Proc. Natl. Acad. Sci. USAVol. 90, pp. 8658-8662, September 1993Neurobiology

A model of human immunodeficiency virus encephalitis in 8cid miceWILLIAM R. TYOR*t, CHRISTOPHER POWER*, HOWARD E. GENDELMANO§, AND RICHARD B. MARKHAM¶

*Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; *Department of Cellular Immunology, Walter Reed ArmyInstitute of Research, Rockville, MD 20850; and IDepartment of Immunology and Infectious Diseases, Johns Hopkins University School of Hygiene andPublic Health, Baltimore, MD 21205

Communicated by John W. Littlefield, June 21, 1993

ABSTRACT Human immunodeficiency virus (HIV)-assciated dementia complex is a common and devastatingmanifstation of the late phases of HIV Infection. The patho-geness of dementia complex is poorly undertood and effectivetreatments have not been developed, In part because ofthe lackof an appropiate anmal model. Mice with severe combinedImmunodeefiiency (scidmice), which accept xenografts withoutrejection, were intracerebrally Inolated with human periph-eral blood momonucear cells and HIV. One to 4 weeks afterinoculation, the brains of these mice contin human macro-phages (some of which were HIiV p24 antigen positive), occa-sional multinucleated ceDs, and sriking giosis by immunocy-tochemical s g. Human macrophages also were fequentiyposidve for tumor necrosis factor tpe a and occasionally forinterleukin 1 and VLA-4. Cultures ofthese brains for HIIV werepositive. Generally, human macrophages were not present inthe brains of control mice, nor was sincant gosis, and HIVwas not recovered from mice that received HIIV only intrace-rebrally. Pathogkally, this model of HIV encephalitis in scidmice resembles HIV encephalitis in hns and the datasuggest that the activation of macrophages by infection withHIV results in their accumulation and persistence in brain andin the development of glids. This model of BINV encephalitisshould provide insights into the pathogenesis and treatment ofthis disorder.

Twenty to seventy percent ofhuman immunodeficiency virus(HIV)-infected individuals will develop HIV-associated de-mentia complex over the course of their illness (1). Once thediagnosis of HIV-associated dementia complex is made, theaverage life-span is =8 months (1). While medications suchas zidovudine and didanosine may improve the symptoms ofHIV-associated dementia complex in some patients, specifictherapies for this disorder do not exist (2).

Clinically and pathologically, HIV-associated dementiacomplex has been well defined, but the pathogenesis of thisdisease is poorly understood (3, 4). The pathologic hallmarksofHIV encephalitis include multinucleated giant cells, HIV-infected macrophages/microglia, and gliosis (4). The associ-ation of two other neuropathological findings with HIV-associated dementia complex is less clear. Diffuse myelinpallor is a frequent finding at autopsy in individuals withAIDS but is present in only 33% of individuals who sufferedwith HIV-associated dementia complex (4-6). Decreasedcortical neuronal cell counts have also been reported in HIVencephalitis (7, 8). However, the precise stereological tech-niques needed to obtain unbiased statistically valid enumer-ation of neurons (9) were not used in these studies; further-more, neither ofthese studies attempted to correlate neuronalcell counts with clinical dementia. Therefore, the role ofneuronal cell loss in the development of HIV-associateddementia complex is unresolved.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The exact relationship between the clinical manifestationsof HIV-associated dementia complex and all of the patho-logical findings of HIV encephalitis has not been elucidated.Most hypotheses for the pathogenesis of HIV-associateddementia complex focus on the production by central ner-vous system (CNS) macrophages/microglia or astrocytes ofsubstances that are toxic for CNS constituents such asneurons, oligodendrocytes, or myelin (10, 11). Candidates forsuch a toxin include metabolites such as quinolinic acid,cytokines such as tumor necrosis factor type a (TNF-a), andHIV proteins such as gp120 or tat (10-19).To investigate these hypotheses and to develop a rational

approach to therapy, an animal model must be used. Currentmodels of retrovirus-induced encephalitis include simian im-munodeficiency virus in monkeys (20), feline immunodefi-ciency virus in cats (20), murine leukemia virus in mice (21),visna-maedi caprine arthritis encephalitis virus in sheep (22),and others (23, 24). However, these models suffer from anumber of drawbacks, including expense, long period ofinfection required to obtain results, awkwardness of handlingand housing the species, difference in pathology from HIVencephalitis in humans, and the use ofviruses other than HIV.

Severe combined immunodeficiency (scid) mice lack func-tional B and T cells due to a defect in T-cell receptor andimmunoglobulin variable chain rearrangement (25). scidmice can accept xenografts without rejection (26) and cells ofthe human immune system, as well as HIV-infected humancells, inoculated into these mice can survive for months (27,28). We injected human peripheral blood mononuclear cells(PBMCs) and HIV intracerebrally (i.c.) into scid mice andexamined their brains pathologically at various time points.We questioned what human cell types would remain in brain,whether they were infected with HIV, and what other CNSpathological effects could be demonstrated in these animals.

MATERIL S AND METHODSAnimal and Tisue Manipulations. Four- to 6-week-old

scid/scid mice, bred and housed under sterile conditions atThe Johns Hopkins University School of Hygiene and PublicHealth, were used. Mice were anesthetized with methoxy-flurane and inoculated in the right cerebral hemisphere. Aftervariable time periods (see below), mice were sacrificed bycervical dislocation. Brains were immediately removed andthe brainstem and cerebellum were placed in phosphate-buffered saline (PBS; pH 7.4) for HIV culture. The cerebralhemispheres were halved coronally, the rostral halfwas snap

Abbreviations: HIV, human immunodeficiency virus; PBMC, pe-ripheral blood mononuclear cell; i.c., intracerebrally; TNF-a, tumornecrosis factor type a; HTLV, human Tlymphotropic virus; TCIDso,tissue culture 50%1o infective dose; mAb, monoclonal antibody; IL-1,interleukin 1; GFAP, glial fibrillary acidic protein.tTo whom reprint requests should be sent at present address:Department of Neurology, Medical University of South Carolina,171 Ashley Avenue, Charleston, SC 29425.Present address: University ofNebraska Medical Center, 600 South42nd Street, Omaha, NB 68199.

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frozen on dry ice to be stored at -70°C, and the caudal halfwas placed in 4% paraformaldehyde.Mononuclear Cells and EIIV Strains. Human PBMCs from

whole blood were obtained from HIV-negative Red Crossdonors, separated on a Ficoll gradient, and resuspended inPBS prior to inoculation. Monocytes were recovered fromPBMCs of HIV and hepatitis B-seronegative donors afterleukapheresis and purified by countercurrent centrifugal elu-triation. Cell suspension was 98% monocytes by criteria ofcell morphology on Wright-stained cytosmears, by granularperoxidase, and by nonspecific esterase. Monocytes werecultured on adherent monolayers (1 x 106 cells per ml in24-mm plastic culture wells) in Dulbecco's modified Eagle'smedium (Sigma) with 10% heat-inactivated AB+ humanserum, gentamicin (50 ug/ml), and highly purified (<0.01 ngof endotoxin per ml) recombinant human macrophage colo-ny-stimulating factor (1000 units/ml) (FAP-809; Cetus) (29).HIV strains included human T-lymphotropic virus type mB

(HTLV-IIIB) (Advanced Biotechnologies), HIV-lBaL (Ad-vanced Biotechnologies), HTLV-IIImN (Advanced Biotech-nologies), and HIV-lAdlaM (provided by H.E.G., Walter ReedArmy Institute of Research), a monocytotropic strain origi-nally isolated from a patient with AIDS.

Inoculations. Experiment 1. scid mice were inoculated i.c.with 30 1,u ofPBS containing 106 PBMCs followed a day laterby 20 p1 i.c. of 1 x 104 TCID5o (tissue culture 50%6 infectivedose) ofHTLV-IIIB in RPMI 1640 medium with 10%6 fetal calfserum (FCS). These mice were sacrificed at 3 weeks (n = 2),4 weeks (n = 2), 10 weeks (n = 1), and 12 weeks (n = 2).Controls consisted of mice receiving 106 PBMCs only i.c. (n= 4) sacrificed at 3, 4, 10, and 12 weeks.Experiment 2. scid mice were inoculated i.c. with 30 pl of

PBS containing 106 PBMCs followed a day later by 20 ;4 i.c.of3 x 103 TCID5o OfHTLV-IIIB in RPMI 1640 medium. Thesemice were sacrificed at 3 weeks (n = 5). Controls consisted ofmice receiving 106 PBMCs i.c. followed a day later by virusvehicle (RPMI 1640 medium with 10%6 FCS) (n = 3), 106PBMCs i.c. only (n = 2), and 3 x 103 TCID50 of HTLV-IIIBi.c. only (n = 3). All controls were sacrificed at 3 weeks.Experiment 3. scid mice were inoculated i.c. with 30 ;4 of

PBS containing 106 PBMCs followed a day later by 20 p1 i.c.Of 5 x 104 TCIDso of HTLV-IIIB in RPMI 1640 medium.These animals were sacrificed at 2 (n = 2) and 3 (n = 2)weeks. Controls included 106 PBMCs inoculated i.c. fol-lowed a day later with virus vehicle sacrificed at 2 (n = 2) and3 (n = 2) weeks, 5 x 104TCIDsoofHTLV-IIIB only sacrificedat 2 (n = 2) and 3 (n = 2) weeks.Experiment 4. scid mice were inoculated i.c. with 30 p1 of

PBS containing 106 PBMCs and 5 x 104 TCID5o of HTLV-IIIB and sacrificed at 1 week (n = 1), 3 weeks (n = 2), and 12weeks (n = 1).Experiment 5. scid mice were inoculated i.c. with 30 p1

of PBS containing 106 PBMCs and 5 x 104 TCIDso ofHIV-1BaL and sacrificed at 1 week (n = 1), 2 weeks (n = 1),and 3 weeks (n = 1). scid mice were also inoculated i.c. with106 purified monocytes and sacrificed at 2 weeks (n = 1) or106 purified monocytes preinfected with HIV-lAdaM andsacrificed at 1 week (n = 1) or 2 weeks (n = 1).Experiment 6. scid mice were inoculated i.c. with 30 p1 of

PBS containing 106 PBMCs and 3 x 103 TCID5o of eitherHTLV-IIIB (n = 4), HIV lBaL (n = 2), or HTLV-IIIm (n =2) and sacrificed at 2 (n = 1 from each strain) or 3 weeks.Controls included mice that received 106 PBMCs only andwere sacrificed at 2 (n = 1) and 3 (n = 2) weeks or thatreceived HIV only (n = 2 for HIV-lBa.L and HTLV-IlHmN, n= 4forHTLV-IIIB) that were also sacrificed at 2 and 3 weeks.Normal scid mice (n = 2) were sacrificed at 6 weeks of age.

Hitochemistry. Frozen serial coronal sections (5 ,m) fromcerebrum were stained immunocytochemically as described(30). Briefly, after blocking for 20 min with 2% normal horse

serum (NHS) in PBS, slides were incubated with primaryantibody (see below) diluted in 2% NHS in PBS for 45 mi.After washing, biotinylated horse anti-mouse IgG, biotinyl-ated goat anti-rabbit IgG, or biotinylated rabbit anti-rat IgGdiluted 1:100in2% NHS inPBS was addedfor30minand thenthe sections were incubated for 30 min in methanol containing0.1% H202. Avidin DH-biotin complex (Vector Laboratories)was added for 30 min followed by diaminobenzidine (Poly-science) at 0.5 mg/mland0.01% H202 inPBS for 20 min. Colorwas darkened with 0.5% CuSO4/0.15 M NaCl for 5 min.Primary antibodies consisted of mouse monoclonal anti-

body (mAb) to HIV p24 core antigen (Olympus) at a dilutionof 1:80, mouse mAb to human macrophages (EBM/11;DAKO) at a dilution of 1:80, mouse mAb to human CD3 (Leu4; Becton Dickinson) at a dilution of 1:50, mouse mAb tohuman VLA4 (AMAC) at a dilution of 1:10, rabbit anti-human TNF-a (Genentech) at a dilution of 1:50, rabbitanti-human interleukin 1 (IL-1) (Endogen) at a dilution of1:50, mouse mAb to gial fibrillary acidic protein (GFAP;DAKO) at a dilution of 1:1000, mouse mAb to mouse CD4(L3T4; Becton Dickinson) at a dilution of 1:50, rat mAb tomouse B cells [RA32C2; American Type Culture Collection(ATCC)] at a dilution of 1:50, ratmAb to mouse macrophages(F4/80; ATCC) neat, and rat mAb to mouse class II antigens(M5/114.15.2; ATCC) at a dilution of 1:50. Control antibodiesconsisted of normal rabbit serum at a dilution of 1:50 and anirrelevant mouse mAb to trinitrophenol (25.11.15; ATCC) ata dilution of 1:10.

Sections (5 pm) were cut from paraformaldehyde-fixedcerebral hemispheres from each mouse and stained with hema-toxylin and eosin, Luxol fast blue, and silver stain. Paraform-aldehyde-fixed, free-floating cerebral hemisphere sections (40pm) were immunocytochemically stained for GFAP. All sec-tions were evaluated by light microscopy (Olympus).For analysis of astrocyte counts, five fields encompassing

the cortex and subcortical white matter of the parietal lobe,contralateral to the injection site, were studied at a magnifi-cation of x 1000 from scid mice that received PBMCs only (n= 5), HTLV-IIIB only (n = 4), and HTLV-IIIB plus PBMCs(n = 8) and were sacrificed at 3 weeks. Only cells that hadGFAP-positive perikarya were counted in each field. Theexaminer (C.P.) was unaware of the section identity. Totalastrocyte numbers for five fields per section were averagedfor each group and subjected to statistical analysis by anal-ysis of variance and Bonferroni corrections (see Results).HIV Culture. Normal human PBMCs were obtained as a

by-product of hemapheresis from the American Red Cross.The PBMCs were suspended in PBS and were depleted oferythrocytes and platelets by passage over a Ficoll/Hypaquegradient (Sigma). The cells were then placed into RPMI 1640tissue culture medium (Sigma) containing 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, Fungizone(1 pg/ml), and phytohemagglutinin (titrated for optimal pro-liferation) (GIBCO) at a concentration of 106 cells per ml.After 48 hr of cultivation, the medium was aspirated from theflask and the cells were recultured at a concentration of S x105 cells in identical medium except that phytohemagglutininwas replaced with recombinant IL-2 (2 units/ml; BoehringerMannheim). Homogenates of the brainstem and cerebellumfrom each mouse that received HIV were placed in 3 ml of thecultured cells in 25-cm2flasks. At weekly intervals, halfof thecells in the culture were replaced with fresh PBMCs from adifferent donor. At the end of 3 weeks, supernatants from thecultures were tested for the presence of p24 antigen by amodified ELISA (HIVAG-1; Abbott).

RESULTSOf the animals that received human PBMCs and HIV, 39.4%(Table 1) were found to contain cells positive for HIV p24antigen by immunocytochemical staining (Fig. 1A). Serial

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sections revealed that these p24-positive cells were humanmacrophages (Fig. 1B) and were also class II positive (Fig.1C). Most human macrophages (79%6) were not p24 positiveand the numbers ofhuman macrophages and p24-positive cellsvaried from mouse to mouse; three mice had >50 p24-positivecells per 5-pm section, most had 5-10 p24-positive cells persection, and three mice had 1 or 2 p24-positive cells persection. These macrophages were located primarily aroundthe needle tract but also were found in the left (opposite)hemisphere. Occasional p24-positive (or EBM/II positive)multinucleated cells were present (Fig. 2A). Human T cellswere occasionally detected but they did not correspond to cellsthat were p24 positive in serial sections. Most (56%) controlmice receiving PBMCs only or PBMCs plus virus diluent hadno human macrophages or T cells visible on sections. Incontrol mice in which human macrophages were identified,0.1 were present per 5-pm section.Immunocytochemical staining of serial 5-pm frozen sec-

tions from the cerebral hemispheres ofscid mice that receivedPBMCs plus HIV also revealed that the majority of humanmacrophages were TNF-a positive (Fig. 2B). The IL-1 stain-ing pattern ofindividual cells was similar to that shown in Fig.2B for TNF-a except that less than half of the clusteredmononuclear cells in a given field were positive and oftenblood vessel endothelium was lightly stained. Staining forVLA-4 differed from TNF-a and IL-1 in that it appeared to bemore clearly confined to the cell surface and only occasionalcells were positive. These VLA4positive cell types alsoappeared to be mononuclear cells that were primarily near theneedle tract, like the TNF-a- and IL-i-positive cells, andtherefore were probably human macrophages.

Paraformaldehyde-fixed cerebral hemisphere sections (40pAm) immunocytochemically stained for GFAP, which iden-tifies astrocytes, demonstrated striking gliosis in scid micethat received PBMCs plus HIV compared to control mice(Fig. 3). Features of the gliosis included an increase inGFAP-positive cells and astrocyte cell body hypertrophywith elongation of cellular processes (Fig. 3C). While somegliosis could be attributed to the trauma of the intracerebralinjection, the extent of gliosis was uniformly greater in thebrains ofmice injected with PBMCs and HIV versus controlsand occurred in areas distant from the needle tract includingthe cerebral hemisphere opposite the injection site. Astrocytenumbers were counted in controls and scid mice that wereinoculated i.c. with HTLV-IIIB and PBMCs (Fig. 4). Therewere significantly greater numbers of astrocytes in the micethat received HIV plus PBMCs versus controls in the hemi-sphere opposite the injected hemisphere. Finally, theretended to be more gliosis in animals that had greater numbersof p24-positive cells, but the differences between mice withlarge numbers of p24-positive cells versus small numberswere not significant.

Histopathological analysis of paraformaldehyde-fixed sec-tions (5 pm) using routine stains showed occasional perivas-cular mononuclear cells in mice receiving PBMCs plus HIV

Table 1. Inoculation of scid mice with human PBMCs andvarious HIV strains

No. of mice p24 positive and cultureHIV strain positive per no. of mice inoculated

HTLV-lB* 8/24HIV 1Ba.Lt 2/5HTLV-MN* 1/2HIV-lAda-M 2/2

See Materials and Methods for description of experiments 1-6.

but no gross neuronal or myelin abnormalities were detectedby silver stain and Luxol fast blue stains. Frozen sectionsfrom cerebral hemispheres were also stained for mouse Tcells, B cells, macrophages, and class antigen. There wereno mouse T and B cells present in any mice. Rare mousemacrophages were present primarily around the site of inoc-ulation and occasional mouse class II-positive macrophages/microglia were present in meningeal areas, subependymally,perivascularly, and, rarely, intraparenchymally.HIV p24 antigen was not detected immunocytochemically

in mice that received virus only; however, p24 antigen wasdetected in 39.4% of mice that received PBMCs plus HIV(Table 1). HIV cultures revealed that only mice that were p24positive by immunocytochemistry were culture positive (Ta-ble 1). There was no clear relationship between the type ofHIV strain used and its ability to infect human macrophagesin scid mouse brains. In fact, there were no apparent differ-ences overall in all the parameters measured (i.e., number ofp24-positive cells, amount ofgliosis, etc.) between mice fromexperiment to experiment (see Materials and Methods) de-spite differences in viral strains, amounts ofvirus inoculated,and inoculation paradigms. Therefore, the results were re-producible from experiment to experiment.

DISCUSSIONIn scid mice that were inoculated i.c. with PBMCs and HIV,immunocytochemistry ofbrain sections revealed p24-positive

FiG. 1. Serial brain sections from a scid mouse that receivedPBMCs and HTLV-IIB stained immunocytochemically for p24antigen and human macrophages. Frozen tissue serial sections (5 pm)from the cerebral hemisphere were stained for HIV p24 antigen (A)and human macrophages (B) and human class II antigen (C). Thesame cells that are p24 positive inA are stained with the antibody tohuman macrophages in B and are class II positive in C. This mousewas sacrificed 3 weeks after receiving PBMCs and HIV and isrepresentative of the mice that had >50 p24-positive cells per 5-pmsection (see Results). (x70.)

*From experiments 1, 2, 3, 4, and 6.tFrom experiments 5 and 6.*From experiment 6.From experiment 5, with preinfected, purified monocytes.

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human macrophages that were primarily distributed aroundthe needle tract but also present in the opposite hemisphere.These macrophages were class II positive and TNF-a positiveand some of them were IL-1 positive and VL4-4 positive,suggesting they were in a state of "immune activation."Furthermore, mice with p24-positive human macrophages hadsign icant gliosis, indicating that mouse cells have initiated thesame type of response to HIV infection of human macro-phages as is seen in HIV-infected human brains (4).HIV p24 antigen was not detected immunocytochemically

in mice that received virus only; however, p24 antigen wasdetected in 39%6 of mice that received PBMCs plus HIV.Macrophages were the predominant (if not exclusive) mono-nuclear cells infected even though they constituted a minorityof the cells injected (when PBMCs were used). This wasapparent regardless of the HIV strain used. HTLV-IIB andHTLV-IIHmN, which are predominantly T-cell tropic strains(31, 32), appeared to establish infection inhuman macrophagesas effectively as HIV-lBa.L, primarily a monocytotropic strainoriginally isolated from bronchial alveolar macrophages (33).This is ofparticular interest since cells ofmonocyte lineage arethe only cell types that have been conclusively demonstratedin human brain to be infected withHIV (4). These data indicatethat (i) there is no evidence ofinfection ofmouse cells by HIV,at least by culture and immunocytochemical techniques; (ii)not all mice receiving human PBMCs plus HIV becomeinfected, regardless ofHIV strain, although two oftwo animalsreceiving purified monocytes preinfected with HIVAd&.M werep24 positive; and (iii) positive HIV cultures from the brains ofp24 positively stained mice indicate persistence of functional

A

FiG. 3. GFAP immunostaining of 40-pm-thick free-floating sec-tions from a scid mouse that received PBMCs alone (A) and PBMCsplus HTLV-IIIB (B). (x230.) (C) Increased GFAPimmunoreactivityand astrocytic hypertrophy was more prominent in the HIV-infectedmice. (x455.) Photomicrographs are representative of each of thepopulations of mice.

FIG. 2. Multinucleated p24-positive cell. Frozen section (5pm) stained immunocytochemi-cally for p24 antigen and counter-stained with hematoxylin. Reac-tion product is brown. (x420.) (B)Frozen section (5 pm) immunocy-tochemically stained for humanTNF-a. (X420.)

virus and not just persistence of viral antigens. The mostefficient method for establishing HIV infection in scid brainsappears to be i.c. inoculation of preinfected, purified mono-cytes because only 39%6 of the mice were p24 positive thatwere inoculated with PBMCs and HIV either simultaneouslyor a day later. The efficiency of establishing an infected graftin the brain, therefore, is probably related to the state ofactivation of infected monocytes since the preinfected mono-cytes were maintained in culture under optimal growth con-ditions, whereas uninfected PBMCs (plus HIV) were i jectedimmediately after their separation from whole blood (seeMaterials and Methods).Human macrophages generally did not remain in the brains

of scid mice unless at least some ofthem were infected. HIVinfection stimulates the production of cytokines in vitro andcytokines in turn exhibit a myriad of functions, includingupregulation of adhesion molecules on cell surfaces andstimulation of the proliferation of cells such as lymphocytes,

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FiG. 4. Astrocyte counts in scid mice inoculated i.c. with HTLV-IIIB plus PBMCs (n = 8), HTLV-Ills only (n = 4), or PBMCs only(n = 5). See Materials and Methods for explanation of how theaverage number of astrocytes was obtained for each group. Analysisof variance revealed significant differences between groups (P =0.0008) and post hoc tests with Bonferroni corrections showedsignificant differences between A and C (P < 0.01) and between Band C (P < 0.05) but not between A and B.

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cells of monocyte lineage, and astrocytes (34-36). A possibleexplanation for the persistence of human macrophages pri-marily in HIV-infected scid brains is that they secrete cyto-kines such as IL-1 and TNF-a, which upregulate adhesionmolecules on their surfaces and thus they are more adherent(36). Our studies demonstrate the presence of TNF-a andIL-1 on human macrophages in the brains of mice inoculatedwith PBMCs and HIV. These cytokines are also capable ofstimulating HIV replication in vitro (34). Macrophages inHIV-infected scid brains were positively stained for VLA-4,a member of the integrin family of adhesion molecules thatbinds fibronectin in the extracellular matrix and, in addition,binds VCAM-1, an adhesion molecule expressed by activatedvascular endothelium (36). These findings suggest that theaccumulation of activated macrophages in the brains ofpatients with HIV encephalitis (16) may be attributable to thelocalization of infected macrophages at the site.TNF-a and IL-1 are capable of stimulating astrocyte pro-

liferation and some cytokines are known to act across speciesbarriers (34, 37). scid mice that were inoculated with PBMCsand HIV demonstrated striking gliosis compared to controlsand this increase in the number of astrocytes and astrocytehypertrophy was noted in the opposite hemisphere frominjection. The data suggest that TNF-a and IL-1 secreted byhuman macrophages, some of which are infected with HIV,are at least in part responsible for the gliosis present inHIV-infected scid brains.We did not find diffuse myelin pallor by Luxol fast blue

stain in the HIV-infected mice. As discussed above, thesignificance of diffuse myelin pallor in AIDS brain is unclear(4, 5) since this was only found in 33% of individuals whosuffered with HIV-associated dementia complex by Glass etal. (6). Furthermore, immunoperoxidase staining for myelinproteins in brain has revealed no differences between con-trols and patients who had HIV-associated dementia complex(38). Diffuse myelin pallor may be found in the HIV-infectedscid mice when they are examined at longer time points.However, the relationship of diffuse myelin pallor to thepathogenesis of HIV encephalitis is tenuous.

This model ofHIV encephalitis in scid mice pathologicallyresembles HIV encephalitis in humans (4). There are humanmacrophages present, some of which are infected with HIVand are occasionally multinucleated. There is also strikinggliosis. This model will permit the investigation of the po-tential pathogenic mechanisms involved in the developmentof HIV encephalitis and thus aid in the development of arational approach to therapy.

We thank Drs. Diane Griffim and Richard Johnson for support andreview of the manuscript; Michael Rho, Pei-Ann Kong, and GeneBarbour for technical support; and Kimberley Collins and SallieBendt for preparation of the manuscript. This work was supported byNational Institutes of Health Grants SNPO1-NS-26643, AI29163, andAI28748. C.P. is a recipient of a Medical Research Council (Canada)Fellowship. W.R.T. is a recipient of an American Foundation forAIDS Research Grant (001809-13-RGT).

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