Proc. Natl. Acad. Sci. USAVol. 83, pp. 2271-2275, April 1986Neurobiology

JC papovavirus large tumor (T)-antigen expression in brain tissueof acquired immune deficiency syndrome (AIDS) and non-AIDSpatients with progressive multifocal leukoencephalopathy

(DNA-binding protein/oligodendroglia/demyelination/myelin basic protein)

GERALD L. STONER*t, CAROLINE F. RYSCHKEWITSCH*, DUARD L. WALKERt, AND HENRY DEF. WEBSTER**Laboratory of Experimental Neuropathology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health,Bethesda, MD 20892; and tDepartment of Medical Microbiology, University of Wisconsin Medical School, Madison, WI 53706

Communicated by Richard L. Sidman, December 6, 1985

ABSTRACT Progressive multifocal leukoencephalopathy(PML) is a JC papovavirus infection of the central nervoussystem in immunocompromised patients. It is well establishedthat demyelination in PML is caused by JC virus infection ofoligodendroglia, but whether the nonstructural regulatoryprotein, large tumor (T) antigen, is detectable in infectedhuman tissue was not known. Using a modification of theperoxidase-antiperoxidase technique, we found T antigen ex-pressed in the nuclei of cells in virus-infected sites in five casesof PML studied, including two with acquired immune defi-ciency syndrome (AIDS). PML occurs in AIDS at a muchhigher frequency than in other immunosuppressive disorders,and PML in AIDS may represent a more severe form of JCvirus infection of the central nervous system.

Serological studies show that JC and BK papovavirusesinfect the majority of the human population by the time ofadolescence (1, 2). These are inapparent infections, and thetissues involved in the primary sites of replication areunknown. However, the shedding of JC and BK virus in theurine with rising serum antibody titers occurs both in kidneytransplant recipients (2-4) and in women late in pregnancy orin the puerperium (4, 5). This implies viral reactivation andreplication in the kidney during both drug-induced andnatural immunosuppression. The persistence of JC and BKviral DNA in the kidneys of healthy individuals supports thereactivation hypothesis (6). In severely immunocompro-mised patients, JC virus causes a very rare, and usually fatal,demyelinating disease of the central nervous system (CNS),progressive multifocal leukoencephalopathy (PML) (7). Themost profound defects in cell-mediated immunity now occurin patients with the acquired immune deficiency syndrome(AIDS) (8), and recently the incidence ofPML has been risingdramatically due to the spread of AIDS (9). In three recentseries of AIDS patients with neurological complications,2-4% developed PML (9-11). It is not clear whether PMLresults from reactivation of a latent infection of the brain ordevelops during a late primary infection.Depending upon the host cell type, papovaviruses may

establish a productive or an abortive infection or maytransform the cell. In each case the DNA-binding regulatoryprotein, large tumor (T) antigen, specified by the early regionof the viral genome, is expressed in the cell nucleus (12).However, the level of T-antigen expression in humanpapovavirus infections was unknown. A previous study fromthis laboratory, which presented immunocytochemical evi-dence for expression of JC virus structural proteins informalin-fixed, paraffin-embedded PML tissue, establishedthe distribution of structural viral antigens within the

demyelinated lesions. However, T antigen could not be detect-ed in those formalin-fixed tissues (13). It remained uncertainwhether T antigen was expressed in human tissue at levelsbelow the threshold of detection or whether there was atechnical problem in its detection, possibly related to fixationconditions.We now have developed methods for detection ofT antigen

in cell lines transformed by JC virus, using the highlysensitive peroxidase-antiperoxidase (PAP) immunocyto-chemical technique on acetone-fixed cell monolayers. Thesame technique readily detects nuclear T antigen in frozensections ofPML brain tissue from both AIDS and non-AIDSpatients. In fact, many more cells express T antigen in thenucleus than express structural viral antigens. The frankexpression of T antigen in human JC virus infections in bothabortively and productively infected cells raises the possi-bility that immunity to T antigen plays a role in controllingpapovavirus infections in man.

MATERIALS AND METHODSPML Tissue. Frozen tissues were obtained from autopsy

specimens from four cases ofJC virus-associated PML referredto the University ofWisconsin Medical School for pathologicaland virological evaluation. Tissue from a fifth PML patient, aswell as control tissue from three auto-accident victims, wasobtained from the Multiple Sclerosis Human NeurospecimenBank, Los Angeles. Patient data are listed in Table 1. Frozentissue blocks were embedded in HistoPrep (Fisher) and 10-1mserial sections were cut by cryostat and placed on chromealum-treated glass slides. Adjacent sections were im-munostained with antibody to T antigen, antibody to virionantigens, or control antiserum. Serial sections were also stainedfor myelin with luxol fast blue and counterstained withhematoxylin/eosin.

Cell Lines. JC virus-transformed hamster glial cells. JC-transformed hamster glial cells (HJC) were derived from ahamster brain tumor induced by JC virus. They were main-tained in Ham's nutrient mixture F-12 supplemented with 8%fetal bovine serum and 25 mM Hepes buffer. Control hamstercells not expressing papovavirus T antigen were BHK-21(baby hamster kidney) cells obtained from the AmericanType Culture Collection (ATCC CCL 10). The BHK cellswere maintained in minimal essential medium alpha supple-mented with 10% tryptose phosphate broth and 10% calfserum.

Abbreviations: AIDS, acquired immune deficiency syndrome; CNS,central nervous system; DAB, 3,3'-diaminobenzidine; HSV-1,herpes simplex virus type 1; PAP, peroxidase-antiperoxidase; PML,progressive multifocal leukoencephalopathy; SV40, simian virus 40;T antigen, large tumor antigen.tTo whom correspondence should be addressed at: Building 9, Room1E-127, National Institutes of Health, Bethesda, MD 20892.

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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.

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Table 1. Patient data

No. ofPatient tissueno. (age, blocks Associated diseaset Method of

sex) (storage*) (duration) diagnosist

1(31, F) 4 (6 mo) AIDS (1 yr) FA, EM, SA, Vir2 (35, M) 1 (2 yr) AIDS (6 mo) FA, Vir3 (58, M) 1 (4 yr) CVI (5 yr) FA, EM, Vir4 (41, M) 1(10 yr) Hodgkin disease (3 yr) FA, EM5 (56, M) 3 (15 yr) CLL (4 yr) LM

*Length of time stored at -70TC after autopsy.tCVI, common variable immune deficiency; CLL, chroniclymphocytic leukemia.tFA, fluorescent antibody; EM, electron microscopy; SA, serumantibody; Vir, virus isolation in cell cultures; LM, light microscopicpathology.

Simian virus 40 (SV40)-transformed human lung cells.SV40-transformed human lung cells (WI-38 VA13 subline2RA) were obtained from the American Type Culture Col-lection (ATCC CCL 75.1) and maintained in minimal essen-tial medium alpha supplemented with 10% fetal bovineserum. The parental line, WI-38 (ATCC CCL 75), which arenot virally transformed, served as the control.

Antisera. Antisera to JC virus. The hyperimmune antise-rum to JC virus, used for immunoperoxidase staining ofvirion antigens, was produced in rabbits as described (14). Inbrief, virus emulsified in Freund's complete adjuvant wasinjected into the hind footpads; 3 weeks later, animals weregiven an intraperitoneal booster injection of virus in 0.9%NaCl. Sera collected 1 or 2 weeks after the booster injectionhad hemagglutination-inhibition titers of 1:1,000,000 andreacted strongly with JC as well as BK and SV40 in immuno-fluorescence tests. For diagnostic purposes, monospecific anti-sera to JC, BK, and SV40 viruses were produced in rabbits bygiving one intravenous injection and collecting serum 10 dayslater (14).Antiserum to Tantigen. Antiserum to T antigen was obtained

from hamsters bearing JC virus-induced tumors. Tumors wereinduced by subcutaneous injection of JC-transformed hamsterglial cells. Sera with high titers against JC T antigen asdetermined by immunofluorescence were pooled. The pooledsera lacked detectable antibody to virus as measured by thehemagglutination-inhibition assay (D.L.W., unpublished data)and did not react with structural antigens of the virus informalin-fixed tissue sections (13). The hamster antiserum is notspecific for the T antigen of JC virus but reacts with the Tantigens of BK and SV40 viruses as well (15).

Control sera. Normal rabbit serum or normal hamsterserum served as controls. Hamster antiserum to herpessimplex type 1 (HSV-1) provided an additional control onspecificity of the immune serum.Immunocytochemical Methods. Fixation. Frozen sections

of CNS tissue and cell lines grown in four-chamber tissueculture slides (Lab-Tek, Naperville, IL) were air-dried 1-2 hrand fixed in acetone at 4°C for 10 min. Other fixatives testedon monolayers of JC virus-transformed hamster glial cellswere formalin (1-10%o), glutaraldehyde (0.5-4%), periodate/ly-sine/paraformaldehyde (16), and methanol/acetone (1:1) at-20°C for 3 min (17).

Indirect peroxidase-labeled antibody for T-antigen local-ization. Acetone-fixed cell monolayers or frozen sectionswere washed three times in 0.05 M Tris C1 (pH 7.6).Antibodies were diluted in the same buffer containing 3%normal goat serum and 2% bovine serum albumin (Tris/NGS). Before application of antisera, the cells were incubat-ed with Tris/NGS alone for 30 min to block nonspecificadsorption of antibodies. After the primary antibody appli-cation, the three washes and a blocking step (10 min) were

repeated. The following antibodies were applied at roomtemperature: (i) hamster anti-T-antigen (1:100) for 60 min (ornormal hamster serum as control); (ii) peroxidase-conjugatedF(ab')2 fragment of goat anti-hamster IgG (heavy- and light-chain specific; Cappel no. 3308-0081; 1:500) for 30 min. Afterthree washes in Tris buffer, the peroxidase-labeled antibodywas developed with 3,3'-diaminobenzidine (DAB) as de-scribed (18), except that the DAB reaction was for 5 min andthe osmium tetroxide treatment for 2 min. The cells weredehydrated and mounted in Permount.PAP methodfor T-antigen localization. Acetone-fixed cell

monolayers or CNS tissue sections were washed three timesin 0.05 M Tris Cl, pH 7.6/0.9% NaCl (Tris/NaCl). Allantisera were diluted in the same buffer containing 3% normalrabbit serum and 2% bovine serum albumin (Tris/NaCl/NRS). Sections were washed three times for 5 min inTris/NaCl and incubated 30 min with Tris/NaCl/NRS forblocking before application of the primary antibody. Thethree washes and a 10-min blocking step were repeated aftereach successive antibody addition. The antibody additions atroom temperature were as follows: (i) hamster anti-T-antigen(1:100) for 60 min (or normal hamster serum or HSV-1immune serum as controls); (ii) IgG fraction of goat anti-hamster IgG (Cappel no. 0208-0081; 1:100) for 30 min; (iii)rabbit anti-goat IgG (Stemberger-Meyer no. 502; 1:40) for 30min; (iv) goat PAP (Sternberger-Meyer no. 402; 1:100) for 30min. Finally, the cells were treated with 4% glutaraldehyde inTris/NaCl containing 0.5 M sucrose for 20 min and washedthree times. The DAB reaction was performed as above for10 min; the osmium tetroxide treatment was either 2% for 2min (monolayer cells) or 0.5% for 2 min (tissue sections).PAP method for virion antigen localization. Tissue sec-

tions were treated as above except that the sequence ofantibodies was as follows: (i) rabbit hyperimmune anti-JCvirus (1:500) for 60 min (or normal rabbit serum as control);(ii) sheep anti-rabbit IgG (Cappel no. 0112-0084; 1:40) for 30min; (iii) rabbit PAP (Sternberger-Meyer no. 401; 1:100) for30 min. The cells were postfixed, stained, and treated withosmium tetroxide as above.

RESULTST Antigen in Virus-Transformed Cell Lines. T antigen was

detected in the nuclei of both JC virus-transformed hamsterglial cells and SV40-transformed WI-38 cells (Fig. 1). Tantigen could be detected both with the indirect peroxidase-labeled antibody technique (Fig. 1B) and with the PAPprocedure (Fig. LA). However, the PAP procedure produceddenser deposits of reaction product in the nucleus, with littlebackground staining in the BHK-21 cells which are not virallytransformed (Fig. 10. Controls using normal hamster serum(or hyperimmune rabbit anti-JC virus antibody) were alsonegative.

Sensitivity ofT Antigen to Fixation Conditions. A variety offixation conditions were tested on monolayers of hamsterglial cells for adequate morphological preservation withoptimum preservation of T-antigen immunoreactivity. Ace-tone at 4°C for 10 min was found to adequately preserve bothcellular morphology and immunoreactivity and was usedroutinely. Methanol/acetone (1:1) at -20°C for 3 min wasfound to be equally good. Periodate/lysine/paraformal-dehyde was inferior to acetone in its ability to preserve thereactivity of JC virus T antigen. Neither formaldehyde norglutaraldehyde preserved antigenic reactivity of T antigenwhen used at concentrations required for adequate fixation.

Expression of JC Virus T Antigen in PML Tissue. Acetone-fixed frozen sections from the tissue blocks of all five casestested showed staining both for virion antigen and for Tantigen. Serial sections stained with the PAP techniqueshowed that T-antigen reaction product was prominent in all

2272 Neurobiology: Stoner et al.

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Proc. Natl. Acad. Sci. USA 83 (1986) 2273

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FIG. 1. JC virus- and SV40-transformed cell lines stained withhamster antiserum to T antigen. (A) JC virus-transformed hamsterglial cells, PAP method. (B) SV40-transformed WI-38 cells (ATCCCCL 75.1), indirect peroxidase-labeled antibody method. (C) Babyhamster kidney cells (BHK-21) stained as in A. (D) WI-38 cells(parental non-SV40-transformed line), stained as in B. (Nomarskioptics; x200.)

foci showing virion antigen. Controls with normal hamsterserum or antiserum to HSV-1 were negative. In contrast tothe results with the PAP procedure, the indirect peroxidase-labeled antibody technique gave weak and variable stainingof T antigen. Therefore, the PAP technique was used rou-tinely for localization of both T antigen and virion antigens.A striking observation on all serial sections was the much

larger number of T-antigen-containing cells compared to thenumber of cells staining for virion antigens (Table 2). Thedifference in counts of stained cells on serial sections washighly significant and was true for both AIDS and non-AIDStissue. This increase was primarily due to a higher density ofcells stained by antibody to T antigen, rather than toenlargement of the lesions defined by virus-infected cells.Two additional populations of cells appeared to stain for Tantigen (see below). In non-AIDS brain tissue a few lesionswere observed in which viral antigens were distributed in a

zone around the edges of the lesion, whereas cells staining forT antigen were scattered throughout the demyelinated area(Fig. 2 A and B). Cell counts were not taken from lesions ofthis type.The reaction product defining T-antigen location was found

Table 2. Cell counts in adjacent sections

Patient no. No. of positive cells*(block no.) T antigen Virion antigen P valuet

1 (3) 160 47 72 ± 30 <0.0051 (4) 184 69 53 ± 14 <0.0052 (1) 172 ± 56 116 ± 41 <0.0053 (1) 215 84 76 ± 38 <0.0054(1) 132 29 91 ± 19 <0.015 (2) 110 45 59 ± 28 <0.01

*Mean ± SD for 10 fields on adjacent sections examined at amagnification of 200x.tWilcoxon matched-pairs signed-ranks test.

exclusively in the nucleus, and, in general, was uniformlydistributed (Fig. 2A, Inset). In contrast, the reaction productdefining the location of virion antigens, although predomi-nantly localized in the nucleus, was characteristically con-centrated at the nuclear margin. Rarely, the staining includeda cytoplasmic component as well (Fig. 2B, Inset).

Positive identification of the types of cells infected by JCvirus is difficult in frozen sections, which show relativelypoor morphological preservation. Nevertheless, the follow-ing observations could be made. The cells containing virionantigens appeared to be predominantly the large, abnormaloligodendroglia which showed characteristic nuclear inclu-sions in sections stained with luxol fast blue plus hematoxy-lin/eosin. In all cases there were few, if any, normal-appearing,small oligodendroglia that were stained. Reaction product couldalso be observed on the plasma membrane of macrophagesinvading some of the demyelinated lesions, but macrophagenuclei were unstained. Except for this staining of macrophagemembranes, the T-antigen reaction product appeared in thesame cells as did staining for virion antigens. In addition, manyof the small nuclei of oligodendroglia of normal appearancewere heavily stained. Since very few normal-appearingoligodendroglia stained for virion antigens, the prominent stain-ingofthese cells by antiserum toT antigen accounts in large partfor the increase in the number of cells that were stained for Tantigen compared to those that were stained for virion antigens.In addition, afew large cells with irregularly shaped nuclei werestained. These cells expressing only T antigen may representlatently infected astrocytes. Staining ofendothelial cells was notobserved with antiserum either to virion antigens or to Tantigen. However, anti-T-antigen reaction product was ob-served occasionally in nuclei of curved, elongated cells notassociated with vessels, possibly activated microglia. This typeof staining was especially prominent in case no. 1 (AIDS).Although the AIDS and non-AIDS tissues were qualita-

tively similar in the distribution of infected cell types, thesamples from the two AIDS patients appeared to be muchmore heavily infected with JC virus than were those of thethree non-AIDS patients (Fig. 2 D and E). In sections fromAIDS patients, more glial cells were stained by both anti-JCand anti-T-antigen sera, and the infected cells were mostlydistributed diffusely, rather than focally. Especially in case 2(Table 1), JC virus-infected oligodendroglia were not asso-ciated with circumscribed areas of demyelination but werewidely scattered through white matter shown to be intact byluxol fast blue staining of adjacent sections.

Control tissues from three auto accident victims werestudied. None showed staining for either JC virus T antigenor virion antigens.

DISCUSSIONThe nuclei of glial cells in and around PML lesions werefound to be readily stained by hamster anti-T-antigen serumas detected by the PAP method. This antiserum also stainsthe nuclei of a cell line derived from a JC virus-inducedhamster tumor and of an SV40-transformed human cell line(ATCC CCL 75.1), but it does not stain BHK cells or theparent line of 75.1, which is not SV40-transformed. Thestaining of 75.1 cells is expected because SV40 and JC virusT antigens have closely related amino acid sequences (19).The acetone-fixation procedure used does not allow detec-tion of that fraction of T antigen thought to be present in theplasma membrane of SV40-transformed cells (20).The detection ofT antigen is unusually sensitive to fixation

conditions. When PML tissue is fixed in formalin andembedded in paraffin, the immunoreactivity of virion anti-gens is recovered in deparaffinized sections, but that of Tantigen is not (13). Similarly, the reactivity ofT antigen in cellmonolayers was found to be destroyed by treatment with

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FIG. 2. T-antigen expression in JC virus-infected CNS tissue from patient 5 (chronic lymphocytic leukemia; A-C) and from patient 2 (AIDS;D-F). All staining was done by the modified PAP technique (see Materials and Methods), using hamster antiserum to T antigen (A and D),hyperimmune rabbit antiserum to JC virion antigens (B and E), normal rabbit serum (C), or normal hamster serum (F). (x80.) (Insets) T antigenis localized in the nucleus (A), whereas virion antigens are stained most intensely at the nuclear margin, and, rarely, in the cytoplasm as well

(B). (Nomarski optics; x430.)

formalin or glutaraldehyde at the concentrations required foradequate fixation of the tissue. However, T antigen is easilydetected in PML tissue when frozen sections are lightly fixedin acetone at 40C or in methanol/acetone (1:1) at -20TC. Itwas also found that additional fixation with glutaraldehydeprior to the DAB reaction improved the recovery ofT-antigenimmunoreactivity, presumably by reducing the loss of PAPcomplexes from the lightly fixed tissue during the developingreaction and subsequent washing. Under these conditions, Tantigen is readily detected in the nuclei of glial cells in thesame areas where virion antigens are found. The conditionsfor T-antigen detection in the nucleus of JC virus-infectedCNS tissue are, in general, consistent with previous findingson detection of SV40 virus T antigen (20, 21) and BK virus Tantigen (22) in cultured cell lines.

In all PML sections, the number of cells expressing Tantigen in the nucleus clearly exceeded the number of cellsexpressing virion antigens. Several factors might contributeto this observation: (i) the temporal expression of virus-

specified proteins, as cells in the initial stages of infectionwould be expected to express only T antigen; (ii) the abortiveor latent infection of additional cells that express the earlyprotein but do not go on to express the structural antigens;(iii) more sensitive detection of T antigen, so that cellsexpressing low levels of virus are picked up only by theT-antigen reaction.

In regard to factor iii, it should be noted that although thePAP technique for T-antigen detection, with its extra ampli-fying step, is extremely sensitive, it seems unlikely that onlythe regulatory protein would be detected in productivelyinfected cells expressing much larger amounts of virionantigens. The contributions from factors i and ii are difficultto differentiate. The probable abortive infection of astrocyteswas previously recognized (23) and has been confirmed here.In addition, this study has now detected a population of smalloligodendroglia of normal appearance that express T antigenbut do not yet express virion antigens. Previously, only theproductive, lytic infection of oligodendroglia was evident (13,

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Proc. Natl. Acad. Sci. USA 83 (1986) 2275

24). Our findings suggest that transformation of smalloligodendroglia into the abnormally large, virus-producingcells may be the slow step. However, what proportion ofthese small cells are in the early stages of a productiveinfection, what proportion are arrested in an abortive infec-tion, and what proportion may be latently infected areunknown.

In the two cases ofPML in AIDS patients studied here, thedistribution of both virion antigens and T antigen throughoutthe sections was much greater than in the three cases ofnon-AIDS PML. The presence of widespread oligodendro-glial infection not confined to demyelinated lesions suggeststhat PML in AIDS may represent a more acute infectiousprocess than that usually seen in classical PML; i.e., PMLsuperimposed on lymphoproliferative disease. Wider sam-pling of PML cases in AIDS patients will be required toconfirm this point. However, AIDS represents the mostprofound defect of cell-mediated immunity known in man (8),and it seems likely that PML in AIDS is a "low-resistance"type of JC virus CNS infection in a spectrum of disease inwhich the previously described "high-resistance" type isPML with relatively long survival times, which may not havean underlying immunosuppressive disorder (25). Pathologi-cally, these high-resistance-PML patients show a character-istic mononuclear/plasma-cell infiltration of the lesions (26).The intermediate type of PML would be the typical PMLoriginally described (27) and usually seen in lymphoprolifera-tive disease.

It has recently been shown that many patients withAIDS-associated encephalopathy have AIDS-associated ret-rovirus present in the brain (28). Therefore, in addition to theindirect effects of AIDS on JC virus replication due to severeimmunosuppression, the possibility that the presence of theretrovirus has a more direct effect on reactivation of latent JCvirus must be considered.Attempts to detect human antibody to T antigen in PML

sera using JC-transformed cells as targets and indirect im-munofluorescence (15) or PAP techniques (unpublished data)have failed. It is clear from the present study that the failureto detect antibody to T antigen in PML sera cannot beattributed to a failure to adequately express T antigen in theinfected human tissue. Possible explanations include specificunresponsiveness or inadequate methodology. Given thehigh levels ofJC virus T antigen expressed during productiveand abortive infection of oligodendroglia, it will be of interestto delineate the full range of immune responses to T antigennot only in PML, but also in normal, asymptomatic primaryJC and BK virus infections in childhood and adolescence. Inaddition, there are the silently reactivating infections thatoccur during pregnancy or in the puerperium (5) and, rarely,in healthy individuals as well (3). The shedding of JC or BKvirus in the urine is accompanied by a rise in antibody titer,but the mechanisms of immunity involved in controlling thereactivated infection, possibly directed in part to T antigen,are unknown.A C-terminal subsequence ofJC, BK, and SV40 T antigens

is shared with myelin basic protein (29). In SV40 T antigenand in myelin basic protein, this sequence appears to functionas a phosphate acceptor site (30, 31). The significance ofthese observations is unknown, but they raise the possibilityof involvement of latent JC or BK virus infections in chronicdemyelinating diseases of unknown etiology (e.g., multiplesclerosis). Therefore, it will be of interest to determinewhether T antigen is expressed in multiple sclerosis braintissue. Alternatively, in the absence of a latent CNS infec-tion, immunopathological damage to myelin based on thesequence similarity between T antigen and myelin basicprotein should be considered.

We thank Dr. Dov Soffer for reviewing the slides and makingimportant observations. The Multiple Sclerosis Human Neurospeci-men Bank, Veterans Administration Wadsworth Medical Center,Los Angeles, is sponsored by the National Multiple SclerosisSociety, the HD Foundation, National Institute of Neurological andCommunicative Disorders and Stroke/National Institute of MentalHealth, and the Veterans Administration. A part of this work wassupported by National Institute of Allergy and Infectious DiseasesGrant A111217.

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