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Kansas State University (if applicable) Office of Naval Research____ ___ ___ ____ ___ ___ ___ N/A Office__ ___f __Naval___Research___

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Division of Biology, Ackert Hall 800 No. Quincy StreetManhattan, KS 66506 Arlington, VA 22217-5000

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11. TITLE (include Security Classification (U) The role of neuropeptides in persistent virus infectionsof the central nervous system

12. PERSONAL AUTHOR(S) Johnson, Terry C.

13a. TYPE OF REPORT 13b TIME COVERED 14. DATE OF REPORT (Year, Month, Day) 15. PAGE COUNT

16.Annual I FROM ..,/1,/R7TOU.Ln1881 July 11, 19881 1516 SUPPLEMENTARY NOTATION

17 COSATI CODES 18 SUBJECT TERMS (Continue on reverse if necessary and identify by block number)

FIELD GROUP SUB-GROUP opioids, lymphocytes, infections, nervous system,

I I virus, immunity, neuropeptides

I .,ARSTRACT (Continuq on reverse if necessary and identify by block number)Tnis study is focused on the development of model systems to study inter-actions between the immune and nervous systems that alter the progress ofinfectious diseases of the central nervous system (CNS). A temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31-KS5 VSV,intracerebrally inoculated into BALB/c (+/+) or Swiss outbred mice yieldean asymptomatic and persistent infection of the CNS. BALB/c athymic nude(nu/nu) mice infected with tsG31-KS5 VSV, however, all perished within 26days of infection. The nude mice were afflicted with a slowly progressinCNS disorder, with symptoms including lethargy, curvature of the spine,hind-leg paralysis ang other neurological disorders. Reconstitution ofnude mice with 5 x 10 syngeneic T lymphocytes, 24 hr prior to their in-fection with ts VSV, led to at least 70% of the animals surviving, andprotection was mediated without a robust humoral antibody response.20 DISTRIBUTION/AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATION

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Dr. J.A. Majde 202/ 696-4055 ONRDr Form 1473. JUN 86 Previous editions are obsolete. SECURITY CLASSIFICATION OF THIS PAGE

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• ' ..

ANNUAL REPORT

The Role of Neuropeptides in Persistent Virus infectionsV of the Central Nervous System

ONR Contract No. N00014-87-K-0217

i .M.

A. Introduction

- During the past decade significant advances have been madein our understanding of the physiological influences of severalneuropeptides, their surprising wide-distribution in non-nervoustissues, and the potential link between these neuropeptides and

Z % the immune system. Although to date most studies are preliminaryin nature, there already remains little question that subtlechanges in neuroendocrine function may play an important role inthe host-parasite relationship associated with infectious dis-eases mediated by viruses.

Central to future advances in our knowledge of the potentialinteractions between the nervous and immune systems of the host--as a mediator of he4lth and disease--is the development of modelsystems of infectious diseases that can discriminate betweencausative and casual relationships. To this end, we have focusedthe first year of this study on the clinical disease caused byintracerebral inoculation of certain ts mutants of VSV in bothBALB/c (+/+) and BALB/c athymic nude (nu/nu) mice. This modelsystem should allow a comparison of immune competent (BALB/c) andimmune deficient (BALB/c nude) animals in warding off a progres-sive CNS disease, the determination of the ability of immunereconstitution (BALB/c nude plus syngeneic T lymphocytes) toalter the clinical course of CNS disease, and the potential modi-fication of the CNS disease by certain neuropeptides that havebeen implicated in immune function..

B. Research Results

1. Survival of BALB/c (+/+) and (nu/nu) to an intracerebralinfection with tsG31-KS5 VSV.

Balb/c nude mice inoculated with tsG31-KS5 VSV all expiredby 26 days after infection (Fig. 1). Before death, all the virusinfected nude mice suffered a severe CNS disorder with symptomsincluding lethargy, loss of appetite, wasting, curvature of the

* spine, and hind-limb paralysis. Nude mice mock-infected with thecarrier alone did not experience any symptoms of the CNS disease

2

and remained healthy for 60 days after the injection at which

tiethe experiment was terminated.

2. Antibody and virus in persistently infected mice.

Nude mice reconstituted with syngeneic T-lymphocyte-enrichedsplenocytes, however, were relatively refractory to the CNSinfection in that over 90% survived for 20 days and 70% livedbeyond 25 days (Table 1). Animals that survived remained healthyfor at least 60 daXs after infection, and nude mice reconstitutedwith either 5 x 10 or 5 x 10 syngeneic T-lymphocytes were pro-vided with a similar degree of protection.

To discern if thesimmung systems of nude mice had been com-pletely restored with 5 x 10 T lymphocytes, reconstituted nudemice that had been infected for 20 days with tsG31-KS5 VSV werechallenged with WT VSV. In Balb/c (+/+) mice, WT VSV infectionwas fatal to all mice by 4 days of infection. WT VSV, however,was not lethal to any Balb/c (+/+) mice that had been infectedwith tsG31-KS5 VSV for 20 days before inoculation with WT VSV.

* Analogous to normal mice, T lymphocyte reconstituted nude miceinoculated with WT VSV all died by 4 days of infection. When T

K., lymphocyte reconstituted nude mice infected for 20 days withtsG31-KS5 VSV were inoculated with WT VSV, only 20% survived theWT VSV challenge. The sera, from the T lymphocyte reconstitutednude mouse that survived the WT VSV chtllenge, had a VSV neutgal-izing antibody titer greater than 1:10 . Injection of 5 x 10 Tlymphocytes into nude mice protected them from the temperature-sensitive VSV induced CNS disease, but their immune functionswere not completely restored since the majority did not survivethe WT VSV challenge.

Like earlier studies with ts VSV and Swiss outbred mice, VSVcould not be detected in the CNS of BALB/c (+/-) mice, even 5-days post-infection (Table 1). However, VSV could easily be iso-lated from the CNS of almgst all nude mice and even thosereconstituted with 5 x 10 syngeneic T lymphocyteg (Table 1).The fact that nude mice reconstituted with 5 x 10 syngeneic T

* lymphocytes were able to survive with significant levels of VSVin their CNS, although they did not mobilize a lasting humoral

'N antibody response suggested that cellular immunity may be the12% most influential factor in the enduring host-parasite relation-ship..x.

*@ 3. Titration of anti-VSV antibody in nude and normal mice.

After 5 days of infection, immune response were detected in8 out of 10 nude mice reconstituted with 5 x 109 T lymphocytes(Table 2). Compared to Balb/c (+/+) mice infected with tsG31-KS5VSV for 5 days, the neutralizing antibody response of T lympho-

." %cyte reconstitute nude mice was weak. Only one mouse reconsti-tuted with 5 x 109 T lymphocytes had a neutralizing antibody

OTIC

',-FCT-r,

3

titer equal to Balb/c (+/+) mice responses. A late humoralresponse was elicited in some of the nude mice reconstituted with5 x 10 T lymphocytes, however, only 30% of the nude miceinfected for 20 or 30 days had antibody responses comparable toBalb/c (+/+) mice. This correlates to the fact that only 20% ofthe T lymphocyte reconstituted nude mice infected for 20 dayswith tsG31-KS5 VSV survived the WT VSV challenge.

Only 4 of 10 tsG31-KS5 VSV infected nude mice that did notreceive T lymphocytes had detectable neutralizing antibody intheir sera, and none had a late humoral response against thevirus (Table 2). When nude mice were injected with 5 x 107 Tlymphocytes, 70% of the mice survived a tsG31-KS5 VSV infection,which is similar to the r sults obtained when nude mice werereconstituted with 5 1 10 T lymphocytes (data not shown). Nudemice receiving 5 x 10 T lymphocytes had vigorous neutralizingantibody responses against tsG31-KS5 VSV (Table 2). In fact, inthe nude mice, reconstituted with 5 x 107 T lymphocytes, earlyand late humoral antibody responses, against tsG31-KS5 VSV,equaled the Balb/c (+/+) mice antibody responses against the

o* virus. The high titers of neutralizing antibody, however, werenot required for protection from the CN9 disease, since most ofthe nude mice reconstituted with 5 x 10 T lymphocytes andinfected for 30 days did not have a vigorous late humoralantibody response, yet survived the tsG31-KI5 VSV infection aswell as nude mice reconstituted with 5 x 10 T lymphocytes.

When sera from nude mice, both those receiving T lymphocytesand those not reconstituted, were tested for antibody thatreacted with tsG31-KS5 VSV in SPRIA, all the sera tested positive

V (data not shown). A correlation existed between the quantity ofneutralizing antibody and the amount of total antibody that boundVSV. The sera of nude mice with elevated titers of neutralizing

%. antibody bound 3 to 5 times more radiolabeled antibody in theSPRIA than did the sera from mice that were negative for neutral-izing antibody. Antibody detected by SPRIA could have been non-neutralizing antibody or neutralizing antibody not detected bythe plaque reduction assays.

4. Titration of VSV in the CNS of BALB/c and nude mice.

By 5 days of infection with tsG31-KS5 VSV, infectious VSVwas difficult to isolate from the brains of persistently infectedBalb/c (+/+) mice (Table 1). Infectious VSV, however was read-ily isolated from nude mice reconstituted with 5 x 10 T lympho-cytes one day before infection or from nude mice only inoculatedwith the virus. In nude mice reconstituted with 5 x 10 T lym-phocytes and infected for 20 or 30 days, VSV was retrieved onlyfrom the CNS of animals that had relatively ninor amounts ofantibody or no neutralizing antibody (Table 3). Infectious VSVwas rarely recovered from nude mice that were reconstituted with5 x 10 T lymphocytes and infected for more than 10 days. The

-,..

4

strong neutralizing antibody responses7in Balb/c (+/+) mice andin nude mice reconstituted with 5 x 10 T lymphocytes probablymade recovery of infectious VSV difficult. All virus isolatedfrom any of the mice was neutralized by antibody made against WTVSV.

infectious VSV was not isolated from the spleens. livers orsera of any of the mice in Table 3, except for the spleens of 2nude mice that had not received T lymp~ocytes and had beeninfected for 5 days. Less than 1 x 10 PFU (plaque formingunits) of infectious VSV per spleen were found. The virusisolated from the spleens was neutralized by antibody against WTVSV. The persistent infection, therefore, largely appeared to belimited to the CNS and was not systemic.

5. Characterization of the cloned VSV isolated from the CNS ofnude mice.

Since infectious VSV was readily isolated from nude micethat were reconstituted with 5 x 10 T lymphocytes, VSV persist-ing in the CNS for 20 days was ?haracterized. The brains of nudemice, reconstituted with 5 x 10 T lymphocytes and one day laterinfected with tsG31-KS5 VSV, were removed after 20 days of infec-tion and virus clones were isolated and plaque purified. Todetermine if the clones could asymptomatically persist in normalmice, the CNS clone viruses were inoculated into Swiss outbredmice. The results of eight clones from the brains of 4 differentmice (BPlA and BP1B are from the same mouse, BP2A and BP2B arefrom the same mouse, etc.) are shown in Table 4. All the CNSisolated clones produced more aggressive diseases than tsG31-KS5VSV in normal mice. In fact, most of the CNS isolated cloneswhen infected into the normal mice were lethal to all the ani-mals. The disease produced by the brain isolated VSV, however,was not like WT VSV infection. WT VSV killed all the mice by 4days of infection, whereas the brain isolated clones produced aslowly progressing, degenerative disease that generally did notkill the mice for at least 10 days. The brain isolated virus wasalso less temperature-sensitive than tsG31-KS5 VSV (Table 4).Induction of the more aggressive disease by the brain isolatedVSV could ostensibly be attributed to the brain isolated VSVbeing less temperature-sensitive than tsG31-KS5 VSV.

6. Inoculation of Swiss outbred mice with pooled VSV isolatedfrom the brains of nude mice.

Selection of CNS isolates that caused more aggressive dis-eases and that were less temperature-sensitive might haveoccurred when the VSV was cloned, thus the CNS clones may nothave been indicative of most of the viruses in the brain pool.Swiss outbred mice, therefore, were inoculated with the virus offour brain pools from which the cloned viruses were derived.Similar to the CNS c1nned isolates, the total brain pools also

-Nj

6m

..............................

5

produced more aggressive diseases than the original tsG31-KS5 VSV(Fig. 2).

7. The modulating influence of P-endorphin.

Similar to our earlier observations with neurotensin andSwiss outbred mice, a single intracerebroventricular injection of100 ng of P-endorphin i BALB/c (+/+) mice, 24 hr prior to aninoculation with 1 x 10 PFU of tsG31-KS5 VSV, dramaticallyaltered the course of clinical disease. The introduction of @-endorphin caused an aggressive CNS disease leading to the deathof 70% of the animals within 15 days while only 3% of the miceinfected with only the ts VSV died (Fig. 3).

The introduction of P-endorphin 24 hr prior to an intracere-bral infection with the ts VSV, however, did not alter theprogression of CNS disease in nude mice (Fig. 3). The inabilityof the neuropeptide to alter the course of CNS disease in nudemice may have been a reflection of a lack of target cells for P-endorphin in the athymic animals. Further experiments with

*_ reconstituted nude mice will be needed to assess the basis for-- this observation.

C. SUMMARY

Nude mice have been used as hosts in many viral model sys-tems, since the role of T cells in the animals defense against

Athe infection can be evaluated. Infection of nude mice withtsG31-KS5 VSV induced a progressively degenerative CNS diseasethat was lethal to all the animals by 26 days of infection. Incontrast, tsG31-KS5 VSV inoculated into normal mice, eitherBalb/c (+/+) or Swiss outbred, produced an asymptomatic, persis-tent infection. Although, the immune cell activity may havecleared the VSV from other organs in nude mice, immune cells werenot effective in clearing the tsG31-KS5 VSV from the CNS of thenude mice.

* A T cell-dependent immune response was required to protectmice from the tsG31-KS5 VSV infection, since nude mice survivedthe infection and remained disease free when they were reconsti-tuted with syngeneic T lymphocytes.

Studies to determine if tsG31-KS5 VSV induces status spon-* giosus in normal and/or nude mice will help determine if the sta-

tus spongiosus, in a manner we previously established with cer-." tain ts VSV mutants in outbred mice, is a requisite for the CNS

disease. Histopathological evaluation of nude mice reconstitutedwith T lymphocytes and persistently infected with tsG31-KS5 VSVand of nude mice only infected with the VSV, will determine if T

*_ lymphocytes can protect the animals from the spongiform changes

04.,,

6

and other neurological lesions that may cause the neurologicaldisease.

How the injected T lymphocytes functioned to protect thenude mice was not clear. The vigorous humoral response elicitedin nude mice reconstituted with 5 x 10 T lymphocytes did notseem to be important in protection of the nude mice from the CNSdisease induced by the pegsistent infection, since many nude micereconstituted with 5 x 10 T lymphocytes did not have a detect-able humoral response after 10 days yet the a9imals remainedhealthy. Nude mice reconstituted with 5 x 10 T lymphocytesplausibly had both primary and secondary humoral responsesagainst thg tsG31-KS5 VSV, whereas most of the nude mice receiv-

A ing 5 x 10 T lymphocytes probably only had primary immuneresponses against the VSV. The injected T lymphocytes probablywere not directly responsible for protection of the nude micefrom the CNS disease.

Since VSV was not consistently found outside the CNS in nudemice not reconstituted with T lymphocytes, an immune mechanism,possibly NK cell activity, must have been functioning in nudemice that cleared the virus from organs other than the CNS.Other viruses have been shown to persist in the CNS after beingcleared from the rest of the animal. In normal mice infectedwith lymphocytic choriomeningitis virus (LCMV), adoptive transferof specific cytotoxic T cells cleared the virus from the animalsexcept for their CNS which remained persistently infected. A Tcell product, such as an interferon or a lymphokine, which couldcross the blood-brain barrier more easily than cells may beresponsible for the protection of nude mice reconstituted with Tlymphocytes from the CNS disease.

The viruses that persisted in the CNS of the T lymphocytereconstituted nude mice were less temperature-sensitive andcaused a more aggressive disease when inoculated into normal micethan tsG31-KS5 VSV. This phenotype may be necessary for the VSVto persist in the CNS of the host. The T lymphocytes injectedinto nude mice may alter the biochemical nature of the VSV, thus

* inhibiting the ability of viruses to induce the CNS disease.Since infectious VSV can e readily isolated from the nude micereconstituted with 5 x 10 T lymphocytes, the biochemical natureof the long term persistent viruses can be evaluated and comparedto VSV isolated from nude mice that have not been reconstituted.

,.'. . '~~~. . ~~ ~~w 4 * L.

7

D. PUBLICATIONS

Doll, S.C., and Johnson, T.C. 1988. Reconstitution with Tlymphocytes protects nude mice from a central nervous systemdisorder induced by a temperature-sensitive vesicularstomatitis virus. J. Gen Virol. 69: (in press).

Doll, S.C., and Johnson, T.C. 1988. A study of persistent viralinfections using nude mice and a temperature-sensitivemutant of vesicular stomatitis virus. N.Y. Acad. Sci. (inpress).

Doll, S.C., and Johnson, T.C. 1988. P-endorphin alters a viralinduced central nervous system disease in normal but not in

.* nude mice. (submitted for publication).

. Doll, S.C., and Johnson, T.C. 1988. The nuclear protein ofvesicular stomatitis virus isolated from the brains of nudemice is responsible for abated viral RNA synthesis at the

* ° normal body temperature of mice. (submitted for publica-tion).

-4.

N

8

Table 1. ANTIBODY AND VSV IN PERSISTENTLY INFECTED MICE

No. of Days Neutralizing VSV inT-Cells Post-Infection Antibody Brain

BALB/C 0 5 4/10 10/10(nu/nu)

10 0/4 7/7

20 0/4 4/4

5 x 106 5 8/10 10/10

10 2/4 9/10

20 1/5 4/4

5 x 107 5 8/10 10/10

10 4/4 2/10

20 6/6 0/6

BALB/c 5 0/6

10 0/5-'+i+)

- 20 0/10

Mice (nu/nu) were reconstituted with the indicated number of

• syngeneic T lymphocytes and 24 hr later inoculated with 10tsG31-KS5 VSV. After 5, 10, or 20 days of infection the animals

- were sacrificed and their sera were measured for neutralizingantibody to VSV by a plaque reduction assay. Animals were scoredpositive if any neutralizing antibody was detected. VSV in brainwas determined by plaque assays of brain tissue homogeneates on

* BHK cell monolayers incubated at 310C, a permissive temperaturefor tsG31-KS5 VSV, and identification of the virus as VSV byantibody neutralization tests.

-...

* 9A .9

TABLE 2. TITRATION OF ANTIBODY IN NUDE AND NORMAL MICE

* NEUTRALIZING ANTIBODY**

No. Mice 50%Positive Neutralization

No. of T-Celis Total Titers of ThoseInjected DAY PI Mice Positive

Balb/c Mice(nu/nu)

0 5 4/10 100(3), 10410 0/10 -20 0/10

5 x 106 5 8/10 10(3}, 10(3),10 10

10 3/10 10(2), 10020 7/10 10J2), 00,

10 ig (3)30 4/10 10, 10 (3)

5 x 107 5 8/10 10- 1004 2),1010-0, 10 (41.-- 0 10/10 10013), 10 13),

10- (3) 6 10-"20 10/10 100, 10 (830 10/10 100, 10 10 (2)

30106(7), 10(2

Balb/c (+/+) Mice

5 4/4 104(2), 105(2)

10 4/4 103(2), 104, 105

20 4/4 10, 106(3)

30 4/4 10 6 , 10 (3)

Nude mice were econstituted with either 5 x 106 T

lymphocytes or 5 x 10 T lymphocytes and 1 day laterinfected with tsG31-KS5 VSV or only inoculated with virus.

Neutralizing antibody against VSV was determined by plaquereduction assays.

.< 10

TABLE 3. ISOLATION OF INFECTIOUS VSV FROM NUDE MICE

VSV BRAIN TITER (PFU)

No. MiceNo. of T Cells Positive Ave. PFU inInjected* DAY PI Total Positive mice

Balb/c (nu/nu)

0 5 10/10 1.5 x 104

10 10/10 5.3 x 104

20 10/10 2.1 x 106

S5x 106 5 10/10 4.2 x 103

10 10 0 4.6 x 104

20 6/10 4.3 x 103

30 7/10 1.2 x 105

5 x 10 7 5 10/10 3.8 x 10 4

10 2/10 150

20 0/10

30 0/10

Balb/c (+/+)

5 0/10

10 0/10

20 0/10

*ude mice were reconstituted with 5 x 106 T lymphocytes or

5 x 10 T lymphocytes, then 1 day later infected with tsG31-KS5VSV or only inoculated with the virus.

.1',-, -'-","--, ""''' - - ' " ; - - .. ."., .""" . . ... .2 " " ' ". " - " " '. v"" "" > " "

Table 4. CHARACTERIZATION OF CLONED VSV ISOLATEDFROM THE CNS OF MICE

-A VIRUS SURVIVAL PARALYZED PFU §(%) (%) (390 C/310 C)

WT 0 0 2 x 10 0

tsG31 97 10 9 x 10- 4

BPlA 0 30 2 x 10-2

BPIB 0 40 2 x 10- 2

BP2A 0 80 2 x 10- 2

BP2B 60 90 7 x 10- 2

BP3A 60 100 2 x 10- 2

BP3B 0 40 1 x 10-2

BP4A 50 60 3 x 10- 2

BP4B 0 30 1 x 10_1

*The brain isolated VSV was double cloned by plaque

* purification

**The clones were inoculated into groups of 10 Swiss

outbred mice and the percent survival was determined after 20days of infection.

* §The temperature-sensitivity of the VSV was determined bycomparing plaque assays done at 390 C and 310 C.

.. ~~% %.

12

'-. FIGURE LEGENDS

K-Fig. 1. Protection of Balb/c nude mice from CNS disease, caused

by tsG31-KS5 VSV with syngeneic splenic lymphocytes. Tennude mice were infected with tsG31-KS5 VSV (0) or 15 nudemice were reconstituted with 5 x i06 T lymphocytes and 1 daylater inoculated with the virus (0).

Fig. 2. Survival of Swiss outbred mice intracerebrally infectedwith VSV recovered and cloned from the brains of tsG31-KS5VSV Pfected nude mice. Nude mice were reconstituted with 5

_. x 10 T lymphocytes and 1 day later inoculated with tsG31-KS5 VSV. After 20 days of infection, the mice were sacri-

* ficed and VSV was isolated from their CNS. The brain iso--'i lated VSV (BP VSV) from 4 different animals, or tsG31-KS

VSV, were inoculated into groups of ten Swiss outbred miceinfected with tsG31-KS5 VSV:

*(tsG31-KSS VSV D BPI VSV ,BP2 VBV ,BP3 VBVBP4 VSVE]).1°

Fig. 3. Mice received a single intracerebroventricular injectionof 100 ng of f-endorphin in 10 Al of sterile distilled water(shaded bars) or with 10 Al of sterile distilled water aIone(open bars). 24 hr later all mice were infected with 10plaque forming units (PFU) of tsG31-KSg VSV. (r), nude(nu/nu) mice reconstituted with 5 x 10 syngeneic T-lympho-cytes one day prior to their injection with VSV.

0 'q

0.

-N"

9

13

FIGURE 1

100

so-

40-

-~ 20-

4 S 2 16 20 I4 as 32

*- DAYS POST-INFECTION

14

FIGURE 2

100-

80-

mm

01 1~ 4402 2

* DAYS POST-INFECTION

15

FIGURE 3

4,.

,

I~00 -

80-

S60

0' 40-% e0

20-

(nu/nu) (r) (nu/nu) (r), +/+ (nu/nu) (r) 1+/+) (nu/nu) (r) 1+1 (nu/fu) (r)

5 10 15 20 25

DAYS POST-INFECTION

4.

0b

ij,

4~n

',4

* DISTRIBUTION LIST

Behavioral Immunology Program

Annual, Final and Technical Reports (one copy each except as noted)

INVESTIGATORS

Dr. Itamar B. Abrass Dr. Sheldon CohenDepartment of Medicine Department of PsychologyUniversity of Washington Carnegie-Mellon UniversityHarborview Medical Center Pittsburgh, PA 15213Seattle, WA 98104

Dr. Walla L. DempseyDr. Prince K. Arora Dept. of Microbiology andNIDDK, Bldg. 8, Rm. 111 ImmunologyNational Institutes of Health The Medical College ofBethesda, MD 20892 Pennsylvania

3300 Henry AvenueDr. Andrew S. Baum Philadelphia, PA 19129Department of Medical PsychologyUniformed Services University Dr. David L. Felten

of Health Sciences, B3050 Department of Anatomy4301 Jones Bridge Road University of RochesterBethesda, MD 20814-4799 School of Medicine

601 Elmwood AvenueDr. Charles A. Bowles Rochester, NY 14642Merrifield Research Lab, Inc.P.O. Box 2362 Dr. John F. HansbroughMerrifield, VA 22116-2362 Department of Surgery

UCSD Medical CenterDr. Karen Bulloch 225 Dickinson StreetHelicon Foundation San Diego, CA 921034622 Sante Fe StreetSan Diego, CA 92109 Dr. Robert L. Hunter

Department of PathologyDr. Michael D. Cahalan Emory Univ. School ofDepartment of Physiology and Biophysics MedicineUniversity of California, Irving WMB 760

.'s Irvine, CA 92717 Atlanta, GA 30322

Dr. Donald A. Chambers Dr. Terry C. JohnsonHealth Sciences Center Division of BiologyUniversity of Illinois at Chicago Ackert HallP.O. Box 6998 Kansas State UniversityChicago, IL 60680 Manhattan, KS 66506

Dr. Christopher L. Coe Dr. Sandra LevyDepartment of Psychology University of PittsburghHarlow Primate Laboratory School of Medicine

University of Wisconsin 3811 O'Hara StreetMadison, IL 53715 Pittsburgh, PA 15213

0%

Dr. Lester Luborsky Dr. Eric M. SmithDepartment of Psychiatry Department of Psychiatry308 Piersol Building/G1 University of TexasUniversity of Pennsylvania Hospital Medical BranchPhiladelphia, PA 19104 Galveston, TX 77550

Dr. Steven F. Maier Dr. Ross R. Vickers, Jr.Department of Psychology Naval Health Research Ctr.University of Colorado Bldg. 346Campus Box 345 P.O. Box 85122Boulder, CO 80309 San Diego, CA 92138

Dr. Diana S. MalcolmDepartment of Surgery, USUHSUniformed Services University

of Health Sciences4301 Jones Bridge RoadBethesda, MD 20814-4799

Dr. Michael H. MelnerDepartment of Reproductive BiologyOregon Regional Primate Center505 N.W. 185th Avenue

* Beaverton, OR 97006

Dr. Vera B. MorhennDepartment of DermatologyStanford University Medical SchoolStanford, CA 94305

Dr. Jose R. Perez-PoloGail Borden Bldg., Rm. 436

,, University of Texas Medical BranchGalveston, TX 77550-2777

Dr. Howard R. PettyDepartment of Biological SciencesWayne State University

Detroit, MI 48202

Dr. Bruce S. RabinClinical ImmunopathologyChildrens HospitalUniversity of Pittsburgh School ofMedicine

Pittsburgh, PA 15213

Dr. Seymour ReichlinDirector, Clinical Study Unit

9New England Medical Center Hospitals, Inc.171 Harrison AvenueBoston, MA 02111

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