Monokine-mediated increase in human immunodeficiency virus type 1 expression in chronically infected promonocyte- and T-cell-derived lines.

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CLINICALANDDIAGNOSTIC LABORATORYIMMUNOLOGY, Sept. 1994,p.517-525 Vol. 1, No. 5 1071-412X/94/$04.00+0

Monokine-Mediated

Increase

in

Human

Immunodeficiency

Virus Type 1

Expression in Chronically Infected

Promonocyte-

and T-Cell-Derived Lines

WEN-ZHEHO,* NATALETOMASSINI, RAVI CHERUKURI, GE SHUN-DI, LI SONG, HAE-RAN LEE,ANDSTEVEND. DOUGLAS

Divisionof

Allergy,

Immunology, and Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, Universityof Pennsylvania MedicalSchool, Philadelphia, Pennsylvania 19104

Received18February 1994/Returnedformodification4April1994/Accepted 20April1994

TheACH-2 cell clone derived froma humanT-cell line and

chronically

infected with human immunodefi-ciency virus 1 (HIV-i)and the Ul cell clone derived fromahuman promonocyte cell line and alsochronically infectedwithHIV-iproduce HIV-1 in responsetostimulation with monokine-enriched supernatantsprepared fromhighly purified populationsofperipheral blood-derived human monocytes. Monokine-mediated expres-sion ofHIV-1 in these cell lines resulted in augmented virus production reflected by increases in reverse transcriptase (RT)activity, production of p24 antigen, and synthesis of major viral proteins. Examination of thecellsby electronmicroscopyrevealed numerousHIV-1 virionsin the cellstreatedwith the supernatants. This stimulation of virusproduction by monokine-enriched supernatants resulted in approximately 100-fold increasesin RTactivityandp24 antigen expression incomparison with those in untreatedUlandACH-2 cells. Absorption of monokine-enriched supernatants with rabbit anti-tumor necrosis factoralphaantibodyremoved most,but not all, of the induced HIV-1 RT activity andp24antigen expression in Ul andACH-2 cell lines, suggesting that tumornecrosis factoralphainthe monokine-enriched supernatants isa majorfactor in the induction of HIV-1 expressionin these cells.

Humanimmunodeficiencyvirus type 1 (HIV-1) infection is characterized by aperiod oflong clinical latency before the developmentof symptomsand HIV-1-related disease(4).The latentperiod of AIDSis influenced byfactorswhich activate HIV-1 replication in different cell types. In addition to the synthesis of viral regulatory proteins, HIV-1 expression and replicationareunder thecontrol ofimmunologically important mechanisms, such as the endogenous cytokine network (20, 25). Cytokines are important constituents of immune cell activation that act as physiologic inductive signals in the regulation of immune responses. Among HIV-1-inductive cy-tokines, tumor necrosis factor alpha

(TNF-a)

up-regulates HIV-1expressioninchronically HIV-infectedTcellsaswellas inmonocyticcell lines(20, 25).Twoothercytokines, interleu-kin 6 (IL-6) andgranulocyte macrophage colony-stimulating factor, enhance virus expression in cells of the monocyte lineage only. Furthermore, TNF-a acts synergistically with either IL-6 or granulocyte macrophage colony-stimulating factor(20, 25).Thesecytokines alsoenhance HIV-1 expression inprimarymonocyte/macrophage cultures(13, 19).Inaddition to up-regulatingHIV replication, somecytokines possess the ability to suppress HIV-1 expression. Alpha interferon de-creases HIV-1 expression in both acutely and chronically infected cellsposttranslationallyby inhibition of the budding of virions (22).Anothercytokine, transforming growth factor

I,

down-regulates constitutive as well as induced expression of HIV-1byblockingbothtranscriptional and posttranscriptional mechanisms (21).Cells of the mononuclear phagocyte system are central elements in antigen presentation, phagocytosis, inflammation, and tumor destruction, and they produce and secrete a vastarrayofdifferent molecules that are involved in

*Correspondingauthor. Mailing address: Division of Allergy, Im-munology, and Infectious Diseases, Children's Hospital of Philadel-phia,34th andCivic Center Blvd.,Philadelphia, PA 19104.

the defenseagainstinfection andmalignancy(16,

31).

Mono-cytesperform manyof their functionsby secreting regulatory cytokines,includingTNF-a,

IL-1,

and IL-6. We havetherefore studied the secretion of these monokines by highly

purified

populationsof human monocytes underessentially endotoxin-free conditions and theeffectsofmonokine-enriched superna-tants derived from a freshly isolated monocyte culture on HIV-1 induction in chronically infected promonocytic and T-cell lines. Our datashow thatmonokine-enriched superna-tants from the primary monocyte culture greatly induced HIV-1expression in these cell lines.

MATERIALSANDMETHODS

Cells.Monocytes fromvenousblood obtained from normal HIV-1 seronegative adult donors were isolated by previously described methods used in our laboratory (9, 10). Briefly, primarymonocyteswereisolatedby the Ficoll-Hypaque tech-niqueandsubsequentlycultured ingelatin-coated flasks for 45 minin5%CO2at37°C. Nonadherent cells were removed by washing the flasks 8 to 10 times with Dulbecco's modified Eaglemedium, and gelatin-adherent cells were detached with EDTA.The purifiedmonocytes werethen plated at a density of2.5 x 105perwellin48-wellplates (Costar)or5.0x

105

per well in 24-well plates (Corning). The cells were cultured in RPMI 1640 medium with10% autologous sera in 5% CO2 at 37°C for7 to 10 days. These cultures were essentially 100% nonspecific esterase positive after cultivation in vitro for 7 days.Other characteristicsof these cells have been previously described (9, 10). In all cases, functionally endotoxin-free media andserawereused. Half of the volume of the medium waschangedevery 5days. The chronically HIV-1-infectedUl and ACH-2 cell lines were provided by Thomas Folks and obtainedthroughthe AIDS Research and Reference Reagent

517

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Q 50000

40000- 30000-

20000->

10000L_

0 o o o00 X

C C

U1 ACH-2

FIG. 1. Inductionof HIV-1 expression inUlandACH-2 cells with monokine-enriched supernatantsfrom freshly isolated human mono-cytecultures.Ul andACH-2cells were cultured for 48 h in complete medium alone (control)orin mediumconditionedwithundiluted or diluted (as indicated) monokine-enriched supernatants. The results show the ability of the monocyte supernatants to induce HIV-1 expression asmeasuredbyanHIV-1 RTactivityassay.

Program Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Ul is a cloned cell line derived by limiting dilutionof a human promonocyte tumor cell line (U937), which survived acute infection with HIV-1 and possesses two integrated copies of HIV-1 proviral DNA. Ul cells constitutively express low levels of the virus as determinedbyimmunofluorescence, cellular and supernatant-associatedreversetranscriptase(RT)activity, HIV p24 antigen capture,and Western blot(immunoblot)and Northern(RNA) blot analyses (6, 7). ACH-2is aclone derived from ahuman T-cell line that survived acute infection and that constitutively produces lowlevels of supernatant RT(2, 5).8E-5 is aclone derived from a human T-cell line and contains a single integratedcopyofproviralDNAdirecting synthesisof defec-tive virusparticles(8).

Human monocyte supernatant preparation. Forthe prepa-ration ofmonokine-enrichedsupernatants,human monocytes were isolated as described above. The supernatants were collectedafter incubation at37°Cin5% CO2 for24 h(unless otherwiseindicated)andcentrifugedat13,800xgfor 5 min in amicrocentrifuge. Supernatantswere aliquoted and storedat -80°C until used. The experiments were conducted under essentially endotoxin-freeconditionsasmonitoredbya

specific

lipopolysaccharide detection test (Limulus amoebocyte lysate assay).

Electronmicroscopy. Cellswere removed fromplastic cul-turewellswithaPasteurpipette.

They

werethen transferredto 15-mlplasticcone-shaped

tubes,

centrifuged

for5 minat600x

g,suspendedinphosphate-bufferedsaline

(PBS),

pelleted,and immediatelyfixed undisturbed with2%

glutaraldehyde

in 0.1 M sodium cacodylate buffer (pH 7.4). After having been washedin 0.1 Mcacodylatebuffer three

times,

thespecimens were fixed in 1% osmium tetroxide for 30 min at room

temperature. Dehydrationwasdone withgraded

alcohol,

and the cellswere embedded inEpon-EM-Bed-812. Thinsections were cutwithanLKBUltratome IIImicrotome and examined withaPhilips300electron microscope.

Detection of HIV-encodedproteins byimmunoblotting. To-tal celllysateswere prepared from untreated and treated

Ul,

8E-5,and ACH-2cells (6 x 106).The immunoblot

analysis

of HIV-1 proteins was carried out as described previously

(5).

Briefly,30 ,ugofproteinextracted from treatedanduntreated

z r

ccnn CM Ct

L7) II I

UJ Li] 0 0

cc CC) < <

z

D D

- 120

.- -- 41

.~~~~~~~~~~~_{_}

~ --24

FIG. 2. Westernblot detectionofHIV-1proteins in Ul, 8E-5,and ACH-2 cell lines with or without monokine-enriched supernatants (SN) from freshly isolated humanmonocytes. Ul, 8E-5, and ACH-2 cells (5 x 106) were cultured incomplete medium alone or in the supernatantsfrom the monocyte culture for 48 h. Total cell lysates were prepared from pelleted cells. Immunoblots were generated as described in Materials and Methods, and the major viral proteins reacting with the IgGfromtheAIDS patient serum areidentified.

Ul, 8E-5, and ACH-2 cells waselectrophoreticallyseparated on7.5% sodiumdodecylsulfate-polyacrylamide gelsand trans-ferred to nitrocellulose. After saturation with a 5% milk solution, thenitrocellulose strips were treated with the anti-body (immunoglobulin G [IgG] purified from HIV-seroposi-tive human serum) againstHIV-1 antigens(diluted 1:100) for 2 h and then with horseradish peroxidase-conjugated goat anti-humanIgG(Cappel) for 1 h. Boundantibody was visual-ized with4-chloro-1-naphthol (Sigma, St. Louis,Mo.).

Detection ofHIV-encodedproteins byindirect immunoflu-orescence.Ul and ACH-2cellswerespundown,airdried,and fixed in 100% coldacetonefor20 min atroom temperature. IgG purified from HIV-1-seropositive human serumwas di-luted in PBS(1:100), added to the slides for 45 min, and then removed bywashingthree times in PBS. Fluorescein isothio-cyanate-conjugated goat anti-human IgG antibody (1:30) (Cappel) was added to the slides for 45 min, and this was followed by three additional washes in PBS. Coverslips were added withaglycerol layer, andthe slideswere observed for fluorescence-positive cells on a Leitz-Wetz Ortholux fluores-cencemicroscopeat a magnificationof X400.

RTassayand ELISA for thep24antigenofHIV-1.TheRT assaywascarriedout aspreviouslydescribed (29),with these modifications. Briefly, 10,ul of eachsupernatantwasaddedto a cocktail containing poly(A), oligo(dT) (Pharmacia, Inc., Piscataway, N.J.), MgCl2, and

[32P]dTIT

(Amersham Corp., Arlington Heights, Ill.) andwas incubated for 20 h at 37°C. Then 30,ul of the cocktailwasspottedontoDE81 paper,dried, andwashed in 2x saline-sodium citrate buffer. The paperwas dried and counted with a scintillation counter

(LS

3801; BeckmanInstruments,Inc., PaloAlto,

Calif.).

Thep24 antigen expressionin the supernatants from treated and untreatedUl and ACH-2cells wasanalyzed with acommercially available enzyme-linked immunosorbent assay

(ELISA)

kit (Coulter, Hialeah,Fla.).

Induction ofHIV-1 expression in Ul and ACH-2 cells.Ul andACH-2 cellswerecultured incompletemedium aloneor in the supernatants from human monocytescultured for24hin vitro. The experiments were carried out either with 24-well

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MONOKINE-MEDIATED EXPRESSION OF HIV-1 519

FIG. 3. Electronmicrograph of unstimulated ACH-2cells.There arefewextracellularHIV-1virions (arrows).Bar

x10,560). Inset:highermagnification of HIV-1 area. Bar = 1

pLm

(magnification, X28,600).

tissue culture plates (5 x 105 cells per well) or with 6-well tissue culture plates (2 x

106

cells per well). As controls, recombinant human cytokines TNF-ot (Genzyme) and IL-6

(Genzyme)

at concentrations ranging between 0.1 and 1,000

U/mlwereadded tothecells. Supernatants were harvested at 48 hpostinductionandassayed forRTactivity and p24 antigen expression.

Blocking of HIV-1 induction by anti-TNF-a antibody. We performed blocking experiments with rabbit polyclonal anti-body (Genzyme) which is directed against TNF-ot. Superna-tantsfromfreshlyisolated monocytes were incubated with the antibodyatdifferent concentrations (10 to 100 ,ug/ml) for 2 h and then addedtoUl andACH-2cells. As a control,normal rabbitserum wasadded at the same concentrations.

1

p.m

(magnification,

Cytokine assays. Supernatants were collected fromfreshly isolated monocyte cultures and centrifuged, aliquoted, and stored at -80°C. The levels of TNF-ot, IL-1, and IL-6 were determined with ELISA kits(Genzyme).

RESULTS

Efects of monocyteculturesupernatants onHIV-1 replica-tion in Ul and ACH-2 cells. Since TNF-ox, IL-1, and IL-6 productionby human monocytes has been reported, we deter-mined whether supernatants from freshlyisolated monocytes hadeffects similar tothose observed with chemically defined TNF-o, IL-1, and IL-6. Freshly isolated monocytes were culturedfor 24 h afterplating. Supernatants were collected and

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CLIN. DIAGN. LAB.IMMUNOL.

r *@X-S;isN's+aW77 .N-.s

v4

-m 9ef*>#4jW

v

A~ t '.' .'*

FIG. 4. Electronmicrographof ACH-2 cellsstimulatedby monokine-enrichedsupernatants.Thereare numerousHIV-1virionsattachedtothe cellmembrane(open arrowheads)andbuddingviralparticles(arrows).Bar= 1 ,um(magnification, X23,140).Theopenrectangleindicates the region in the inset. The inset shows onevirionexhibiting spike-like projections attached tothe cell membrane (thickarrow). Bar = 0.1 ,um

(magnification, X84,550).

stored without furthertreatment.When the supernatantswere added to the Ul and ACH-2 cells, HIV-1 replication was enhanced(Fig. 1).Inordertodetermine whetherthe induction of HIV-1 expression in Ul and ACH-2 cells by monocyte-enriched supernatants isaconcentration-dependentresponse, weexposedthe cellstothe undiluted and diluted supernatants

(1:1,000 to 1:10) for 48 h. Of the untreated Ul and ACH-2 cellsexaminedbyimmunofluorescence, only1to5%expressed detectable amounts of proteins reactive with the anti-HIV-1 IgGantibody, whereas 50to80%of the cellsshowedpositive immunofluorescence 48 h after treatment with monokine-enriched supernatants fromfreshly isolated monocytes

(data

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FIG. 5. Electronmicrographof unstimulatedUlcells. NoHIV-1virionsweredetected.Atotal of 200 cellcrosssectionswereexamined. Bar

= 1 P,m(magnification, x14,400).

notshown). Thismonokine-mediatedexpression of HIV-1 in all three cell lines resulted in augmented virus induction as demonstrated by an increase in the synthesis of major viral proteins, including gpl20, gp55, gp4l, and p24 (Fig. 2). In addition, whenUl and ACH-2cellswereculturedwith super-natants derived from freshly isolated monocytes for 48 h and

examinedbyelectronmicroscopy, numerousHIV-1 viral par-ticleswereobserved incomparison with those in unstimulated Ul and ACH-2 cells (Fig.4 to6).

Effect of anti-TNF-a antibody on HIV-1 expression in Ul andACH-2 cells.Experimentswere performed todetermine whether neutralizing antibody to TNF-a has the capacity to VOL. 1, 1994

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FIG. 6. ElectronmicrographofUlcells stimulatedbymonokine-enrichedsupematants.NumerousextracellularHIV-1virionsarepresent.Bar= 1

p.m (magnification, x11,160).The inset showstwomodes ofHIV-1particleattachmenttothe cell membrane. Bar=0.1p.m

(magnification, X88,350).

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FIG. 7. Effectsofanti-TNF-ax antibodyonHIV-1expression inducedwithmonokine-enriched supernatantsin theUl (A)and ACH-2(B)cell

lines. Supernatants from freshly isolated monocytes were incubatedwith a rabbit anti-TNF-a antibody or normal rabbit serum at different

concentrations(6.25to100,ug/ml)for 2 hat37'Candthen addedtoUl and ACH-2 cells.Supernatantswereharvested for determination ofHIV-1

RTactivityat48 hposttreatment.The results shownarerepresentative of results of threeindependent experiments.

block the effect of TNF-ot in the monocyte-derived

superna-tantsoninduction of HIV-1expressioninUl and ACH-2cells.

The inducedexpressionof HIV-1 in both Ul and ACH-2 cell culturesby monocyte-derived supematantswasinhibited, but not completely, by preincubation of the supernatants with rabbit anti-TNF-otasassayed byHIV-1 RTactivity (Fig. 7).No significant blockingwasobserved when theantibodywasadded at a concentration of less than 2.5 ,ug/ml (Fig. 7). Thus, the efficiencyof theblockingofproductiveHIV-1 replicationwas

dependentonthe concentration of anti-TNF-ctantibodyused duringthepreincubation.

TNF-a, IL-1, and IL-6 production by primary cultures of peripheral blood-derived monocytes/macrophages. The cul-turesupernatantsofprimarymonocyteswerecollectedatdays 1, 2, 4, and 7 during the first week and at weekly intervals afterwards. The supernatantswerethen assayedfor the

pres-enceofTNF-a,IL-1,and IL-6.Highconcentrations ofTNF-a, IL-1, andIL-6 were detectedduringthe first 72 h (Table 1). The levelsof cytokinesdeclined steadilyduringthesubsequent days. Byweek2, TNF-a,IL-1,andIL-6werenotdetectableby

theassaysperformed.

DISCUSSION

Mononuclearphagocytes, eitherascirculatingblood

mono-cytes or as tissue macrophages, influence host defense

re-sponses through their capacity to present antigens and to

release several types of soluble mediators(30). These

mono-cyte/macrophage-derived cytokinesactinaparacrine-like

fash-ion in the local environment to stimulate immune responses

but also in an endocrine-like fashion on distant organs that participate in inflammatory responses. IL-1 andTNF-axwere

the first monocyte-derived cytokines shown to have diverse regulatory properties in immunity and inflammation (3, 16, 31).We have shown thatmonocytes secretelarge quantitiesof TNF-a and IL-6ondays1to3 of the first week in vitro(Table 1). Secretion of IL-1 andTNF-oa occursin human monocytes

followingadherence of the cellstosurfaces (1, 3, 16). HIV-1 induces production of IL-1, IL-6, and TNF-a from peripheral blood mononuclear cells and monocytes/macro-phages(14, 15, 27, 28). This effectwas observed with live or

inactivated virus as well as with purified HIV-1 envelope

glycoprotein (gpl20), suggesting that interaction of HIV-1 gpl20with itsreceptor, the CD4molecule, onthe surface of monocytestriggeredtheproductionof thesecytokines(14, 15). The role of the CD4 molecule in triggering the cytokine

response was demonstrated by blocking studies with soluble

CD4 aswell asbyinductionstudies with anti-CD4 antibodies (27, 28). Furthermore, elevated levels of TNF-a have been found in the sera and in the macrophage supernatants from

patientswithAIDS(11, 23, 26). Folksetal.previously reported that TNF-ot induces expression of HIV-1 in ACH-2cells (5).

TABLE 1. Monokines released by normal unstimulated or stimulated adultmonocytes/macrophagesa

Concn(pg/ml) of:

No.ofdaymonocytes IL-1 IL-6

TNF-a

cultured invitro

Unstimulated Stimulated Unstimulated Stimulated Unstimulated Stimulated

1 1,292 15,137 521 1,569 3,500 17,272

2 1,192 14,142 201 1,211 2,025 15,101

4 412 10,212 72 710 1,121 10,112

7 187 987 <10 238 127 1,250

14 >10 <10 >10 >10 >10 >10

21 NDb ND ND ND >10 >10

aHumanmonocytes isolatedbythegelatin-coated-surfacetechnique were cultured in 24-wellplatesat a concentration of 5 x106cells per well and eitherwereleft

unstimulated or were stimulatedwithlipopolysaccharide (10ng/ml).Production of monokineswasdetermined in thesupernatantsby monokine-specificELISA.The

results shownare meansoftriplicate culturesand are representative of results of twoindependentexperiments (two different donors).

bND,notdone.

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production inUl cells (6). Other investigators, however, have described the enhancement of HIV-1 replication byTNF-oL in the U937 cell line, including Ul cells, as well as in primary macrophage cultures (12, 19). Our results demonstrated that monokine-enriched supernatants from freshly isolated, highly purified, and unstimulated human monocytes had effects sim-ilar to those observed with chemically defined

TNF-ot,

IL-6, and IL-1 on HIV-1 expression in both chronically infected T-cell- and promonocyte-derived cell lines. However, we did not observe the enhanced effects of the monokine-enriched supernatants on HIV-1-infected primary monocyte-derived macrophages (data not shown), although the supernatants up-regulated HIV-1 expression in chronically infected promonocytes(Ul), indicating that a different mechanism(s) is involved in primary monocytes/macrophages. We did not ob-serve significant rises in HIV-1 RT activities in either the Ul or the ACH-2 cell line within 24 h after stimulation with mono-kine-enriched monocyte supernatants. The optimal effects were observed at 48 or 72 h after monocyte supernatants were added to the cell cultures, although only the 48-h results are illustrated (Fig. 1). In addition, the supernatants collected from macrophages in culture for at least 14 days (no detectable monokine secretion; Table 1) did not stimulate HIV-1 RT activities in either Ul orACH-2 cells (data not shown). The mechanism(s) by whichTNF-cx enhances HIV-1 replication in vitro has been investigated by several groups (17, 18, 24). One possible mechanism is that TNF-ox may up-regulate HIV-1 expression in vitro byactivation of nuclear factor KB(NF-KB), and HIV-1 could therefore augment its own expression (18, 24). The enhancing effect ofTNF-oton HIV-1 expression in T lymphocytes and monocytic cell lines has been shown to involve transactivation of the HIV-1 long terminal repeat promoter, in part through the binding of NF-KB to the two responsive elements located in the enhancer region of the HIV-1 promoter (17, 18). In contrast, IL-6 and granulocyte macrophage colony-stimulating factor induce HIV-1 expres-sion predominantly by posttranscriptional mechanisms (19).

Our experiments demonstrate that most of the HIV-1-inducible activity is removed by absorption of the monokine-enriched supernatants with a rabbit anti-TNF-ot antibody, indicating that TNF-oL secreted from highly purified human monocytes has effects on HIV-1 expression in the presenceof other monokines such as IL-1 and IL-6 similar to those of TNF-ot alone. On the basis of published studies, we assume that IL-1 and/or IL-6 may also be involved in the increased expression of HIV-1 in Ul and ACH-2 cells, although anti-bodies against these two monokines were not tested in these experiments for the ability to block the effects of monocyte supernatants on HIV-1 expression. The importance of mono-kines, in particularTNF-ot, in the activation ofclinically latent HIV-1 infection and disease progression in vivo is unknown. AIDS patients and individuals with AIDS-related complex haveelevated levelsofcirculatingTNF-ot compared withthose ofasymptomatic HIV-1-infected individuals andpatients with lymphadenopathy syndrome. A vicious cycle in which latently orchronically infected cells gradually expressthevirus,leading to progressive immunosuppression and subsequent opportu-nisticdiseases, could conceivably exist. These eventswould in turn result in the increased secretion ofTNF-ox,whichinduces further expression of HIV-1 and accelerates the process of lymphocyte depletion and immunosuppression.

ACKNOWLEDGMENTS

We aregrateful to MaryReynolds for her secretarial assistance.

27405, NIH-UO1-AI 32921, the Pediatric AIDS Foundation grants 500-185-11PGand500-270-13-PGR,andtheCHOP-Weizmann Insti-tuteCollaboration. Ravi Cherukuri was the recipient of the Pediatric AIDS Foundation Student Summer Fellowship in 1992.

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