Fostering Communication and Collaboration

The nihCatalystA Publication for NIH Intramural Scientists

National Institutes of Health j Office of the Director m Volume 8 Issue 1 Q January-February 2000

Dye and Let Live:Magtags EmergingAs Cellular Compass

by Celia Hooper

The Clinical Center’s Laboratoryof Diagnostic Radiology Re-

search (LDRR) is on to some-thing big, and the excitement sur-faces quickly when you talk to labchief Joe Frank or staff scientist JeffBulte. Along with colleagues at NIHand at the University of Wisconsin,Bulte and Frank have developed anelegant New Age version of whatold-school mi-

croscopistscalled a vital

dye—that is, asubstance thatmarks cells with-

out damagingthem.

The New Agetwist is that the

“dye” in thiscase is a mag-netic label that allows researchers

to image tagged cells in living ani-mals, noninvasively, withoutradioiosotopes, at a microscopiclevel, using magnetic resonance im-aging (MRI).

If LDRR’s work pans out, it couldyield the label of choice for track-

ing cells in vivo. Beyond the track-ing of oligodendrocyte progenitor

cells injected into the rat—LDRR’sstarting point—Frank envisions thetechnique being used to trace a va-riety of stem cells and transgeniccells that have been harvested, cul-tured, or manipulated in some wayoutside the body and then injectedback in. This could include “anystem cell, tumor cell, or other trans-plantable cells—for instance, isletcells or specific subpopulations of

T cells to evaluate temporal-spatial

continued on page 10

Magtag“magic —seepage 10

Gene Therapy Trial and ErrorsRaise Scientific, Ethical, and Oversight Questions

by Fran Pollner

Current Event

N early a decade after it permit-ted NIH intramural researchersto pioneer this country’s firsttwo human gene therapy trials—andsome 350 diverse human gene therapyprotocols later—the NIH RecombinantDNA Advisory Committee (RAC ) helda three-day assembly centered on thefirst reported death of a gene therapyresearch patient attributed to the

therapy itself.

The death last September of JesseGelsinger, an 18-year-old patient withpartial ornithine transcarbamylase(OTC) deficiency, injected a soberingpause in a field rushing toward the 21stcentury’s promise of an avalanche ofgene therapy applications inspired bythe completion of the Human GenomeProject and related discoveries.The death also precipitated an FDA

freeze on the clinical trial in whichGelsinger was enrolled—a safety andbiological efficacy study of the recom-binant adenoviral vector-OTC gene de-livered to the liver via the intrahepatic

artery. Gelsinger was the scheduledpenultimate patient in what was to havebeen an 18-patient study and the sec-ond one to receive the highest proto-col-defined dose in this dose-escalationtrial that was begun in 1997 at the Uni-versity of Pennsylvania in Philadelphia.

The pivotal speakers at the RAC pro-ceeding, held December 8 through 10,1999, on the NIH campus and underbright media lights, were the OTC study

Fran Pollner

University ofPennsylvania gene therapyresearcher James Wilson reads a prepared

statement at a press briefing after thefirst dayof the RACproceedings in December. The RAC

meeting was convened to review the trackrecord ofadenoviral gene therapy vectors and

the conduct ofthe UPenn protocol thatresulted in the death ofpatientJesse Gelsinger.

Wilson was introduced by Paul Gelsinger(right), Jesse Gelsinger’sfather, shown exiting

after delivering his own statement andanswering reporters’ questions. Wilson

declined to entertain questions.

investigators: veteran gene therapy re-searcher James Wilson, director of theInstitute for Human Gene Therapy, andsurgeon Steven Raper, both of LTPenn,

continued on page 4

CONTENTS

1

Gene TherapyTrial and Errors

Magtags:The Dye is Cast

2From the DDIR:Talk is Dear

3Ethics Forum:For the Record

4

5Gene Trials Oversight

7Gene Therapy:Preclinical WorkAt 5 Research Court

8-10

Harold Varmus:Like a Rolling Stone

12-15Recently Tenured/Cartoon

-KETT£P>

Harold Varmus on the road again. See p. 8. Gene Therapy:OTCD Picture

16Catalytic Questions

The NIH Catalyst

From the Deputy Director for Intramural Research

Improving the Postdoctoral Experience at NIH?Two Words to the Wise

Postdocs are critical to NIH. They have a handin most NIH intramural research and are ourlargest group of trainees. And critical to

postdocs is mentoring. A previous column on theexpectations that a postdoc should have about train-

ing at NIH led to the “Guide to Mentoring and Train-ing in the Intramural Program.” This booklet, whichI hope you have all read carefully, provides anoutline of the responsibilities of both mentor andtrainee at NIH. This column targets what I con-sider to be the most basic first step in mentoring:

Whether you are a supervisor or mentor—or both

—

I encourage you early on, to approach your fel-lows and begin a dialogue that opens with a simpleinvitation:

“Let’s talk.”

Starting shortly after a

postdoc arrives at a lab,

and then continuing on aregular basis, the point of

discussions between men-tor and fellow should be

to set goals and assessprogress toward them,provide a framework for

the training experience at

NIH, clarify expectations

on both sides, and providecareer advice for the fel-

low.

These discussionsshould be frank and fair,

or they will be of little

value. If necessary, discus-

sions can be initiated by

the fellow; but, in any

case, they should occur.

Where it is helpful, meet-ing details could be put in

writing.

When a fellow enters a lab, his or her supervisorshould spend some time discussing the researchproject or projects currently underway in the lab;the role of the fellow in each project; expectations

about independence, authorship (including the like-

lihood of there being publishable work from aproject), and collaborations; any prior agreementsthat could affect the fellow’s work; and any rulesthat govern conduct in the lab.

A first meeting is also a time to provide and dis-cuss the “Guidelines for the Conduct of Science in

the NIH Intramural Program” and the “Guide toMentoring and Training.” There should be explicit

mention of the duration of the appointment, the

experience of previous fellows in finding jobs, and

what the expectations of fellow and mentor arewith respect to careers in biomedical research. In a

recent survey conducted by the NIH Fellows’ Com-mittee, about one-quarter of the fellows who re-sponded were unable to identify their mentors. (SeeThe NIH Catalyst

,March-April 1999, pages 4-5.)

The first meeting is an excellent time for the super-visor to offer to be a mentor and to suggest otherscientists who could also offer useful advice to thefellow.

Beyond this first talk, there should be regularmeetings to discuss science, conduct of science,

and career-related issues. Data sessions should beheld frequently—perhaps weekly—especially earlyin the training of a postdoc. The same Fellows’ sur-vey suggested that about 20 percent of the fellows

who responded meet lessthan once a month withtheir supervisors.

There should also be an

annual meeting specifically

to evaluate the work of thefellow and indicate whether

he or she is on track toachieve research and career

goals. This is an important

opportunity to begin a dis-

cussion about jobs after the

postdoctoral experience, to

indicate that some goals areimpractical, and to help a

fellow choose a reasonable

course of action.

In the year before a fel-

low leaves, more specificcareer planning has to take

place. Is another postdoc a

reasonable option? To whatjobs should a fellow apply?

What scientific work can betaken from the lab and what should stay? It is very

helpful to fellows to have someone critique theircover letters, their CVs, and their applications to

specific jobs. Will the mentor be willing to makesome telephone calls on behalf of the fellow?

Finally, the fellow should have an exit interview

with the mentor to outline what went well, what

could use improvement, and what should be

avoided in the future. These kinds of discussion

are difficult, but very important if we want to fulfillour duties as mentors and improve the postdoctoral

experience at NIH.

—Michael GottesmanDeputy Directorfor Intramural Research

These discussions [between

MENTOR AND FELLOW] SHOULD

BE FRANK AND FAIR, OR THEY

WILL BE OF LITTLE VALUE. If

NECESSARY, DISCUSSIONS CAN

BE INITIATED BY THE FELLOW;

BUT, IN ANY CASE, THEY

SHOULD OCCUR. WHERE IT IS

HELPFUL, MEETING DETAILS

COULD BE PUT IN WRITING.

2

January — February 2000

Ethics Forum

A Matter of Integrity:Fabrication in Research and Administrative Records

byJoan P. Schwartz, Ph.D., NINDSAssistant Director, OIR

Science is built on trust. Eveiyoneexpects that data published in the

literature are real data, and webase new experiments on that assump-tion. But occasionally an instance ofmisconduct is uncovered in which anauthor of a scientific paper has falsified

or fabricated the reported results. Aproposed new definition of researchmisconduct—which will apply govern-ment-wide for the first time—was pub-lished for comment in the Federal Reg-ister [64 (198): 55722-55725, October

14, 19991. The definition is:Research misconduct is defined as

fabrication, falsification, or plagiarism

in proposing, performing, or reviewing

research, or in reporting research re-

ports.

Fabrication is making up results andrecording or reporting them.

Falsification is manipulating re-search materials, or changing or omit-ting data or residts such that the re-search is not accurately represented in

the research record.

Plagiarism is the appropriation of

another person’s ideas, processes, re-

sults, or words without giving appropri-ate credit, including those obtainedthrough confidential review of others’research proposals and manuscripts.

Research misconduct does not in-clude honest error or honest differences

of opinion.

Research is defined as all basic, ap-plied, and demonstration research in allfields of science, engineering, and math-ematics. The research record is definedas the record of data or results that em-body the facts resulting from scientificinquiry, and includes, for example, labo-ratory records, both physical and elec-tronic, research proposals, progressreports, abstracts, theses, oral presen-

tations, internal reports, and journalarticles.

How NIH handles allegations of sci-entific misconduct within the intramu-ral program will also be modified. Thelargest change is that NIH will cany outits own investigations, rather than turn-ing them over to the Office of ResearchIntegrity (ORI). The NIH Committee onScientific Conduct and Ethics is currentlyrewriting the Guidelines that describe

NIH Policies and Procedures for Inves-tigation of Scientific Misconduct—a sub-ject for a future Catalyst column.Over the past few months, instances

of falsification have turned up at NIH

involving radiation con-

tamination survey data,

animal weight records,

and publications listedin the bibliography of

a scientist’s CV. In pon-dering the ramifications

of these events, the spe-

cific offices involved, as

well as the NIH Com-mittee on ScientificConduct and Ethics, firstconsidered whether thematerial in question constituted a part

of the research record as defined above,

in which case the falsification or fabri-cation would be deemed scientific mis-conduct. In each case, it was concludedthat, technically, no scientific misconducthad been committed but the scientist in-volved had behaved unacceptably anddeserved some type of sanction.

Radiation contamination surveys mustbe done monthly in any laboratory thatuses radioisotopes. The Nuclear Regu-latory Commission (NRC) requires themas a condition of the NIH’s license be-

cause they ensure that no staff are ex-posed to radiation of which they areunaware. Twice recently, NIH lab staffmembers fabricated surveys in responseto notifications that survey results for cer-

tain months were missing. Because suchrecords are not a part of the research

record per se and are never publishedas research results, this fabrication doesnot reach the bar for scientific miscon-

duct. Rather, surveys of this sort might

fall into a category of administrative

records. Nevertheless, such fabricationis a violation of NRC regulations and theindividual(s) involved could be subjectto criminal penalties. The “authorizeduser” responsible for the laboratory

could be baixed from using NRC-licensedradioactive materials for up to 3 years,and NIH could deliver an official repri-mand that becomes a part of the individ-ual’s personnel record. As all NIH staffwere recently notified in a desk-to-deskmemo, such falsification of radiation con-tamination surveys is unacceptable.

The second incident involved fabrica-tion of weights for a research animal that

had been placed on a restricted diet

—

instead of weighing the animal and re-cording the actual weight weekly. As aconsequence, the animal lost signifi-cantly more weight than acceptable,thereby endangering its health. Theweight data were used neither in a sci-

entific publication norin a specific research

protocol and, again,could be considered anadministrative record.

However, these actionsclearly violated the NIHGuidelines for AnimalCare, and the scientistinvolved therefore re-

ceived an official rep-rimand and was pro-hibited from participat-

ing in animal protocols for the duration

of his appointment at NIH.The third example involved the iden-

tification of a series of irregularities in

five references included in the bibliog-

raphy of a researcher’s CV. For two ofthem, actual references existed in the lit-

erature, but the first author’s name hadbeen replaced with the name of the per-son accused of the falsification; the yearhad also been changed. Two other ref-erences cited papers that do not exist;the fifth had the correct authors and titlebut the wrong journal. A CV is also atype of administrative document, usedfor applications for jobs and grants. Ifthis CV had been used to apply for afederal job, the falsification would havebeen a criminal act. If used in a grantapplication, it would have constituted re-search misconduct. In this case, the NIHappointment of the errant CV author wasending and was simply not renewed. Amajor issue, however, was what a su-pervisor should say if asked for a letter

of recommendation for this individual.It is ultimately a decision each supervi-sor must make, but NIH has recom-mended that the fact be disclosed that aCV was received from the individual inwhich certain bibliographic citationsproved to be erroneous. Such a state-ment advises the next employer of po-tential problems with the applicant.Too often, when a supervisor encoun-

ters such behaviors, he or she is eagerto get the offender out of the laboratory

but avoids coming to grips directly withthe issues of fabrication and falsification.But we cannot turn our backs on theintegrity of either the research record or

the administrative record that supports

it. The public trust—reflected in our li-censes to conduct research with radio-isotopes, animals, and human subjects,as well as the tax dollars that supportNIH—is contingent on our responsiblebehavior as a community. H

Joan P. Schwartz

3

The NIH Catalyst

Gene Therapy Trialcontinued from page 1

and pediatrician Mark Batshaw, chiefacademic officer at the Children’s Na-tional Medical Center in Washington. All

three apologized for communicationgaps with oversight agencies and lapsesin complying with adverse event report-ing requirements, which, had they beenadhered to, might have led to a proto-col modification or a reevaluation of

Gelsinger’s candidacy for trial participa-

tion. Gelsinger’s precipitous and ulti-mately irreversible response to the ex-perimental therapy included signs ofdisseminated intravascular coagulation

(DIC), massive cytokine release, adult

respiratory distress syndrome (ARDS),and multiple organ failure.The purpose of the meeting, federal

officials and RAC officers emphasized,was largely educational: to pool infor-mation on the biology and toxicity ofadenoviral vectors; to scrutinize the con-

duct of the OTC trial with a view to-ward identifying red flags and improv-ing gene therapy trial design in general;and to revisit federal reporting require-ments, interagency communications, andthe role of the RAC in gene therapy over-sight. Patient advocates, however, voiced

worries that gene therapy research mightbe stopped or curtailed; industry repre-sentatives cautioned against increased

public scrutiny that might bare tradesecrets or misleading information; andreporters wanted to know the specificsof protocol violations and any otherproblems in the OTC study, as well assteps planned by overseers to enforcecompliance with scientific and ethicalguidelines in the conduct of human genetherapy research.

RAC chair Claudia Mickelson, a bio-safety officer at the Massachusetts Insti-

tute of Technology in Cambridge, andmolecular biologist and former RAC chair

Inder Verma, of the Salk Institute for Bio-logical Studies Laboratory of Genetics

in San Diego, served as moderators.

Adenoviral Vector ExperienceAccording to Amy Patterson, director

of the NIH Office of Biotechnology Ac-tivities (OBA, formerly the Office of Re-combinant DNA Activities), about 4,000patients have participated in about 350gene therapy trials in the past decade;adenoviral vectors have been used inabout one-quarter. Immediately afterOTC investigators notified OBA ofGelsinger’s death, Patterson said, OBAnotified the more than 70 other research-ers currently using similar methods intheir own gene transfer studies.

After Gelsinger’s death, the FDA con-ducted a database search of all genetherapy protocols with adenovirus vec-

tor and found no similar ARDS, aplasticanemia, or extensive DIC findings, anFDA official said.A refresher course in adenovirus ba-

sics was presented by experts sum-moned to the RAC proceedings. Amongthe relevant facts noted were that aden-ovirus can cause fatal hepatitis in im-munocompromised individuals; and thatadenovirus attaches to the coxsackie-

adenovirus receptor (CAR) in the liver,a receptor whose function and level ofexpression in different tissues are not

well known and whose abundance inthe liver varies among humans and ani-mal models. To render adenovirus suit-able as a gene vector, viral genes re-quired for replication are deleted; thus

enfeebled, adenovirus generally serves

well as a vector. There are, however,some concerns regarding viral infection:In simian species, adenoviral replication

is enhanced in the context of co-infec-tion with another virus, such as SV40;

OTCD in General and in the Case ofJesse Gelsinger

O rnithine transcarbamylase deficiency(OTCD) is an inborn error of me-tabolism in which the second enzyme inthe urea cycle is missing; it affects one in40,000 to 80,000 children. The inabilityto excrete urea can precipitate an acuterise in ammonia concentrations, braindamage, coma, and death. Neonatal mor-tality within the first month is 50 percent,and 50 percent of early survivors diewithin the first five years. Without a livertransplant, 75 percent of survivors willsustain severe neurological dysfunction.

Patients with partial OTCD have some

residual enzyme activity and can be man-aged on a strict low-protein diet and the “al-ternative pathway” regimen (intravenous so-dium benzoate, sodium acetate, and argin-ine hydrochloride), co-developed by MarkBatshaw, principle investigator in the UPennstudy. They are always at risk of coma, how-ever, and the therapy, says Batshaw, is “in-complete at best."

Jesse Gelsinger’s partial OTCD was diag-nosed at 33 months, when he presented le-thargic, incoherent, and with elevated am-monia and glutamine concentrations. He washospitalized at age 10 and again in Decem-

ancl adenoviais can also function as ahelper virus, for instance, potentiating

the growth of parvovirus. Still to be un-derstood is the ability of certain integrins

that facilitate adenoviral integration into

target cells to induce cytotoxic andapoptotic responses.

Verma recounted his own lab’s adap-tation of adenovirus for use as a genevector, with the deletion of the Ela, Elb,

and E4 adenoviral genes to blunt itsimmunogenicity and the summoning ofcytotoxic T cell lymphocytes. The easeof disarming these replication compo-nents and of growing billions of virusparticles quickly, as well as adenovirus

infectivity of both dividing and nondi-viding cells, he said, make it an “attrac-tive” vector.

University of California at San Fran-cisco (UCSF) surgical oncologist RobertWarren offered observations from a trialinvolving adenovirally packaged p53 de-livered via the intrahepatic artery to the

liver of colorectal cancer patients with

hepatocellular metastases. Postinfusion

transaminase elevations invariably camedown and were never dose limiting; hy-potension, he said, was the dose-limit-ing toxicity, with liver toxicity “signifi-

cantly less than expected.” Comment-ing on questions later related to thepossibility that high doses could mobi-lize proinflammatory cytokines and leadto intravascular coagulation, Warren saidthat duration of exposure to the vectorin his trial may have been too short toobserve adverse cytokine events. “Wewould expect adenovirus induction ofcytokines in the liver,” he said.

Margaret Rick, hematology chief in theNIH Clinical Center pathology depart-ment, noted that acute hepatic failure is

among possible triggers of DIC. She sug-gested that patients be screened for he-

ber 1998. In June 1999, he met screeningcriteria for the UPenn trial, but his ammo-nia concentration the day before actualdosing three months later exceeded thecutoff for entry eligibility. He was placedon the alternative pathway regimen, hy-drated, and the next day, September 13,dosed as per protocol. He died on Sep-tember 17. Though the relationship of hisdeath to his liver function at dosing had

not been established, co-investigator SteveRaper asserted that “We agree that futureinclusion criteria” should address standards

of patient status directly before dosing.

4

January— February 2000

Gene Therapy Oversight

Overseers Answer to the Press (left toright): Amy Patterson, director NIH OfficeofBiotechnology Activities; Lana Skirboll,director, NIH Office ofScience Policy; PhilNoguchi, director, FDA Division of Cellularand Gene Therapies; and Kathryn Zoon,

director, FDA Centerfor BiologiesEvaluation and Research, respond to

questions during a news briefing after the

first day of the RAC meeting. Nearly allquestions centered on FDA 's oversight rolein the OTC study, protocol violations, andFDA ’s steps to uncover, punish, andpreventirregularities in gene therapy clinical trials.

The rules and roles of federal agenciesinvolved in overseeing gene therapyexperiments are undergoing review andmay be revised in the wake of JesseGelsinger’s death. Specifically, the role of

the NIH Recombinant DNA Advisory Com-mittee (RAC) is being revisited, and pro-posed amendments to the NIH Guidelines

for Research Involving Recombinant DNAMolecules (NIH Guidelines) have been pub-lished. The harmony—or lack of it—betweenNIH and FDA adverse event reporting re-quirements in the conduct of clinical trialsis also being scrutinized.

Revisiting the RAC. Before vacating theoffice of NIH director, Harold Varmus an-nounced the formation of a subcommitteeof the director’s advisory group to recom-mend further actions NIH might take to mini-mize adverse events in gene therapy trials.Although the group's charge also places therole of the RAC back on the table, Varmusstands by his actions in 1995 to recast theRAC from a quasi-regulatory body with ap-proval authority over every gene therapyapplication. to a public policy forum on novelmethods in gene therapy clinical trials andthorny issues related to gene therapy (seeinterview, p. 10).

NIH Guidelines. NIH clarified the defi-nition of adverse events and investigators’reporting obligations in a proposed actionto amend the NIH Guidelines, published inthe Federal Register November 22, 1999-The proposal reaffirms that investigators

must report serious adverse events immedi-ately to NIH, so NIH may rapidly notify theRAC and others involved in gene transferstudies. “Immediately” is defined as no laterthan 15 days from the event. A “serious ad-

verse event” is defined as any “expectedor unexpected adverse event, related orunrelated to the intervention, occurring at

any dose” that results in death, a life-threat-ening event, hospital admission or pro-

longed stay, or disability.It also rejects recent claims by some

gene therapy investigators and sponsorsthat human gene transfer protocols andserious adverse event reports are trade

secrets. Informed consent documentswould reflect the necessarily public na-ture of RAC discussions of adverse events.The NIH Guidelines require the princi-

pal investigator to report serious adverse

events to local review bodies and the FDAas well as to NIH and the federal Office ofProtection from Research Risks. FDA re-porting requirements require the studysponsor (who has presumably been in-formed by the investigator) to immediatelyreport serious and unexpected adverseevents to FDA. NIH proceedings are pub-lic; FDA proceedings are often closed.

During the RAC proceedings, industryrepresentatives accepted the need for im-mediate reporting only of “related and un-expected” serious adverse events, andsome RAC members voiced skepticism thatthey could deal meaningfully with reports

of all adverse events. H —F.P.

patic vulnerabilities to tissue factor

upregulation due to infection- or cytokine-induced endothelial cell damage.RAC member Estuardo Aguilar-Cord-

ova, of the Baylor College of Medicinein Houston, suggested that dose-related

adenoviral toxicity may not be linear.“Perhaps there is a threshold effect at a

certain point where only a slight increasein dose results in a great increase in tox-icity,” he said, noting that different spe-cies exhibit different levels of inflamma-

tory response to the equivalent dose and,to make matters more complicated, titerand dose specifications are not standard-ized. “It’s not clear we are all even talk-ing in the same language,” he com-mented, a lament later answered bySavio Woo, president of the AmericanSociety for Gene Therapy, who offeredhis group’s services in developing stan-

dards for vector quantification.

Phase 1 adenoviral vector-genetherapy studies have been ongoing atthe University of Pennsylvania for the

past five years and have involved 95 pa-tients, 18 of whom participated in theOTC study. Prior to his detailed accountof that trial, Wilson summarized find-ings among the 77 other gene therapypatients enrolled in trials addressing cys-

tic fibrosis and various types of malig-nancy. “In general,” he said, “toxicitiesseem to be dose-dependent, time-lim-ited, and confined to the target site."

However, he added, there is a sugges-tion of “broader cytokine release” in the

presence of fever.

Ron Crystal, chief of pulmonary andcritical care medicine at New York'sCornell Medical Center, summarizedsafety data from gene therapy studiesinvolving El/E3-deleted adenoviral vec-

tors of three different transgenes. Among90 patients with cystic fibrosis, metastatic

colon cancer, or coronary or peripheralvascular conditions that would benefitfrom angiogenesis, there have beenmore than 140 gene administrations,more than 44,000 follow-up days, and13 deaths—all related to the patients’disease and unrelated to therapy dos-age. One serious adverse event in acystic fibrosis patient was linked tobronchoscopic delivery and has not re-curred since a switch to aerosol spray,

he said. Adverse pulmonary events werefound to be dose related in animal stud-ies conducted by the Genzyme Corpo-ration (Cambridge, Mass.).

The OTC TrialThe prelude, conduct, and aftermath

of the OTC trial were presented byBatshaw, Raper, and Wilson. Using thesparse fur mouse as a model, they de-livered what would have been a fatalammonia dose to animals pretreatedwith the adenoviral OTC gene vector.The ensuing rapid drops in glutamine

and ammonia concentrations matchedthe speed the investigators sought to pre-vent the irreversible brain damage sus-tained by children who manage to sur-vive coma longer than 72 hours. The ad-enoviral vector evolved from an El -de-leted to an El-deleted/E2-mutated and,finally, to an El/E4-deleted construct.Although newborns with full-blown

OTCD were the population for whomthe gene therapy was ultimately targeted,it was decided that the study cohort forthis first experiment to determine safetywould be stable adults with partial dis-ease who could give informed consent.The investigators submitted their pro-

tocol application to the RAC in March1994, when the RAC still had approvalauthority, along with the FDA, for allgene therapy protocols. In 1995, the RACapproved the protocol contingent onwhat members concluded would be asafer route of administration: intrave-

nous, rather than intrahepatic. The FDA,however, later opted for intrahepaticdelivery, in part to lessen the possibility

of unintended delivery of the transgeneto the gonads. The RAC was never in-formed of the protocol modification.“We recognize that we probably

should have gone back to the RAC todiscuss this,” Batshaw said, “but RAC’sresponsibilities were changing. . . . Weapologize.” Wilson issued the same apol-ogy during his presentation later that day.

5

The NIH Catalyst

-

FDA approved the protocol—with theintrahepatic artery route—in 1996; thefirst patient was dosed in April 1997; andJesse Gelsinger, the last patient, wasdosed in September 1999.

All told, four men and 14 women par-ticipated in the trial. High fever post-infusion was frequent, as were backacheand nausea. Drops in platelet counts andphosphates were dose related and in-creased over the course of the cohorts

(the protocol called for six cohorts, with

three patients in each—the first twowomen and the last a man—to receivedoses escalating in half-log increments

beginning with 2 times 99 particles/kg

and ending with 6 times 10n ). Similarly,transaminase elevations appeared to bedose related, but not consistently.An odd and surprising finding related

to antibody response to the vector: ad-enoviais-naive patients developed anexpected lymphoproliferative response(LPR), but those who had antibodies tobegin with experienced a “transient LPRloss,” a phenomenon some panelistsviewed as a safety concern in the con-text of inadvertent exposure to aden-ovirus infection during LPR downtime.This loss of T cell ability to respond toadenovirus, Wilson said later that day,was not seen in any other clinical oranimal trials.

In general, the team was concernedabout platelet lowering and its relationto vector dose. In collaboration with

FDA, the team began DIC monitoringwith the second cohort, tracking plate-let counts and fibrin split products be-fore, during, and after infusion and keep-ing an eye out for the presence of vec-tor in the systemic circulation.

But it was not until the fourth cohortthat there were recurring problems thatthe investigators were required by pro-tocol to report to FDA before proceed-ing with subsequent dosing. These in-volved marked elevations in liver func-tion assays, two of which were reportedin a timely manner and two of whichwere not. Raper acknowledged theselapses: “We should have called FDAbefore dosing patient 14, and we shouldhave made another call before proceed-ing to the next dose level,” he said.Other irregularities mentioned by FDA

officials during the meeting but not ad-dressed publicly by the investigators in-cluded the investigators’ failure to getFDA permission to have a man (JesseGelsinger) replace a woman as the sec-

ond patient in his cohort and that infor-mation in the consent form submittedto FDA—that primates had died on highdoses in preclinical studies—was re-moved from the final consent document.When Raper turned to the specifics of

Jesse Gelsinger ’s clinical course, he didnot address a question raised during the

meeting: whether the investigatorsshould have pro-ceeded to infuseGelsinger in the face of

abnormal liver func-tion tests that required

alternative pathwaytherapy prior to dos-

ing. He later impliedthat patient status be-

fore infusion shouldbe rethought.

Gelsinger’s CourseGelsinger’s status

eight hours postinfu-

sion was not unlikethat of other study par-

ticipants. Raper said:he had a fever, normalliver function tests, andno evidence of DIC; hewas given intravenous potassium phos-phate for low phosphate concentrations.The morning after, however, nurses no-ticed an altered mental state; his ammo-nia concentration had risen, and he wasjaundiced and tachycardic. A coagula-tion workup showed increased pro-thrombin time and fibrin split productsand decreased platelets.He became “progressively obtunded”

and by the second evening he was co-matose. Chest X-ray showed infiltratessuggestive of ARDS. Raper described aseries of developments in Gelsinger andactions taken by medical teams to off-set or reverse what eventually becamewhat he called a “desperate lung situa-tion.” These measures did not save thepatient.

Commenting generally on Gelsinger’sclinical course and the efforts to savehis life, UCSF’s Warren later observedthat “each intervention seemed logicaland appropriate” but did not prevailagainst the “cascade of irreversible

events.”

With the Gelsinger family permission,

Raper said, a postmortem examinationwas performed. There was little vector-induced hepatitis and no significant signsof DIC. Jesse Gelsinger died of intrac-

table ARDS, with anoxia evident in theliver, kidney, brain, and spleen. Theteam speculated that ARDS was second-ary to “secondary inflammatory response

syndrome,” referred to by Raper as “im-mune revolt.”Bone marrow yielded “perhaps the

most unexpected findings: an absenceof erythroid precursors and mature

granulocytes, suggest-

ing acute insult.”

The team specu-lated that the anemiacould be an idiosyn-cratic reaction to the

medications or a sign

of human parvovirusinfection. One RACconsultant noted dur-

ing discussions later

that the one diseaseassociated with such

acute and strikingbone marrow aplasiais parvovirus infec-

tion.

Questions“We in no way ex-

pected to see what wesaw in Jesse Gelsinger. Our animal stud-ies never demonstrated these pulmonaryevents,” said Wilson, who presented achronology of the team’s vector manipu-

lations and animal studies—includingmice, newborn and adult rhesus mon-keys, and baboons—and ruminated onthe cytokine findings in Gelsinger’s case

and the questions remaining to be an-swered.

Test animals, he said, had demon-strated self-limited, dose-dependenthepatitis, a transient decline in platelets,

and transient transaminase elevations,with an apparently biphasic dose-toxic-

ity relationship. Dose-limiting toxicities

were liver damage and DIC at signifi-cantly higher doses, he said, than that

received by Gelsinger. UCSF’s Warrencommented: “We have not seen the pul-monary dysfunction in our 60 patientsthat you saw with Jesse Gelsinger.”Wilson elaborated on Gelsinger’s im-

mune response to the vector, as well asvector biodistribution on autopsy. At thehigher vector doses received by patients

in the last two study cohorts, there wererapid, dramatic cytokine increases, spe-

cifically of interleukin-6 (IL-6) and IL-10. Recovery followed in all cases but

Gelsinger’s, whose IL-6 trajectory never

James Wilson details gene therapytrial events to RAC members,scientists, and the public.

6

January — February 2000

returned to base line. “Maybe we are ac-tivating some aspect of the immune sys-tem,” Wilson said, suggesting that some“inciting event of acute cytokine release”

from macrophages and monocytes hadoccurred. “What was different about thispatient?” he asked—and listed hemodi-alysis, intubation, and external ventila-tion among the atypical proceduresGelsinger had undergone.The investigators used PCR to track the

vector after hepatic artery infusion. Thehighest DNA concentration was foundin the liver—first in the macrophages(Kupffer cells) and then the hepato-cytes—with significant amounts also ap-pearing in the spleen, lymph node, andbone marrow. Biodistribution, Wilsonremarked, “was not as hepatospecific asI had hoped.” RAC co-chair Verma laternoted that animal models are generallynot good enough to pinpoint biodis-tribution. He cited the fact that rhesusmonkeys and humans do not have thesame CAR distribution.

After the death, the team studied thespecific vector lot Gelsinger had received,as well as the equivalent of the preced-

ing lot, and tested them at the same dosesin rhesus monkeys. “Clinically, the ani-mals did fine,” Wilson said.Among issues to be investigated, he

said, are what role, if any, Gelsinger’sunderlying condition and medicationsplayed in his outcome; whether he hada genetic predisposition to such an out-come; why the vector distributed beyondthe liver and whether this affected out-come; and what stimulated cytokine re-lease and, again, whether this affectedoutcome. “We don't know the roles ofIL-6 and IL-10,” he emphasized.Asked what he would have done dif-

ferently, Wilson replied, “Including nothaving done this at all, the trial couldhave been designed differently [with re-spect to] the half-log increments. The in-crements should be smaller.” He notedthat the dose-toxicity relationship ap-

peared to be “elbow shaped” and that atthe half-log increment the difference in

dosage between the later cohorts is sig-nificantly greater than that between theearlier ones and “may be breaching thatelbow.” A RAC member agreed that thereappeared to be a “narrow window be-tween early and severe toxicity, requir-ing meticulous measures.”

FDA’s investigation into the conduct

of the UPenn trial was ongoing at Cata-lyst press time

.

Gene Therapy:Preclinical Work at 5 Research Court

My purpose here,” says Robert Donahue, in his head-quarters at 5 Research Court in Rockville, Md., “is todemonstrate that novel and potentially therapeutic vec-tors are both safe and efficacious”—after they have beenevaluated in small animals and before they are tested inhuman gene therapy trials. “It’s important to make ourprogram visible,” Donahue said in an interview. “Preclini-cal testing enables us to predict whether a vector has thepossibility of being pathogenic in humans.”Donahue directs the NHLBI program on gene transfer

and bone marrow transplantation in nonhuman primatesand, with NHGRI collaborators Rick Morgan and Jay Lozier,has been exploring the potential of an adenoviral vectorfor human coagulation factor IX as a therapeutic agent inhemophilia B. He is more enthusiastic about other viralcandidates than adenovirus as a vector for gene transfer in the treatment of chronicdeficiency disorders.

Retroviral vectors of murine origin and weakened or “gutless” human lentiviralvectors appear more promising as vectors that can safely target multiple cell types,he said. He and his team currently have six gene therapy projects underway, witha variety of collaborators, pursuing these vectors as conduits for gene expressionin multiple lineages of hematopoietic cells. They're currently looking at green

fluorescent marker genes, provided by jellyfish, that enable them to track both thegene and the protein product. They anticipate moving on to genes for drug resis-tance, factor IX, chronic granulomatous disease, the gamma chain for the T cellreceptor in severe combined immunodeficiency disease, and proteins that couldinhibit replication of such viruses as HIV.

The work with factor IX packaged in an adenoviral vector was originally aproject in Morgan’s lab, where Lozier developed the vector; it was brought to 5Research Court for evaluation in nonhuman primates, Donahue recounted. Deliv-ered alone, the factor IX protein was not immunogenic; delivered in the adenovi-ral package, however, both the protein and the vector were immunogenic. Thissuggests, he said, that an adenoviral vector could be quite useful in a vaccineconstruct, enhancing the immunogenicity of an antigen, but less as a therapeuticagent for delivery of a protein on a chronic basis. The team determined—andLozier reported more than a year ago at a meeting of the American Society ofHematology and again in June 1999 at an American Society of Gene Therapymeeting—that expression of the gene for human coagulation factor IX could beachieved in rhesus monkeys using an El/E3-deleted adenoviral vector. But theprice was acute liver toxicity and coagulopathy.The study dose ranged from 1 times 10 10 to 1 times 10 11 plaque-forming units

(pfu)/kg. At the low doses, there was no gene expression; at the high doses, geneexpression was accompanied by severe liver pathology in response to the vector.Among the findings were increased interleukin-6, decreased serum iron, and sig-nificant derangments in liver enzyme, albumin, and bilirubin concentrations andclotting time. These findings were formally published in the December 15, 1999,issue of the journal of the American Society of Hematology (Jay Lozier, MarkMetzger, Robert Donahue, and Richard Morgan. “Adenovirus-mediated expres-sion of human coagulation factor IX in the rhesus macaque is associated withdose-limiting toxicity.” Blood 94:3968-3975, 1999). They were also cited duringthe meeting of the NIH Recombinant DNA Advisory Committee to review aden-oviral vectors and the death of a patient in a University of Pennsylvania genetherapy trial (see “Gene Therapy Trial and Errors,” p. 1).

“In our study,” Donahue said, “administration was intravenous; in the Pennsyl-vania study, a higher dose was administered directly to the liver, which led toeven more damage.” He speculated that vector distribution beyond the liver inthat study could be attributed to the high dose and noted, too, that the liver’sKupffer cells are a type of macrophage that processes antigen and releases he-matopoietic growth factors that activate an immune response. “There’s a verynarrow window between efficacy and toxicity,” he observed, noting that convert-ing animal to human dosages in this context demands precision and must takesurface area into account. Formulas for computing comparable doses in rodents,dogs, monkeys, and humans can be found in an article by former NCI directorVincent DeVita in the text Cancer: Principles and Practice ofOncology, he said. H

—Frail Pollner

7

The NIH Catalyst

Harold Varmus: Like a Rolling Stone (with Receptors)

by Fran Pollner

In the lastmonth of thetwentieth

century, HaroldVarmus took hisleave of NIH. Heoffered “valedic-

tory thoughts” to

his scientific advi-

sory committee athis final meetingwith them; he bidfond adieu to theNIH communitygathered in MasurAuditorium tolaugh away thepangs of parting(see “Coda,” page

9); and he obligedreporters fromThe NIH Catalyst

,

the NIH Record,

Nature,Science & Government Reports,

Washington Fax,and the Blue Sheet With

a farewell interview.

Time To GoWhy are you leaving? asked a reporter.

“It began to feel repetitive," Varmus said,especially the annual appropriationsprocess. And, he added, there was alsothe matter of “timing”: “To have gonedeeply into my sixties would have re-duced the chances of getting anotherreally good job—and this particular jobbecame available now,” he said of theopportunity to head the Memorial Sloan-Kettering Cancer Center (MSKCC) inNew York City. “In an ideal world, Iprobably would have left [NIH] one yearafter the next administration came in”

—

in which case he would have gonethrough two more of those appropria-tions shuffles he does not relish butwhich he counts among the prioritiesfor the NIH leadership.

It’s up to NIH leaders to chart and pro-mote the “right flight path” for futurebudgets, he said. It's also up to them torecognize that technologic advances andthe “genomic revolution” could drive“deeper divisions in access,” in whichcase, he told his advisory group, “wewon’t have achieved the NIH missionof improving the public health and wewill look like an institution that servesthe elite.” Similarly, he told reporters ofhis concern that “we’re going to cut asignificant proportion of the populationout of the benefits of certain approaches

8

to health that are

paid for with pub-lic money and thatought to be pub-licly accessible.”

Current NIH initia-tives related to re-

search into health

disparities should

help, as shouldtapping the wis-

dom of the NIHdirector’s Council

of Public Repre-

sentatives (COPR),

an activity his suc-cessor would bewell-advised toengage in seri-ously and often,he said.

“I was slow toappreciate the im-

portance of a nonsectarian council like

COPR, a good sounding board,” he said,for scientific, political, and ethical mat-ters, especially equity and health dispar-ity issues.

Infrastructure

Varmus repeated his suggestion thatNIH be reconfig-ured into perhapsfive “clusters” that

would logicallyorganize existing

centers and insti-tutes and discour-age add-ons, but

such a proposalfrom him or anyNIH official, hesaid, would ap-pear self-serving.

Rather, he hopesCongress or theNational Academyof Sciences will

some day arrive ata similar conclu-

sion based on in-dependent study, and then such a reor-ganization could be accepted and imple-mented. His own dim view of prolifer-ating NIH institutes is well known, henoted. Far better, from both scientific andadministrative viewpoints, are NIH’s newinterdisciplinary Neuroscience Center

and the new transinstitute Office ofBioimaging and Bioengineering (askedfor and received in the appropriations

bill).

But there are still some componentsof the intramural research program that“are too far removed from the centersof activity. These [geographically] out-lying groups are a worry,” he said.Nearly out the NIH door, Varmus

nonetheless used the word “we” fre-quently in addressing future courses of

action for NIH. It was also clear, how-ever, that his future at the biomedical

complex on New York’s East Side wasclose upon him and he was eager for it.

The Varmus Lab“I will be at a place,” he said, “where

cancer research and cancer treatmentcoexist—at a strong institution, with in-teresting neighbors (New York Univer-sity-Cornell Medical Center, Rockefeller

University) and many good friends andcolleagues.” And, of course, he’ll be tak-ing his lab with him—a lab, he proudlysaid as the interview opened, that had“just received a quite favorable review”

from NCI. He noted with bemusementthat someone had asked, “Who’s goingto run the Varmus lab now?” as if it werea permanent entity at NIH and not thework-in-progress of the person whobrought it to NIH and would be taking

it with him whenhe left.

Indeed, at the

MSKCC, Varmuswill have moretime to spend inthat lab than hedid at NIH,where, he said,he really couldn’t“settle in” in the

hour or so a dayand off-hours thathe had there. Half

his time in NewYork, he said, will

be in his hospitalexecutive office

and the other halfwill be in his lab.

The administrative aspects of the newjob may actually be more onerous thanhis counterpart tasks as NIH director,which dealt less with administrative de-tails than with the broader policy issues

he enjoys grappling with. Nonetheless,

it’s the science that’s most compelling,and the science in the Varmus lab, hesaid, has been centered on “two majorthemes”:

Fran Pollner

Harold Varmus:“I'm goingfrom one very goodjob to another

very goodjob. ”

Fran Pollner

“When recombinant DNA came to the table25 years ago, society was shocked; no one hadthought much about ethical issues in biology.In general, we’ve become much better at this. ”

January — February 2000

Getting better models for studyinghuman cancer in the mouse. No longerconfined to making transgenics andcrossing them with knockouts, theVarmus team has been using viral vec-tors to deliver conditional genes to or-gans expressing target receptors. Therepertoire has included oncogenes, tu-

mor-suppressor genes, marker genes,and recombinases, or genes pro-grammed to be rearranged. They’vebeen working with tumors characteris-tically hard to grow in cell culture, suchas gliomas and breast tumors, and arecrafting models for lung, pancreatic, andovarian cancer. Ultimately, knowledgeof gene abnormalities in human cancershould yield therapeutic and preventiveinterventions that are less toxic andmore effective than currently availableagents. It should also yield a more pro-found understanding of metastasis.

Understanding how growth and dif-ferentiation factors (such as WNT pro-teins, fibroblast growth factors, andhedgehog and TGF-p genes) signal thecell nucleus to alter the way the cellbehaves. Varmus’s interest in signaling

was “activated” about 20 years ago, hesaid, when he and his colleagues dis-covered the first WNT gene and traced

the role of WNT1 incancer developmentafter the insertion of vi-

ral DNA from themouse mammary tu-mor vims.

The World at LargeAsked if he would

miss his role in the in-

ternational arena,working on such prob-lems as malaria, he re-plied: “I’d miss it if I

lost it, but I don’t think

I will.” He’ll likely be a

consultant to the World

Health Organization and

will soon be traveling to

India; he’ll also continue

his involvement inPubMed Central, which,he said, is an “important issue in interna-

tional health and the support of science inthe developing world.”

Beyond that, he’ll be rejoining the steer-ing committee of the American Society for

Cell Biology, from which, he said, he wasapparently granted a “six-year leave of ab-

sence.” And he’s been asked to join the“p53 Club” in New York, as well as many

other clubs, athletic and

scientific, that he will

consider once he fig-ures out how muchtime his job leaves himfor other pursuits.Among other things helooks forward to are

New York’s artistic of-ferings, having to an-

swer to “fewer bosses,”and “more flexible in-teractions with the pri-

vate sector,” although

he intends to “serveonly on nonprofits.”

Recollections

But he will leavebehind the NIH intra-mural program, which“from the first day I ar-

rived here, I’ve really enjoyed”— his sci-entific collaborations, the seminars, the

interest groups, the “esprit,” and thecampus building boom. He lauded thespeed with which NIH was able to re-spond to a perceived national need

—

both physically and intellectually—withthe building of the Vaccine Research

Center and recruitment of Gary Nabel

“Iplan to make a tape [ofsome ofthe ‘goodbyeparty) see below] to

show at Memorial Sloan-Kettering,so people can see how we havefunhere and how Iplan to interact. ...”

Coda

Fran Pollner

The Party’s Over: As the NIH troopsfiled out, afewremainedforparting photos (left to right): HHS

secretary Donna Shalala, Tracey Rouault (NICHD.keyboardist), now acting NIH director Ruth

Kirschstein, Chuck Allerson (NICHD, drums), NIAIDdirector Tony Fauci, Francis Collins (NHGRI director,

“lead guitar"), Harold Varmus, Connie Casey,Richard Klausner (NCI director, “lead guitar’’), Steve

Katz (NIAMS director, “lead guitar"), andJohn O'Shea(NIAMS, bass, “the only one who knows how to play a

guitar”).

—band hierarchyprovided by Richard Klausner

C elebrating Harold Varmus and, at the same time, saying goodbyeto him is what “bittersweet” is all about, said Tony Fauci, NIAIDdirector and emcee at a farewell sendoff for Harold Varmus in themonth before the NIH director would officially leave his positionto head to New York City and the Memorial Sloan-Kettering Can-cer Center.

“It’s been “six years and 23 days and counting,” Fauci said, as ifwhen that ball dropped 15 days later—at midnight in TimesSquare—the roar from the crowd would not be “Happy New Year"but “Welcome, Harold!”Paeans mixed with jokes for a couple of hours as farewell mes-

sages rained down upon Harold Varmus. HHS Secretary DonnaShalala was on hand to voice her deep respect for Varmus’ bril-liance—and his unique sense of fashion.And a videotaped “documentary” from the “troops” showed that

however much Varmus may inspire awe from the world at large,he’s more than fair game for the inspired wit of his NIH colleagues,about a dozen of whom deadpanned hilarious answers to suchquestions as: “Why did Harold Varmus hire you?”The event rocked to a close with a stellar performance by “The

Directors,” NIH’s unparalleled rock band, and their doctored lyricsof sixties hits that ended with the exhortation “Oh, won’t you stay. .

.just a little bit longer .... please, please, please, please . . .

say-ay you-ou will. ...”

But he wouldn’t.—F.P

.

9

The NIH Catalyst

as its director.

For none of these aspects of NIH forwhich he expressed appreciation didVarmus take credit. He even punctuatedhis praise of the Clinical Center’s activ-

ity level and clinical research on cam-pus in general with an aside that thoughhe himself did not contribute to the clini-cal side of NIH studies, his research hadbenefited from its resources, such as thesmall animal imaging facility.But he had some good things to say

for his handling of several matters as

NIH director.On patents: "I chose not to pursue

intellectual property rights on anony-mous cDNAs, and I think that was theright decision. Why prosecute a claimyou don’t think should be made? I’mpleased the Patent Trademark Office iscloser to our position now: that the spe-cific utility must be known in order topatent a squence.”

On material transfer agreements:“We’re not trying to lay down a strictlaw but to move the scientific commu-nity to a more generous mode of be-havior. We had a major success with Cre-Lox technology, and although our holdis less secure with corporate entities that

don’t have NIH funding, the movementis in the direction of greater sharing.”

On PubMed Central: “Once it's un-veiled, the public interest will soar.”

On stem cell research: “This issue willcontinue to heat up. I look back with asense of pride at the Human EmbryoResearch Report of 1994, which was pre-scient [regarding issues raised in 19991.”

On his past actions to recast the roleof the NIH Recombinant DNA Advisory’Committee (RAC): “I have no objectionto its being put on the table [that RACagain approve gene therapy protocols],but I think it’s the wrong way to go.Jesse Gelsinger’s death would not havebeen prevented if the RAC had approvalauthority, and NIH is not a regulatoryagency [see Catalyst coverage of the RACmeeting in December on these matters,page 1], I don’t think we should respondto this unfortunate episode by return-ing RAC to what was a somewhat dys-functional state. Now it’s working well.”Asked if he had any disappointments,

he said he rued his lack of visible suc-cess in increasing the numbers of in-vestigators from minority backgrounds."I haven’t seen major changes. . . . Per-haps you couldn't in just six years.” g

Magtags

continued from page 1

migration in autoimmune disease,” Franksuggests.

Many types of cells, he says, will takeup the nanoparticles of oxidized iron inLDRR’s magnetic tagging system. If theappropriate number of cells are theninjected back into a living body and thenhome in on any soft tissue, Bulte andFrank predict they will generate detect-

able signal changes on MRI.“People have wanted to do this for a

long time,” saysBulte, and he himselfcould barely believe

it when the researchteam first injectedand imaged mag-netically tagged cells

in an animal.“I thought I was

looking at charcoal

particles” that hadbeen used to markthe injection site “or

some other artifact,”he recalls. The keyto LDRR’s success,according to Frank,

was getting a highdegree of magnetic

labeling of cells.

The LDRR groupconstructed theirfirst-generationmagtag by attachinga nanoparticle ofmaghemite to amonoclonal anti-body to the rat’stransferrin recep-tor— a receptorfound on almost all cells in all speciesof animals. The cell surface receptorbinds the antibody and then rapidly in-ternalizes the iron, stashing it away inendosomes. The iron oxide in the tag issuperparamagnetic, the LDRR scientistsexplain. This means that cells loadedwith the tag are magnetized only whenthey are in the strong magnetic field of

an MR scanner. Tagged cells retain nomagnetic memory after being scannedand will not clump with one another orwith metallic materials.

The researchers believe the nano-par-ticles of iron eventually get used by thecell and then recycle through the

animal’s iron pool. The team has beenable to follow magtagged oligodendro-cyte progenitor cells for 42 days aftertransplantation into the rat. They havewatched the cells slowly migrate fromthe injection site.

Bulte says there is almost no chanceof overloading an experimental subjectwith iron. Humans, for example, typi-cally have 4 grams of iron in their bod-ies, and experimental dosing would bein the microgram range. Bulte found thatthe iron tag did not impair the migra-

tion or other functions of labeled cells.

In fact, he says, “youcan take these cellsback and put themin culture” and theywill thrive. Hepoints out that birds,

butterflies, bacteria,

and other organismswith innate direc-tional sense all use

nanoparticles of iron

in their internal

compasses.

In their initial ex-

periments, Frank,Bulte, and Peter vanGelderen of the NIHIn Vivo NMR Centerviewed the mag-tagged cells with a

4.7 Tesla MR instru-ment. But they have

since shown thatthey can follow in-

jected cells with a

1.5 Tesla instrument

—a field strengththat can be usedclinically in humans.

This field strength

showed signal changes from a few mil-limeters of rat spinal cord bearing 50,000

or so tagged cells injected during the

group’s experiments.

These days, Frank says, clinical MRIhas a resolution of 1 mm in the humanbrain, and the team expects to be ableto reach this level of resolution, or bet-

ter, when magtagged cells make theirway into clinical applications.

For animal imaging, the potential reso-

lution could be much greater, however,with the higher field strength MR scan-ners now being installed in the In VivoNMR Center. With the current 4.7 Teslainstrument, the team can resolve patches

Original magnetic tagging system

developed by the LDRR team and theircolleagues shuttles nanoparticles of iron

oxide (MION-46L) into cells via amonoclonal antibody (OX-26) to the

cell's transferrin receptor. Cells internal-

ize the superparamagneticnanoparticles, ultimately leading to the

visible marking ofthe cells duringmagnetic resonance imaging.

10

January — February 2000

Three-dimensional reconstruction

magnetic resonance image of rat spinalcord shows distribution of magnetically

tagged cells 1 0 days after the taggedoligodendrocyteprogen itor cells were

injected into a myelin-deficient animal.This image was captured by Peter van

Gelderen ofthe NTH In Vivo NMR Centerusing a 4.7 Tesla MR imager.

as small as 78 micrometers. At least in

animal models, they expect to be able

to resolve as few as five to 100 taggedcells in living animals, depending on thelocation of the cells within tissue. Bulte

says one factor boosting the system’ssensitivity is a “blooming” or magnetic

susceptibility effect—an amplification ofsignal from surrounding water molecules

that occurs when tagged cells are ex-cited in the MR field.

Fittingly, the first research employingthe new technique involved a centralnervous system study that appeared inthe final issue of the Proceedings ofthe

National Academy of Science in theDecade of the Brain [PNAS 96:15256-15261, 19991- The work sprang fromLDRR’s research on repair ofdysmyelination and demyelination

—

damage or loss of the myelin sheatharound axons seen in multiple sclerosisand other neurological diseases.Su-Chun Zhang and Ian Duncan of

the University of Wisconsin’s School of

Veterinary Medicine injected oligoden-

drocyte precursor (CG-4) cells into the

spinal cord of myelin-deficient rats. Be-

cause the injected cells had beenmagtagged, the researchers were, for

the first time, able to follow the migra-

tion and integration of the cells into thenervous system noninvasively.

The distribution of magtagged cells ontheir three-dimensional MR images cor-related closely with myelination in the

rats, shown subsequently by sacrificingthe rats, dissecting the spinal cord, andlooking at the distribution of the cells

with traditional histopathological tech-

niques 14 days post-transplantation.

Bulte says the high resolution, MRI-com-puter-generated sections of tissue wereactually more helpful in interpreting thetraditional anatomic sections, rather than

vice versa.

Since establishing the efficacy of their

technique, the LDRR scientists have goneon to document the migration of oligo-dendrocyte precursors in young normalrats. Along with their collaborators, theyare planning studies to track migration

and remyelination of stem-cell derivedoligospheres injected into a myelin-de-

ficient dog developed by the Wisconsingroup.

Frank says he foresees no major tech-nical barriers that would block use ofmagnetic tags in humans. “It’s just amatter of time.” Tracking neural stem

cells inserted in the brain or spinal cord

to repair neurodegeneration or trauma-

related injury would be natural exten-sions of LDRR’s magtag work.

In tandem with their substantive re-search, LDRR scientists have also beenimproving upon and developing theoriginal magtag technique, for whichthey filed a patent application. They nowhave a second-generation magtag thatis not dependent on species-specificmonoclonal antibodies or even on thetransferrin receptor. Like the first-gen-

eration tag, the new marker—a coatedpolymeric iron compound—shows highaffinity for cell membranes and will workon any cell type.Although the magtags have been de-

veloped to be used on a broad range of

StaffscientistJeffBulte showed thispicture ofone ofhis daughter’sPokemon game cards at a recent

professional meeting, but strenuously

denies that the Pokemon character wasthe inspirationfor LDRR’s maghemite

cell-tagging system.

cells and to applied ex vivo, Frank saysit should be possible to adapt the tag-ging system to target individual cell types

via specific receptors and to label thesecell populations in vivo. But targeted

delivery of magtags would be anotherfrontier with additional hurdles andprobably couldn’t be explored for a fewyears—at least until the researchers havemapped the research continent they justdiscovered.

Bulte observes that the group is notthe first to use magnetism in navigatinguncharted territory: “Human explorersmade their greatest discoveries only af-ter the compass was invented.”

Averting Violence

C IVIL, a newly established co-ordinated NIH resource, isnow available to help the NIHcommunity prevent and respondto workplace threats and violence.

If you feel there is IMMEDIATEDANGER:

Dial 911, if on campusDial 9-911, if off-campus

Call CIVIL if:You need help assessing the

potential seriousness of a threat-

ening situation;

You are experiencing a threat-ening situation at work and needintervention from trained staff;

You become aware of a work-place situation involving intimidat-

ing, harassing, or other unproduc-tive/dangerous behaviors andneed consultation; or

A situation involving threatsor aggressive acts already has oc-

curred and you need assistancemanaging the aftermath and itseffect on staff.Anyone can call CIVIL di-

rectly by dialing C-I-V-I-L or402 -4845 .

Callfor AbstractsThe NIH Bioengineering Con-

sortium (BECON) symposium on“Nanoscience and Nanotechnol-ogy” is set forJune 25-26. For info,contact

.

11

The NIH Catalyst

People

Recently Tenured

Henry Levin

Henry Levin received his Ph D. in thelab ofHoward Schachman at the Uni-versity of California, Berkeley, in 1987.He did his postdoctoral work at JohnsHopkins Medical School before joiningthe N1CHD Laboratory ofMolecular Ge-netics in 1993 He is now a senior inves-tigator in the Laboratory of Eukaryotic

Gene Regulation.My interests lie in the

proliferation of retroele-

ments, the most medicallyimportant class of whichare the retroviruses. Thegenetic complexity of thehost vertebrates forretroviruses, however,complicates the study of

particle assembly, reverse

transcription, nuclear en-

try, and integration intochromosome.Our approach to understanding these

events is to study retrotransposons, a

family of elements closely related toretroviruses that offer a significant ad-

vantage: They exist in yeast, a host thatcan be studied using the powerful tech-niques of molecular genetics.

The retrotransposon we study is theTfl element of fission yeast,Schizosaccbaromyces pombe. Likeretroviruses, Tfl encodes a protease, re-verse transcriptase, and integrase. Wehave developed a genetic assay for Tfltransposition that has allowed us to study

several aspects of its propagation.

One of our early results was perhapsthe most surprising. Although all longterminal repeat retroelements werethought to initiate reverse transcription

from specific tRNA primers, we showedthat tRNAs are not essential primers ofreverse transcription. We found that Tflundergoes an unusual mechanism of self-primed reverse transcription that definesa new family of retroelements. In placeof a tRNA primer, the first 11 bases ofthe Tfl mRNA anneal to the primer bind-ing site, and a nucleolytic cleavage atthe 12th base allows the first 11 ribo-nucleotides to prime reverse transcrip-tion.

A series of genetic and biochemicalexperiments identified a complex struc-ture in the Tfl mRNA that is essential forpriming. Despite the novelty of thismechanism, we identified structural fea-tures within the self-priming mRNA thatbear surprising similarity to sequences

12

in the mRNA of retroviruses. The simi-larities of the self-priming mRNA toRNAs of many other retroelementshave motivated us to continue ouranalysis of the self-priming mecha-nism.

One of the principal goals of ourresearch is to identify host functions

that are necessary for retrotransposi-

tion. As we characterize thesebasic features of cellular biol-

ogy, we can simultaneouslyidentify potential targets for

antiviral therapies. Wescreened randomly muta-genized strains of S. pombeand identified genes that arenecessary for transposition.

Much of our effort is focusedon Nupl24p, a nuclear porefactor that is required for the

nuclear import of Tfl. Strains

with mutations in nupl24 showed nor-mal growth rates and exhibited nodefects in the nuclear im-

port of other nuclear pro-

teins or in the nuclear ex-

port of poly(A) mRNA.The specificity of this im-

port pathway for Tfl islikely due to the direct in-teraction between Nupl24pand Gag that we detected.The contribution ofNupl24p to the nuclear im-port of Tfl bears significant

similarity to the nuclear

transport of HIV in that the Vpr pro-tein of HIV mediates the import of HIVvia an interaction with specific nuclear

pore factors. This particular similarity

suggests there may be common prop-erties of large viral complexes that re-quire a specialized form of nuclearimport, and we are continuing ouranalysis of Tfl import to test this pos-

sibility.

In addition, we are exploring ourcollection of mutant strains for evi-dence of other genes that contributeto the import of Tfl.

Charles Rabkin received hisM.D.fromBrown University (Providence

,R.L.) in

1981 and bis M.Sc. in epidemiologyfrom the London School ofHygiene andTropical Medicine in 1988. He trainedin internal medicine at the University

ofColorado in Denver. He was an Epi-demic Intelligence Service officer and

Charles Rabkin

medical epidemiologist at the CentersforDisease Control and Prevention beforejoining NCI in 1989 and is now a seniorinvestigator in the Viral EpidemiologyBranch

,NCI.

My research combines epidemiologicapproaches to cancer in human popula-tions with the evolving tools of molecu-lar genetics. My primary interest is themolecular mechanisms of HIV-associatedcancers, focusing on non-Hodgkin’s lym-phoma and Kaposi’s sarcoma.AIDS-related cancers are an importantmodel for investigating the role of spe-cific mutations in carcinogenesis.

With colleagues in NCI’s Laboratory ofGenetics, we demonstrated that HIV-infected-but-lymphoma-free individuals

frequently harbored circulating lympho-cytes with the t(8; 14) chromosomal trans-location characteristic of Burkitt’s lym-

phoma. Translocation prevalence in-creased with duration of HIV infection,and aberrant clones could persist for

many years without evolu-tion into non-Hodgkin’s lym-

phoma. We also found thatthese individuals did not

have increased prevalence offollicular (that is, non-AIDS-related) lymphoma-associ-ated t(l4; 18) translocations,

demonstrating the specificity

of HIV’s effect.

Having identified somaticmutation as an early event, Iam now concentrating on ab-

normalities in advanced HIV infectionthat may control the rate of tumor devel-opment.With collaborators in the NCI-Frederick

Cancer Research and Development Cen-ter, we have also examined germ-linemutations and recently found that a com-mon polymorphism in the SDF-1chemokine gene strongly increases lym-phoma risk in AIDS, whereas a variantof the chemokine receptor gene CCR5 ishighly protective. Because the SDF-1 vari-

ant is four times as common in whites asin blacks, these data may explain racialdifferences in AIDS-lymphoma risk thatour group and others previously re-ported.

My Kaposi’s sarcoma studies have ex-amined the neoplastic nature of this dis-order. In collaborations with several labo-

ratories, we found that Kaposi’s sarcomatumors appear to derive from clonal rep-

lication of a single cell. By combining

January — February 2000

microdissection with the clonality assay,

we then found that spindle cells frommultiple Kaposi’s sarcoma lesions fromthe same patient appeared to be clonallyrelated, suggesting the disease derived

and disseminated from a single cell.These experiments have been based onthe X-linked androgen receptor(HI 'MARA) clonality assay, and I am cur-rently attempting to validate and extendthese findings with more robust tech-niques.

My other major interest is in infectiousmechanisms in gastrointestinal cancers.Helicobacter pylori infection is associ-

ated with both gastric cancer and non-malignant duodenal ulcer disease. Thereasons for these divergent clinical out-

comes are not clear, but the gastric physi-ological response is influenced by theseverity and anatomical distribution ofH. pylori-induced gastritis. Our groupis focusing on polymorphisms in thegenes regulating inflammation and im-munity as possible determinants of gas-tric cancer and its precursors.Another endeavor is to better deter-

mine the long-term prognosis of hepati-tis C infection—currently a difficultproposition. The risk of liver cancer inparticular is controversial. In collabora-

tion with NIDDK, we ascertained thehealth outcomes after 45 years’ follow-upof initially healthy hepatitis C-seroposi-

tive young men. Liver-related morbidityand mortality were low, suggesting thatthe risk of progressive disease may beless than is currently perceived.

Heavy use of alcohol and other sub-stances may alter this natural history,however, and we are extending thesestudies with a unique collection of seraand liver biopsy specimens of hepatitisC-seropositive injection drug users fol-lowed for as long as 25 years.

Infection-related cancers are fruitful

areas for research of fundamentalmechanisms of carcinogenesis. Betterunderstanding of these processes should

help in the development of targeted in-terventions for cancer prevention.

Juan Rivera received his Ph.D.from theCatholic University of America (Wash-ington, D.Cjin 1990 and did hispostdoctoral work in the Arthritis andRheumatism Branch ofNIAMS, where heis now a senior investigatorand head ofthe Signal Transduction Group.

My interests are in the area of recep-

tors that bind the crystallizable fragment

of an antibody (Fc receptors) and theirrole in inflammation, with a focus onhow these receptors transduce signalsthat result in gene expression. The un-derlying objective is the discovery of the

receptor-proximal molecules that link

the immune complex activation of Fcreceptors to expression of particular

cytokine genes. I see this as an impor-

tant step in understandinghow Fc receptors contributeto the process of inflamma-

tion in health and disease.Hopefully, these studies will

facilitate development of bet-ter therapeutics that target

those mediators that contrib-

ute to inflammatory disease,

without affecting the produc-

tion of others that may bebeneficial for recovery.

Fc receptors can either activate or in-hibit cell effector functions. The sub-family of activating receptors share in

common the Fc receptor gamma signal-ing subunit. My colleagues and I studythe molecular signals initiated by the ac-tivating Fc receptor with high affinity

for immunoglobulin E (IgE) that is ex-pressed on mast cells and is involvedin allergy and inflammation. We focuson identifying IgE Fc receptor-activatedsignaling molecules that could modu-late cytokine gene expression.We found that selected members of

the protein kinase C family participatein IgE Fc receptor induction of the early

response genes c-fos and c-jun andcause a selective induction of IL-2 andIL-6. In addition, we discovered thatanother protein kinase C isoform selec-tively phosphorylates the IgE Fc recep-

tor and that this event is important increating an appropriate surface for thebinding and activation of Syk, the ki-nase critical to propagating signaling andmast cell effector function. We observedthat in the absence of this protein ki-nase C-mediated phosphorylation thereis a loss of cytokine production. Thesestudies demonstrated that Fc receptorsuse different members of the proteinkinase C family to either selectively orgenerically influence the production of

cytokines.

We are now investigating how theformation of macromolecular signalingcomplexes contributes to the regulationand specificity of Fc receptor cytokine

gene expression. Much of our effort isfocused on Vav, a guanine nucleotideexchange factor that is selectively ex-pressed in hematopoietic cells. Ourprogress in this area relied on the de-velopment of a versatile gene expres-sion system based on the Semliki Forestvirus that can be used to overexpress orrestore proteins of interest in most pri-mary and immortalized cell lines tested.

We found that Vav modu-lates selected cytokine ex-

pression in mast cells. Wealso found that Vav movesfrom the cytosol to deter-gent-insoluble plasma mem-brane domains, or rafts,where activated IgE Fc re-ceptors also reside. Inhibi-

tion of the redistribution of

Vav results in inhibition ofc-jun NH2-terminal kinase

activity, an activity required for expres-sion of selected cytokines. In addition,

we found that Vav co-immunoprecipi-tates with the raft-localized scaffold pro-

tein linker of activated T cells (LAT) inmast cells. In recent collaborative stud-

ies, we found that LAT-null mast cellsshow ablation or decreased productionof most cytokines tested. Thus, macro-molecular signaling complexes areseemingly comprised of constituents thatindividually influence selected genes but

collectively affect many.We are currently investigating how Fc

receptors engage the macromolecularsignaling complexes and are trying todetermine the importance of each sig-naling complex constituent in cytokinegene expression. We expect to explorethe role of the constituent proteins in

inflammatory disease in animal models.

Catalytic ReactionsAn anonymous reader responded to

two questionsposed in the last issue: 1

)

Wloat’s the biggest challengefor the nextN1H director? and 2) Can you suggestany new rides as a basisfor biomedi-cal researchfor the next 100 years?

1) Living up to the high standardset by Dr. Varmus.

2) Requiring Pis to actually fol-

low the guidelines for authorshipset forth in the NIH Fellows hand-book. It’s a shame when onepostdoc analyzes and writes an-other postdoc’s paper.

13

The NIH Catalyst

People

Recently Tenured

Sandra Swain received her M.D. fromthe University of Florida in Gainesville

in 1980. She did an internal medicineresidency at Vanderbilt University in

Nashville, Tenn., and an oncology fel-lowship at NCI, where she supervisedbreast cancer clinical trials until 1988.After serving as director ofthe Compre-hensive Breast Center at GeorgetownUniversity's Lombardi Cancer Center(Washington, D.C.Jand then as medicaldirectorfor Salick Health Care, Inc., she

returned to NCI, where she is deputybranch chief of the Medicine Branchand a senior investigator in theDevelopmental TherapeuticsDepartment.

My primary area of inter-est is breast cancer clinical

research. I have designed,implemented, participatedin, analyzed, and publishedfindings from numerousclinical trials, ranging fromPhase 1 to large, multicenter

Phase 3 trials. Crucial to myunderstanding of transla-tional research issues was my work ona project in the laboratory of MarcLippman that led to the discovery ofpleiotropin, which is implicated in an-giogenesis.

I have participated in several nationalcooperative group trials and am cur-rently on the breast cancer committeeof the National Surgical Breast andBowel Project (NSABP). I am also na-tional principal investigator for the node-

positive NSABP clinical trial (NSABP B-30), which is accruing 4,000 patients atthe rate of 105 patients a month. Thetrial has three treatment arms and is de-signed to determine the effect ofdocetaxel on survival and quality of lifein women who are node-positive atbreast cancer diagnosis. This study will

capture the effect of menopause by in-cluding and following women who arepremenopausal at diagnosis. As nationalPI, I review ongoing trials, with particu-lar attention to toxicities.

Earlier in my career, I supervised anNCI study of the effect of chemotherapybefore local therapy in patients with lo-

cally advanced breast cancer, a studywhose results led to a larger version ofthe trial in the NSABP B-18. I plan tocontinue research in this area to evalu-

ate new agents that could be used inthe context of pre- and posttreatment

biopsy for gene discoveiy and proteinexpression analysis.

The cardiotoxicity of some anticanceragents is another of my research inter-ests. I participated in the design, imple-

mentation, conduct, and data analysesof two national, multicenter, placebo-controlled, randomized studies thatshowed that dexrazoxane, an iron-chelating agent, could decreasecardiotoxicity in breast cancer patients

taking doxorubicin.

These findings were the basis for ap-proval of the daig by the Food and DaigAdministration in 1995, and dexrazoxane

remains the only drug ap-proved for cardioprotectionin oncology. I plan to con-

tinue working in the area ofcardiotoxicity.

My service on the FDA’sOncology Advisory Commit-tee, as a member from 1994to 1998 and continuing as aconsultant, has provided in-

valuable understanding of

the scientific and regulatoryprocesses that underlie the

determination of safety and efficacy ofnew therapeutic modalities. This expe-rience will serve me well in my ongo-ing work in drug development and clini-cal trial design.

David Wink received hisPh.D. in chem-istry at the University of California,Santa Barbara in 1985. Following apostdoctoral fellowship in biochemistry

as a National Research Service Awardrecipient at the Massachusetts Institute

of Technology in Cambridge, be joined

the Laboratory ofComparative Carcino-

genesis at NCI-FCRDC as a stafffelloiv.In 1995, he joined NCI’s Radiation Bi-

ology Branch, where he isnow a senior investigator.My general aim is to eluci-

date the chemical reactions

of small redox molecules andtheir importance in different

physiological and cellularmechanisms. Since comingto NCI, I have been particu-larly interested in the role of

nitric oxide (NO) in regula-tory mechanisms as well asin pathophysiological condi-

tions. Appreciation of the importance of

NO in biology has increased exponen-tially, making it one of the fastest grow-ing fields in biomedical research.

My early work concentrated on eluci-dating different chemical mechanisms ofNO and how these reactions might beinvolved in different pathophysiologi-

cal conditions. We began to define thechemistry of NO in toxicological andcarcinogenic mechanisms and to iden-tify different molecular targets that are

modified by NO and related chemicalspecies. My current research interestscenter on the chemistry and biochemis-try of free radicals in cellular and tissuedamage and how these reactions canbe modulated. We have explored someof the basic chemical mechanisms ofreactive oxygen species (ROS) forma-tion from metal-peroxide interactions aswell as reactions involved with NO andthe effect these reactions have on bio-logical systems.

One important discovery was the se-lective inhibition of specific DNA repairenzymes by NO. These mechanismswere shown to prevent the repair ofDNA lesions induced by alkylatingagents as well as ROS. In these studies,we have catalogued and defined theeffects of the interaction of different radi-

cals on cytotoxic mechanisms. For in-stance, NO seives to protect mamma-lian cells from hydrogen peroxide andxanthine oxidase-mediated cytotoxicity.

Yet in prokaroytes, NO in combinationwith peroxide and xanthine serves asan impressive bactericidal agent. Corre-

lation of the different effects NO has onthe cytotoxicity of other agents with the

chemical mechanisms has been de-scribed in what I refer to as the “chemi-cal biology of nitric oxide.”

This concept has provided a guide to

understanding the free radical chemis-

try of NO in biological systems (D.A.Wink and J.B. Mitchell. “The chemicalbiology of nitric oxide: insights into regu-

latory, cytotoxic, and cyto-protective mechanisms ofnitric oxide. FreeRadic. Biol.Med. 25:434-456, 1998). Wehave collaborated with sev-

eral groups to explore the

role of NO in infectious dis-eases, ischemic reperfusion

injury, and neurological dis-orders.

We became particularlyinterested in the effect of NO

on increasing the toxicity of chemothera-

peutic agents and radiation. This has re-

sulted in a major new challenge—toapply what has been learned in the

Fran Pollner

Sandra Swain

Fran Pollner

David Wink

14

January — February 2000

chemical biology of NO to improvingcancer treatment. We have investigatedthe use of free radicals to modulate mo-dalities of cancer treatment. Nitric ox-

ide can radiosensitize hypoxic cells. In

addition, recent results show that NOdramatically enhances the cytotoxicityof some chemotherapeutic drugs includ-ing melphalan and cisplatin. A major ob-jective is to selectively target tumor sitesas opposed to normal tissue. We havetaken two approaches. The first is theuse of exogenous chemical agents thatcan specifically target tumors and de-liver NO. Second, because there are re-ports that certain tumors may exhibitnitric oxide synthase (NOS) activity, wehave begun to explore different strate-gies of inducing NOS directly in tumors,thereby providing a source of NO to thetumor directly.Throughout this research, we have

had to explore different methods todeliver the NO redox chemistry to cel-lular models as well as in vivo. This hasrequired the development of an analyti-cal repertoire and synthetic systems thatmimic redox reactions. Using a varietyof analytical methods, we can determinethe production of NO, RNOS, and ROSderived from different stimuli of the im-mune system. We can then develop acorresponding chemical model that canbe used to treat cells and probe differ-ent molecular aspects of redox chemis-try. We have been mapping the chemi-cal effects on molecular targets to un-derstand in vivo mechanisms of NO andto define the redox chemistry requiredto improve therapeutic outcomes.

Going Places

T he Office of Research Servicesannounces that a new perform-ance-based contract has been awardedto WorldTravelService (WTS). Thetransition began November 15, with fullimplementation and performance stan-dards effective January 18, 2000.

WTS bring NIH:•Reservations via e-mail and fax•Lowest fare guarantees• Meeting planning sendees (for a fee)•Upgraded telephone and fax system•Web site dedicated to NIH travelers• 24-hour emergency customer support•Electronic booking (in the future)A new requirement for this contract

entails a nonrefundable service fee,which will be charged for each trans-action that results in issuance of anairline or train ticket. A fee-for-servicefact sheet and other information canbe found at , oryou may call 301-402-8180 to requestcopies.

WTS is