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New Clues to Asthma Therapies Identification of major players in the inflammatory cascade that damages the lungs in asthma offers targets that may produce better treatments for the disease A risi.ng pollen count means itchy eyes, scratchy throats, and stopped-up sinuses for many allergy sufferers, but for those whose allergies trigger asthma, this means more than just discomfort. For them, exposure to usually harmless pollen or other allergens can set offa life-threatening attack in which the airways leading to the lungs close up-- ma~ing the sufferers feel, they say, as if they are trying to breathe with a full-grown per- son standing on their chest. These frighten- ing attacks are becoming more and more common. Since |980, the prevalence of asthma has almost doubled in the United States. Today, it afflicts more than 1.at mil- tion people in this count,'y alone, and costs almost 5000 lives each year--with no signs o~ ~eveling off. Researchers don't have a clear idea of what is causing this increase (see sidebar). Nor have they worked out what predisposes some ,people to asthma ir~ the first place. But on one frot~t, dley are making real headway. They are beginning to pinpoint many of the key biological players el~c ~ake part in asthma a~tacks. And that in turn is providing searchers with openings for new ways to treat asthma, some of which are just entering clini- cal use. "There is a story that's coming out," says Yale .puhnonologist Jack Elias. "It's be- ginning to hang together." The main dmme of the atot~] is inflamma- tion. Doctors have known for years that asthma attacks are often triggered by aller- gens, such as cockroaches, dust-mite feces, pollen, or animal hair. Now, researchers are working out the exact .cascade of events that these allergens--and other nonallergen trig- gets sudx as cokt air, viral infections, anti exercise--set in motion in the lungs. At the top of the cascade is a particular type of im- mune cell called tl~e T lymphocyte, which responds to the noxious substances by send- ing out more than a dozen chemical signals: so-called cytokitxes, which attract inflamma- tory cells ~o the airways of tile lungs. These warriors, in particular those called the eosinophils, release chemical weapons of their own. This second wave of signals, including histamine and small, flirty mol- ecules called leukotrienes, causes blood yes- se'ls to leak and lung tissues to swell, con- ~raccs the smooth muscles of the airways~ cutting off the air supply like squeezing a hose--and encourages mucus production, further dogging already constricted airways. Each crisis causes an immediate difficuk3, in breathing, and repeated crises over time lead to permanent lung changes that may make the next attack even worse. Current ~,sthma treatments are aimed at the end rest, It. Bronchodilators open t!xe air- ways, and antihistamines and steroids reduce the inflammatory cascMe are a few years away from patients' medicine cabinets, two have already made it to the shelves. Both drugs; which were approved lace last year by the U_.S. Food and Drug Administration (FDA), got ahead of the crowd for the simple reason that _their c.argets, the [eukotrienes, were impli- inflammation. But by dissecting the chain . cated ip the cascMe nearly 50 years ago. of command that leads to an attack, re- Released by activated eosinophils and searclxers have idelxtified a whole new set other immune-system soldiers recruited to 11-5 and others Allerge.~.n" ---,-~ '1~.~ Ant'g n:~r senting.~-=Z: 11-4 and others Histamines Leukotrienes Overzealous warriors. The T lymphocyte helps command the immune cells--including mast cells and eosinophils--that react to pollen, cold, exercise, and other stimuli to trigger an asthma attack. of promising targets for asthma drugs. "The therapy is moving back closer and closer to ~he beginning of the inflammation cascade," says Harold Nelson, an allergist and immu- t~ologist at the National Jewish/vledica[ and Research Center in Denver. The hope is that these therapies, because of their improved specificity, will be more effective and less liable to cause dangerous side effects than current treatments. Aithough most of the treatments aimed at: asthmatic lungs, the leukotrienes have several effects that contribute to the airway constriction and inflammation of asthma. They recruit other inflam- matory ceils, for example. But they are particularly effective in contract- ing the smooth muscle of the broncixi, the tubes carrying air from the trachea into the lungs, bdolecule for molecule, says pulmonologist Jeffrey Drazen of Brigham and Women's Hospital in Boston, the leukocrienes are the mQ~jt potent bronchoconstrictors ever de- scribed--a fact, he adds, "that was not lost on the drug companies," which set out to develop inhibitors. The two approved last year are Zi- leuton, which blocks a vital enzyme needed for leukotriene synthesis and is marketed by Abbott Laboratories in Chicago, and Zafir[eukast, which btoc~ the lipid's receptors on smooch muscle and ocher cells and is produced by Zeneca Pharmaceuticals, based in the United Kingdom. Two more receptor blockers, from lVlerck and from SmithKline Beech- am, are awaiting FDA approval. The drugs have worked miracles in some bard-to-crest patients, Drazen says. "In some patients it's like manna fl'om heaven. We treated a veterinar- ian allergic to dogs and cats who was just miserable. On this treatment, he is a normal person again." But for reasons that are currently unclear, only about half of all patients respond to the drugs; in the other half, rhere is almost no change, says Sally Wenzel ~f National Jewish, who helped conduct several of the prcapproval clinical trials. Stopping inflammation early Patients who receive no relief from ~:he [eukotriene inhibitors still have reason to 'hope, however. While the leukotrienes act late in the inflammatory cascade, other el- www.sciencemag.org • SCIENCE * VOL. 276 - 13 JUNE 1997 1643

forts are aimed at interrupting it before it ge~s established. One development propelling that research is the recognition that a particular subset ofT lymphocytes seems to be a major culprit in asthma and other allergic diseases, responding with undue vigor to apparently harmless invaders. In work done nearly a decade ago, re- searchers working with T ceils from mice found they could divide the cells into two groups based on the cytokines they produce. Members of one set, which they called cells, produce a set of signals that orches- trate attacks on unfiami[iar ceils, protecting the body against bacteria ,.< and tumor ceils. Those in = the other set--the TH2 cells--produce inflamma- tory signals normally di- rected against parasitic in- vaders. They also encour- age the antibody-producing B cells to secrete IgE anti- bodies, the hallmark of al- lergies, which help trigger the inflammatory responses. As researchers learned more about these activity patterns, it became clear that TvI2 overactivity is a major factor in asthma. High levels of IgE, for example, are common in asthma patients. And one of the key TH2 cytokines, called interleukin-5 (I1-5 for short), helps trigger the eosinophils that can wreak havoc in asthmatic lungs. Although the dis- tinction between T~f 1 and TH2 cells is not as cut-and-dried as many might like--many hu- man T cells seem to produce both TH1 and T~t2 signals~Yale's Elias says the concept "has opmxed doors in thinking about asthma" and about potential new therapies. Some of those efforts are aimed directly at thwarting the effects of Ta2 cytokines. For exaraple, I[-5 appears to be a good target. If the cymkine's action-in mice is blocked, ei- ther by inactivating the gent that codes for the protein or by giving the animal antibod- ies that prevent II-5 from binding to and ac- tivating eosinophils, the animal's airways do not react to allergens, says immunologist David Huston at Baytor College of Medicine in Houston. This suggests several possible ap- proaches to asthma treatments. Two pharmaceutical companies, Schering- Plough and SmithKiine Beecham, have been working on the development of human ver- sions of mouse anti-[l-5 antibodies, and have been getting promising results in trials with animals--including primates. In the animal trials, the anti-II-5 antibodies have pre- vented both eosinophil inflammation and airway constriction. Human trials "are immi- nent," Htlsto~. says. In addition, Huston and his colleagues, as well as a number of industry groups, are work- ing to engineer an inactive version of II-5 itself that would bind to the cytokine's re- ceptor on eosinophils without triggering the ceils, while also preventing the native mob ecule from binding. Perhaps closer to pharmacy shelves is an antibody that blocks IgE itself. After TI~2 signals trigger B-cell production of an IgE witlx a particular specificity, the antibody attaches to mast cells, and when it encoun- ters a protein it recognizes as threatening, it triggers the mast cells to unleash their weap- ons, including leukotrienes and histamine. If there were some way to block the IgE trigger, researchers reasoned, the whole battle could be avoided. But disarming lye has proved to be a tricky business. When researchers tried to no indication of any side effects," Jardieu says. Results from a second round of trials, which te~ted the antibody's ability to protect 400 asthma patients from natural exposures to allergens, should be published later this summer, says Jardieu, and she expec_ts phase III trials--testing anti-IgE against the best available treatment--to begin this fail. Terrible trio. House dust mite (top left), Altemaria mold (center), and birch pollen (left) are all common triggers of asthma attacks. inactivate it with antibodies, some of their efforts turned out to have just the opposite effect, even triggering fatal allergic reac- tions. "I've accidentally killed animals with [the wrong kind of] anti-IgE," says Paula Jardieu of Genentech, who has led her com- pany's efforts to develop the therapy. The problem was that these antibodies attached to the same part of IgE that binds the aller- gen, thus triggering, rather than blocking, IgE's effects on mast ceils. Recently, however, researchers have iden- tified the specific region of lye that binds to the mast cell receptor, enabling them to produce antibodies that block only that site. Buoyed by promising results in mice, they went on to build a human version of the mouse antibody. Through DNA ma- nipulation, they were able to transplant the IgE-binding region of the mouse mol- ecule onto the base of a human antibody. They tested the resulting "humanized" an- tibody by giving it to monkeys allergic to ragweed and found that it prevented the typical skin sensitivity to the pollen. Initial trials, designed to test the safety of this antibody in humans, have been very positive, says Jardieu. The 40 patients who received doses of the antibody suffered only mild reactions when the research team blew allergens into their lungs, with "absolutely Tipping the balance against asthma Another therapeutic strategy currently be- ing investigated aims to short-circuit mis- placed Tt~2 attacks. T~I anti Tt~2 activities are mutually suppressive: Signals from one ceil type inhibit the activity of the other. So several researchers are attemp~:ing to take advantage of certain bacteria that induce vigorous THI responses, causing the im- mune system to pump out messengers, such as interlet, kin-12 a,xd interferon-% ithat inhibit Tll2 cell activity. Some, incit,ding Steven Holgare of Southampton University in the Un.ited Kingdom, Julian Hopkin of Oxford University, and Gra- ham Rook of University College, London, are working with whole bacteria. They have just begtm a series of studies in which they will attempt to prc~tect allergic volun- teers from theDerils of allergy sea- son by injecting them with a harm- less bacterium of the Mycobacte- rium genus, which--like many bac- teria-is a strong Tnl inducer. The hope, says Holgate, is that "if we give this to asthmatic subjects, maybe it can switch off the allergies." Immunologists found a few years ago that it is particular sequences in t'he bacterial DNA that induce such a strong Tt~ 1 respense. Those sequences plW a key role in a therapy under development by tmmu,xologists Eyal Raz and Dennis Carso,~ and allergist David Broide of the University of California, San Diego. The temn is attempting to devise a more effec- tive means ~f desensitizing people to their allergies, which currently involves repeat- edly injecting them with small amounts of the allergen, often for years. To bolster this effect, the researchers have designed a smal[ circular piece of DNA, called a plasmid, that includes both the DNA encoding any of sev- eral common allergen proteins and fragments of bacterial DNA. In early tests, the team injected the pIasmids into mice, whose skin cells took up the DNA. There the p[asmid started pro- ducing the antigen protein. "It's like immu- notherapy," says Raz, "but instead of having to give it repeatedly, you give it nnly twice or three times and ir is there permanently." At the same time, the researchers hoped, the bacterial DNA in the plasmids woukt crank up the suppressive effects of the treatment by 1644 SCIENCE • VOL. 276 ° 13 JUNE 1997 • www.sciencemag.org

Asthma is a disease of the industrialized 20th century. First described in the mid- 18005, it ,nay have existed before that time, but was very rare. It is still rare in developing countries. But in the developed world in the last 2 decades, asthma rates have skyrock- eted~doubling in the United' States since 1980. "Asthma and allergies have become representative of the westernization of our society," says William Busse, an allergist at the University of Wisconsin, Madison. Researchers do not yet know why. The}, have come a long way of Immunology and Allergy in Rome and his colleagues found that soldiers who tested positive for antibodies to hepatitis A virus--a sign of more childhood infections in general, say the authors-~-had significantly fewerallergies. (Theresults appeared in the April British Medical Journal.) Researchers haye._also turnedup other hints that early im- munological experience can affect a child's chances of devel- oping asthma~very early experience, if Jill Warner at the University at Southampton in the United Kingdom is right. in dissecting the sequence of events that leads to individual .. When she and hes__col~le_agues studied immune cells from pre- asthma attacks: the activation by an allergen or other trigger of mature and terminated fetuses, they found that cells from certain immune ceils, which in turn marshal other cells that ruses as young as 22 w~el~ ~zould multiply when exposed to mount inflmnmatory attacks on the lungs (see: main text). BUL hgu_se dus~ m!tgs!a~_.d bi_rch pol!en suggestingsl_aa~_they ~_eso_g-=~ why some people are predisposed to such attacks--and why their nized the allergens from a previo{~s ex~osur~ W~r i~ Ed~-= numbers are now increasing--remain mysteries, although re- rently studying whether |imiting a mother's-exposure to corn- searchers have some clues. Iqcreased exposure to environ- mental allergens and immune sys- tem changes due to fewer child- hood infections may play a role, say some_And geneticists are dosing in on a host of genes that have been linked to increased asthma suscep- tibility. The search for a cause is urgent, says Busse, because it might point to ways of preventing chil- dren from developing the disease in the first place. For the moment, he says, "we are treating the conse- quences of the disease, not prevent- ing it from occurring." :: Ofie of the most popular theories holdsthat asthma has increased partly 16 14 12 10 8 6 4 2 0 '82 '83 '84 '85 .'86 '87 !88..'89 '90 '91 '9_2:93__'94 Asthma ascend!rig. Researchers are struggling to explain asthma's dramatic increase~ " mon allergens can prote~t her. unborn child from later develop- ...... ing allergies and asthma. Still, environmental influences can't be the full answer, because asthma susceptibility is well "known to run in tiunilies. Ant, mber of all- out hunt~ are now under way for asthma-susceptibility genes, which might be interacting with environ- mental factors to drive the rising incidence. So far, only one team-- at Sequana Therapeutics Inc. in San Diego---says it has pinpointed a gene, and team members are keep- ing details of their find under wraps (Science, 30 }via}, 1997, p. 1327). But several more puhlic searchm are clos- ing in on genes. because of greater exposure to aller- A team led by Carol Ober, a ge- gens such as hot,se dust mites or cockroaches. Alle_rgist Thomas neticis( at the Un.iversity.of Chicago, reported at the recent Plaits-Mills of the University of Virginia notes ~at _np_waday_s _ American Thoracic Society meeting that its work with the South children spend more time indoors in front of th<television= in Dakota~H{~{te-rf~Ta~@lixious group of 5000 descended from 64 close contact with caq~ets and upholstered furniture crawling with dust mites. Still, Plaits-Mills says, this "Annette Funicello" effect, as he calls it, "can't explain the rise by itself." The asthma increase is just too great and has occurred even in dry regions i~{where the dust mite is uncommon. '~> Another feature of modern life might also be contributing: the fall in childhood infections. Early infections, say proponents !;g:of this idea, may stimulate a kind of immune response that '~suppresses later allergic reactions. Earlier this year, Oxford pulmonologist Julian Hopkin and colleagues at the Wakayama "Medical Center in Wakayama, Japan, found that children who i,'~i.res~onded strongly to a skin test indicating that they had been. :{[g~posed to tuberculosis are less likely to suffer from asthma or :~-:- ot_b_er allergic diseases (Science, 3 January, p. 77). Similarly, in a ;::s.tu.dy of1600 Italian soldiers, Pao[o Matricardi of the Laboratory 18th-century ancestors, has linked asthma or asthmalike condi- tions to specific regions on chromosomes 2, 13, and 2 I. And in a wider stud}, of the general population, the multicenter Collabora- tive Study on the Genetics of Asthma reported in the April issue of Nature Genetics that its researchers have linked asthma in various ethnic groups to a half-dozen different chromosome re- gions. Other studies have found linkages to regions on Chrom0~ somes 11 and 12 containing genes known to code for important players in the inflammation that is part of asthma pathology. Thelinkages, like all the other clues, are a long way from solving the asttima fiddle, but they are a start. "Everyone knew [the gene search] was a black hole," says Susan Banks-Schlagel, manager of asthma research at the National Heart, Lung, and Blood Institute. "They said, 'Oh, you'll never find anything.' But some interesting things are starting to happen:" -G.V. eliciting production ofinterferon-gand other TH2 suppressors. Again, initial results are promising. Mice receiving the novel immunotherapy have less IgE in their blood, fewer eosinophils in their lungs, and less evidence of-TH2-type cytokines when they are exposed to the sub- stance to which they were allergic. Raz and his colleagues have formed a company, called Dynavax, and plan to begin human trials in collaboration with researchers at Johns Hopkins University as soon as they receive FDA approval---expected "within the year," says Raz. But the TH2 model that has inspired these new treatments may not be a complete an- swer to the asthma puzzle. Viral infections, for example, have been blamed for 80% of severe asthma attacks, says Daniel Rotrosen of the National Institute of Allergy and In- fectious Diseases. But viruses have usually www.sciencemag.org • SCIENCE • VOL. 276 • 13 JUNE 1997 1645

been considered a trigger of a THl-type response. Some researchers believe that vi- ruses are not the immediate trigger, but contribute to asthma susceptibility by at- tacking the lining of the lungs, leaving the inner layers more exposed to environmen- tal allergens or other traditional asthma triggers--which would then activate TH2 cells and the other responses they orches- trate. Others believe the viruses may have an inside role, activating certain genes in the nucleus that exacerbate o~ trigger the inflammatory cascade. Those unanswered questions might ex- plain why new treatments such as the [eu- kotriene inhibitors will not work for every- one. But the fact that the drags don't help some patients may be as important: as the help they do give some people: "That is where it gets really interesting," Drazen says. "Up until, now, we have graded asthma as mild, moderate or severe," whiclx is only of limited help to physicians trying to deter- mine the best course of treatment. Patients' different responses to the vari- ous drugs may help doctors sort out what many suspect is the case:'Asthma is not a single disease. Like pneumonia or anemia, Brigham and Women's Drazen says, asthma is a set of ~ymptoms that has varied causes. The new treatments, by getting closet to those causes, may help doctors divide pa- tients into subgroups based on how they respond to treatments, he adds. That, in turn, will help researchers determine how to treat each patient most effectively---a de- velopment, certainly, that wil.l help mil- lions breathe easier. -Oretchen Vogel IMMUNOLOGY New Lead to Safer Marrow Transplants Bone-marrow transplants have become a mainstay of medicine's battle against blood- cell cancers, such as leukemias and lym- phomas, as well as against certain noncan- cerous blood diseases. But in at least half of all patients, the donor immune ceils turn against the recipient's own tissues, triggering a deadly ailment called graft-versus-host dis- ease (GVHD). Now a team of doctors led by hematologist Claudio Bordignon at the San Raffaele Scientific Institute in Milan, Italy, may have found a solution to this problem. On page 1719, the group reports the first successful human test of a gent therapy designed to halt the attack of the donated cells on the recipient's tis- sues. The researchers genetically en- gineered the transplanted cells with a self-destruct button that enables doctors to kill them selectively with a drug if they turn mutinous. This allowed the team to wipe out GVHD in two of the three patients who de- veloped it, and partially eliminate it in the third--without using immuno- suppressive drugs. That success is a boost for the strug- gling field of genetic therapy, says im- munologist Drew Pardoll of the Johns Hopkins University School of Medicine in Baltimore, who calls the work "one of a very small cohort of examples in which gent therapy has been shown to have clinical util- ity." Indeed, if further studies bear out the early promise of the technique, it could make bone-marrow transplants much safer and more effective. Doctors might even start using such transplants more broadly, in pa- tients with less advanced disease. The tech- nique is "very exciting," says immunologist Philip Greenberg of the Fred Hutchinson Cancer Research Center in Seattle. "It has the potential to improve substantially the out- come of [bone-marrow] transplantation." The strategy's seeds were planted in 1990, when Bordignon first heard about the prob- lems with GVHD that were cropping up in the top bone-marrow transplant centers, par- dcuiarly in patients who relapsed and re- quired infusions of donor lymphocytes, lviar- row transplants are needed because the high doses of chemotherapeutic drugs and radia- tion given to leukemia and lymphoma pa- tients in an effort to rid them of all cancer cells also destroy the patients' bone marrow, the vital source of both the red cells and the infection-fighting white ceils of the blood. But unless the donor is an identical twin, the transplant may turn on a patient, caus- ing GVHD, as the foreign white blood ceils Under attack. Multiple lymphccytes are invading the epidermis of human skin with graft-versus-host disease. attack essential organs such as the liver, gut, and skin. Clinicians have sought to avoid this attack by sifting out all of the mature T lymphocytes from the foreign mar- row befbre infusing it. Those are the cells that trigger GVHD, but their removal leaves the patient more vulnerable to infections or can- cer relapse. If infection or cancer does de- velop, tile patient can be infused with the do- nor T cells~again running the risk of GVHD. Bordignon, a doctor trained in gent therapy, recalls that he asked himself, "How might one take advantage of gent-transfer technology to control this problem?" He set out in early 1992 to test whether he could introduce a "suicide gent" into tlxese cells, tlxen use the gent to kill the cell8 if they triggered GVHD. Results with cultured ceils looked promising: Lymphocytes engineered with the gent for the enzyme thymidine ki- nase died when he doused them with the antiviral drug ganciclovir, which the enzyme converts to a.deadly poison. After showing that ganciclovir also kills the suicide gene-bearing lymphocytes in mice, Bordignon and colleagues began ~heir pilot study in humans. In 1993, they infused donor lymphocytes bearing the thymidine kinase or suicide gent into 12 patients who, after re- ceiving bone-marrow transplants, had suf- fered complications such as cancer relapse or virus-induced lymphomas. The [ymphocytes survived in the patients for up to a year, bat- tling the tumors to achieve complete or par- tial remissions in five of the eight patients for whom results are available. Of the three patients who developed GVHD, ganciclovir totally shut down the immune attack in two; in tile third, tile disease was attenuated. The success may have been limited in the third patient, Greenberg specu- lates, because some of the infused cytes may not have borne th~ suicide gent. Still, if the new gent-therapy procedure helps ~wo out of every three patients, it will be an improvement. Researchers caution, how- ever, that tests in many more patients will be needed to determine just how effective the therapy is. Toward this end, Bordignon is or- ganizing a muldcenter European trial that he hopes will start by the end of 1997. But even that may not settle the question, says Pardo[l, because transporting the Italian group's tech- nique to other centers may be difficult: "I c~n count on one hand, with a couple of finge/rs missing, the number of groups that could do this [gent-transfer procedure] with high effi- ciency." He adds, however, that developing simple, reproducible protocols for the proce- dure could boost that number. One thing is certain. The therapy has al- ready shown sufficient promise, says Richard O'Reilly, a marrow-transplant pioneer at New York City's Memorial Sloan-Kettering Cancer Center, to ensure that it "will be looked at by many people." -Ingrid Wickelgren 1646 SCIENCE • VOL. 276 • 13 JUNE 1997 • www.sciencemag.org