to halt the spread of mefloquine resistance that way: “It's a disaster that we didn't get them out 5 years ago.”

18. GENOME SEQUENCING

# Closing In on a Deadly Parasite's Genome

1. Elizabeth Pennisi

Extremely difficult to decipher, the Plasmodium genome is already providing targets for new drugs. Next project: the mosquito genome

There's no better way to locate the soft underbelly of a pathogen than through its genome. The genome provides a view of all the key proteins involved in infection and in the pathogen's life cycle. And those proteins make good drug targets. Sequencers are now barreling through the genome of the most virulent malaria parasite, Plasmodium falciparum, and they hope soon to turn their sequencing machines loose on the genome of the Anopheles mosquito that transmits it. Combined with the emerging data on the human genetic code, “the three [genomes] will go a long way in helping us understand this disease,” says Michael Gottlieb, a parasitologist at the National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland.

Unexpectedly, the smallest genome of the trio may be the toughest to sequence. Although relatively puny (fewer than 30 million bases compared to, say, the 100-million-base genome of the nematode Caenorhabditis elegans), the parasite's genome has put up quite a fight. In 1996, work on the genome began on both sides of the Atlantic. The sequencing groups, each with its own source of support, have coordinated their effort, dividing up the genome and talking often about their progress—or lack thereof.

Almost as soon as they started, the teams ran into trouble. Compared to the genomes of other organisms tackled to date, P. falciparum is chock full of adenines and thymines, two of the four nucleotide bases that are the building blocks of DNA. The proportion of A's and T's averages 82% and soars to 97% in spots, says molecular biologist Sharen Bowman of the Sanger Centre near Cambridge, U.K. This not only makes copying the DNA a challenge but also bogs down the sequencing and analysis. P. falciparum “has been much more difficult” than “every other organism we've experienced,” says Bowman. Indeed, “most of the malaria community said we'd never be able to do it,” recalls Malcolm Gardner of The Institute for Genomic Research (TIGR) in Rockville, Maryland.

Nevertheless, the first two of P. falciparum's 14 chromosomes were completed fairly quickly, in 1998 and 1999, proving that it could be done (Science, 6 November 1998, p. 1126; Nature, 5 August 1999). And even before these chromosomes were finished, there was a groundswell of support for tackling the entire genome. NIAID; the Military Infectious Diseases Program of the U.S. Department of Defense; the Burroughs Wellcome Fund, based in Morrisville, North Carolina; and Britain's Wellcome Trust have kicked in a total of about \$23 million for various sequencing efforts over the past 3 years. Stanford sequenced chromosome 12; TIGR and the U.S. Naval Research Center did 14, 10, 11, and 2; and Sanger tackled the rest.

The Sanger group may have landed the toughest task: deciphering the “Blob”—three chromosomes that have to be treated as one. To work on any chromosome, each lab separates the DNA by allowing the entire genome to migrate through a gel. Because lighter chromosomes travel farther, each can be identified and be cut out of the gel for sequencing. But chromosomes 6, 7, and 8 are so close in size that they cannot be separated.

Sequencing of all the chromosomes—including the Blob—is virtually complete, but the groups are now struggling to assemble the thousands of pieces of sequence for each chromosome in the right order. “It's a long, hard slog,” says Gardner, who, with Leda Cummings, has led TIGR's effort. The problem is that lots of pieces don't seem to fit anywhere because of long, difficult-to-sequence stretches of the same base. Nevertheless, Bowman expects that within 9 months most of the genome will be finished and annotated, with the Blob coming a year or so later. In the interim, all the labs are releasing their raw data daily, and malaria researchers have already identified new drug targets from those data. If the Anopheles genome project, being spearheaded by Frank Collins of the University of Notre Dame in Indiana and colleagues in Crete, Germany, and France, gets off the ground, drug designers may soon be able to find still more chinks in malaria's armor.

19. MOSQUITO ENGINEERING

# Building a Disease-Fighting Mosquito

1. Martin Enserink

In a futuristic scheme, researchers are trying to engineer a malaria-resistant mosquito to replace natural populations

She lands on your arm so softly she doesn't even wake you up; neither do her frantic attempts to find a blood vessel during the next minute and a half. Repeatedly, she thrusts her needle-sharp, double-barreled mouthpiece into your skin, trying her luck. As she sucks through one barrel to see if anything comes up, she spits a sophisticated mix of drugs down the other to prevent blood from clotting and your vessels from constricting. When she finally hits a vein, she quickly fills up her gut. Then she leaves. One meal is enough: As she flies away, suddenly three times heavier, she has enough proteins on board to produce about 100 eggs a few days from now.

Meet the female Anopheles mosquito, arguably the most dangerous animal in the world. It's not those 3 or 4 microliters of blood that she steals; it's the sinister gift that she sometimes leaves behind, some of the Plasmodium parasites that cause malaria. Within weeks you may come down with a fever; if you're a child or a pregnant woman, the encounter may kill you, as it does 1 million or more people every year.

For decades, scientists have tried to stop her from spreading disease. And now, with resistance against insecticides on the rise and a U.N.-backed push to phase out DDT, several labs have embarked on the most ambitious and futuristic of all approaches to combat malaria: They hope to replace billions and billions of mosquitoes in the world's endemic areas with new strains, created in the lab, that would be “refractory,” or unable to transmit the parasite.

The idea is not that farfetched, these researchers claim. In a paper scheduled for publication in the American Journal of Tropical Medicine and Hygiene, for instance, a team led by Anthony James of the University of California, Irvine (UCI), reports that it has created mosquitoes that produce antibodies against Plasmodium. In lab studies, these antibodies reduced the number of parasites in the insects' salivary glands—their last stop in the mosquito's body—by 99.9%.

Granted, says James, the researchers used Plasmodium gallinacium, which infects chickens, not humans, and its mosquito host, Aedes aegypti, rather than the feared Anopheles. And they cut corners by infecting the mosquitoes with a smartly designed virus that expresses the antibody gene rather than creating a true transgenic—in other words, they did not insert the gene into the mosquito's genome. But creating a transgenic insect is feasible, says James, one of several researchers trying to reach that goal. Indeed, most researchers optimistically predict that the first malaria-resistant Anopheles larvae will crawl out of their eggs in a lab somewhere within a few years.

Others agree the study is an important step. “It's the first time anybody has shown you can actually make a mosquito refractory to a malaria parasite,” says Joe Vinetz of the University of Texas Medical Branch (UTMB) in Galveston. “I think it's really neat.”

It's also a welcome boost for a field that still faces enormous hurdles. To realize the grandiose plan of building a “better mosquito,” researchers have to overcome three problems. First, they have to find genes that frustrate the parasite's life cycle, complete with switches to turn them on in the right place and time in the mosquito's body. Next, they need a way to stick those genes into the mosquito. Finally, they have to devise a way to give their winged creation a flying start in the real world, so that it can beat out natural populations—not in a centuries-long, evolutionary struggle, but in a few years.

The second step—finding a way to genetically engineer mosquitoes—has long stymied researchers. In the 1980s, they had hopes of borrowing a technique used for years to equip the fruit fly, Drosophila, with new genes. In the fly, researchers simply attach the desired gene to a so-called transposon, a short piece of DNA that knows how to worm itself into the fly's genome. Then they inject the transposon into a fly egg; with luck, it lands somewhere on a chromosome where it works and doesn't harm other genes. But however hard they tried, researchers couldn't get the standard Drosophila transposon, simply called P, to deliver a gene—any gene—in mosquitoes. “It put a cloud over the whole research area,” says James. “At meetings, we were always talking about things that didn't work.”

That started to change in the 1990s with the discovery of a series of new transposons, such as Hermes, a house fly transposon discovered by David O'Brochta of the University of Maryland, College Park, and Peter Atkinson of the University of California, Riverside. In 1998, James's team, collaborating with Frank Collins (who is now at the University of Notre Dame in Indiana) and others at the Centers for Disease Control and Prevention (CDC) in Atlanta, used Hermes to insert an eye color gene in Aedes aegypti. And last June, Andrea Crisanti's team at the European Molecular Biology Laboratory in Heidelberg, Germany, reported slipping the gene for a green fluorescent protein into Anopheles stephensi, the mosquito that transmits malaria in India, with yet another transposon called Minos. Now, several research groups are feverishly trying to tinker with the biggest killer of all: Anopheles gambiae, the main malaria vector in Africa. Success is just months away, predicts CDC's Mark Benedict.

Meanwhile, other researchers have been making considerable progress on the first step: finding genes that can thwart the parasite. In theory, every stage of Plasmodium's life cycle in the mosquito is fair game. After being sucked up during a blood meal, Plasmodium's male and female gametes merge in the blood droplet inside the mosquito's gut. Eventually, they form a so-called ookinete, which pierces the sturdy sac surrounding the blood meal and nestles inside the gut wall. There, the ookinete develops into an oocyst, which eventually bursts open on the other side of the gut, releasing thousands of minuscule sporozoites into the mosquito's circulatory system, which is filled with a fluid called hemolymph. The sporozoites travel to the salivary gland, force its wall open, and take up residence in the mosquito's saliva—ready for the next round in humans.

Alexander Raikhel and colleagues at Michigan State University in East Lansing want to attack the sporozoites as they float in the hemolymph. To do so, they recently took the gene for defensin, an antimicrobial compound that occurs naturally in Aedes mosquitoes, and coupled it to a promoter—a stretch of DNA that determines when and where a gene is switched on—that activates the gene only after the mosquito has eaten. When they built this ensemble into Aedes mosquitoes, the researchers reported 2 months ago, it boosted defensin levels in the mosquito's hemolymph. They're currently testing whether this also drives down P. gallinacium sporozoite numbers.

UCI's James also attacks sporozoites in Aedes mosquitoes, but with a different strategy. As they describe in their upcoming paper, the researchers first coaxed mouse cells to produce antibodies against CSP, a protein known to occur on the sporozoites' outer coat. Next they created an artificial gene coding for a slightly altered version of that antibody and used a virus to infect the mosquitoes with the construct. The insects started producing antibodies, which apparently attack the sporozoites very efficiently, leading to the 99.9% decrease in the salivary glands. James is now trying to do the same in Anopheles.

UTMB's Vinetz, meanwhile, is eyeing a much earlier stage of the parasite's life cycle—its piercing of the blood meal sac—using a Plasmodium enzyme he discovered last year.

With so many options, researchers are confident they'll be able to engineer a refractory Anopheles gambiae one way or the other. But step three—replacing existing mosquito populations with that humanmade critter—is the real sticking point, says Marcelo Jacobs-Lorena of Case Western Reserve University in Cleveland. “That's not going to be easy at all.”

Many mosquito engineers hope the very same transposons they use to slip a gene into the mosquito's chromosome can also perform this trick. That's because some transposons have a thoroughly selfish way of spreading: Whenever two individuals mate, one of which has the transposon, all of their offspring inherit it. Researchers know, for instance, that transposon P has conquered Drosophila melanogaster populations all over the world in just the past century.

But researchers have no idea whether Hermes, Minos, and other transposons used to transform mosquitoes today could have the same effect. Nor do they know if a transposon and an artificially attached gene would stay together as the transposon started spreading.

Others are banking on a different way to convert the mosquito population: by enlisting Wolbachia, a bizarre bacterium that lives inside the cells of many insects. To favor its own reproduction, Wolbachia ensures that whenever an infected and an uninfected partner mate, either all their offspring will be infected or there will be no offspring at all. As a result, Wolbachia can spread like wildfire.

If you could stick some of the genes that can make mosquitoes refractory into Wolbachia, then infect mosquitoes with the bug and set them loose in a natural population, you might make it refractory in a relatively short time, speculates Scott O'Neill, a Wolbachia researcher at Yale University. The catch is that researchers don't know of any Wolbachia species that naturally inhabit Anopheles, but they could probably figure out a way to trick one into doing so, says O'Neill.

But even if these dreams come true, more problems loom. Entomologists don't know all that much about Anopheles populations, but recent studies by UTMB's Gregory Lanzaro and his colleagues in Mali have shown that what looks like one Anopheles gambiae population is in fact several different subpopulations that don't interbreed much. That could complicate matters considerably, as researchers would have to fix each population separately.

A host of other questions must be answered before one could even think about a field trial, says Kathryn Aultman, program officer at the Parasitology and International Programs Branch of the National Institutes of Health. Some people think transposons may “escape” and sweep through other insect populations, with unknown effects. The first field trial would probably be held on an island to contain the transposon, says Aultman. “If it turns out to have been a disastrously terrible idea, you can just spray the place down with malathion,” she says.

Further complicating matters, it appears that each transposon can only sweep through a population once. If so, researchers would have to choose the attached gene very judiciously, lest they blow their only chance. And finally, the idea of releasing transgenic mosquitoes may prove highly controversial, if the recent backlash against transgenic crops is any indication. “We're planning some meetings to start discussing all of these issues,” says Aultman. With so many uncertainties, spending money on a vaccine or new drugs may seem a safer bet. But James defends this unconventional approach: “The malaria vaccine has been predicted to be just around the corner for at least 2 decades. I think spending a few percent on something else is a reasonable investment.” Fortunately, adds Vinetz, researchers would not have to wipe out each and every natural mosquito. Models have shown that even when only part of the population becomes refractory, transmission may drop enough for the epidemic to peter out.

If they're right, mosquitoes will still keep hunting for your blood, as they have for at least 150 million years. But there's a chance they will leave you with just an itch and not a malaria parasite.