Progressive multifocal leukoencephalopathy

Context.-Progressive multifocal leukoencephalopathy is a fatal demyelinating disease of the central nervous system frequently seen in patients with impaired immune systems, particularly acquired immunodeficiency syndrome. JC virus (JCV), a human neurotropic polyomavirus, is the etiologic infectious agent of this disease.

Objective.-The significantly higher incidence of progressive multifocal leukoencephalopathy in patients with acquired immunodeficiency syndrome than in patients with other immunosuppressive conditions suggests that molecular interactions between human immunodeficiency virus 1 and JCV, via the Tat protein, are responsible for the activation of the JCV enhancer/promoter and the development of progressive multifocal leukoencephalopathy. An indirect mechanism through activation of cytokines, such as transforming growth factor [beta]1 and Smads 3 and 4, may also be responsible for the enhancement of JCV gene expression.

Design.-Immunohistochemical analysis in progressive multifocal leukoencephalopathy samples and chloramphenicol acetyl transferase assays on cell cultures were performed to corroborate this hypothesis.

Results.-The JCV capsid protein VP-1 was found in the nuclei of oligodendrocytes and in the nuclei and cytoplasm of bizarre astrocytes. Human immunodeficiency virus proteins, including p24 and Tat, were detected in the cytoplasm of astrocytes. Tat, but not p24, was detected in oligodendrocytes, suggesting that extracellular Tat accumulates in the nuclei of oligodendrocytes, where JCV gene transcription takes place. High levels of transforming growth factor [beta]1 and Smads 3 and 4 were detected in JCV-infected oligodendrocytes. Results from in vitro studies confirm activation of the JCV early and late promoters by Smads 3 and 4.

Conclusions.-These observations support our model, suggesting that the induction of transforming growth factor [beta]1 by human immunodeficiency virus 1 Tat can stimulate its downstream factors, including Smads 3 and 4, which in turn augment transcription of the JCV promoter in glial cells.

(Arch Pathol Lab Med. 2004;128:282-291)

Human immunodeficiency virus 1 (HIV-1) infection of the brain results in a variety of clinical syndromes and opportunistic infections.1 Progressive multifocal leukoencephalopathy (PML), a subacute demyelinating dis ease of the central nervous system, is one such disease, caused by the opportunistic human neurotropic JC virus (JCV). JCV, a member of the polyomavirus family, is widespread among the adult population of the world, as proven by serologic studies in which more than 80% of tested populations exhibit antibodies against the virus.2

PML is histologically characterized by extensive areas of demyelination in the subcortical white matter, due to the active infection and cytolytic destruction of oligodendrocytes, the myelin-producing cells of the central nervous system. Other histologie features of PML include giant bizarre astrocytes, eosinophilic nuclear inclusions in oligodendrocytes, microglial nodules, and foamy activated macrophages within the demyelinated plaques.3-5

JCV consists of a 38- to 40-nm icosahedral capsid enclosing a closed, circular, double-stranded DNA genome of approximately 5 kilobases. The viral genome of JCV reference strain MAD-1 (GenBank J02226) can be divided into 3 regions, including a regulatory noncoding region composed of two 98-base pair repeats, which divides the early coding region that contains information encoding the transforming protein T-antigen, and the late coding region, which encodes the capsid proteins VP-1, VP-2, and VP-3 as well as the accessory Agnoprotein.6,7 HIV-1 encodes for a 14-kd transactivator protein, Tat, which is produced early in HIV-1 infection and plays a key role in transcription and replication through interaction with the HIV-1 long terminal repeat.8-10 Tat activation leads to the increased expression of transcripts encoding all viral proteins, including Tat itself, resulting in a positive feedback cycle and massive induction of HIV-1 viral gene expression.

Approximately 4% to 8% of HIV-1-infected patients will develop PML, which, on the basis of this fact, is now considered an acquired immunodeficiency syndrome (AIDS)-defining condition.11,12 The significantly higher incidence of PML in AIDS patients than in any other immunosuppressive disorders has suggested that the presence of HIV-1 in the brain participates, directly or indirectly, in the pathogenesis of this disease.13 In support of this concept, previous in vitro studies have demonstrated cross-communication between HIV-1 and JCV through the HFV-1-encoded regulatory protein Tat. Specifically, it has been shown that Tat has the ability to bind to specific sequences within the JCV control region, which results in the enhancement of JCV promoter transcription in glial cells.14-17 The activation of JC viral replication may be initiated by Tat stimulation of an HIV-1 transacting responsive-like region within the JCV control region.18 Furthermore, Tat is capable of increasing JCV DNA replication.19 Having established that Tat might be secreted from HIV-1-infected cells and subsequently taken up by neighboring uninfected cells, which may harbor the opportunistic JCV,20-23 it might be concluded that the reactivation of JCV through HIV-1 Tat does not require the coinfection of glial cells with both viruses.

An indirect pathway that may also be involved in the activation of the JCV promoter involves the transforming growth factor [beta]1 (TGF-[beta]1), which, through downstream signaling, modulates the Smad family of transcription factors. The TGF-[beta]1 family comprises a large number of structurally related polypeptide growth factors, each capable of regulating a wide variety of cell processes, including cell proliferation, lineage determination, motility, adhesion, and cell death.24,25 TGF-[beta]1 family members initiate signaling from the cell surface by binding to a heteromeric complex of 2 distinct but related serine/threonine kinase receptors. Binding of the ligand to the type II receptor results in the recruitment and phosphorylation of the type I receptor. This activates the type I receptor, which propagates the signal to a family of intracellular signaling mediators known as Smads.26 Smads are a novel family of signal transducers that have been implicated as downstream effectors of TGF-[beta]1 signaling. These proteins translocate to the nucleus, target specific genes, and generate transcriptional complexes of specific DNA-binding ability.25,27 In the basal state, Smads exist as homo-oligomers or monomers that reside in the cytoplasm. Upon ligand activation of the receptor complex, the type I receptor kinase phosphorylates specific Smads, which then form a complex with Smad 4 and translocate into the nucleus, where these complexes, either alone or in association with a DNA-binding subunit, activate target genes by binding to specific promoter elements.

Previous work by our laboratory and other laboratories has indicated the ability of HIV-1 to up-regulate TGF-[beta]1 expression and has directly implicated HIV-1 Tat in this up-regulation.28-10 Such direct mechanisms, via Tat transactivation of the JCV promoter, and indirect effects, via up-regulation of the TGF-[beta]1/Smad pathway by secretion of Tat from neighboring cells, suggest potential targets for the modulation of JCV replication and control of PML in HIV-1-infected individuals.

MATERIALS AND METHODS

Clinical Samples

A total of 12 formalin-fixed, paraffin-embedded autopsy samples of PML were collected from the archives of the Pathology Institute, University of Laussane, Switzerland (6 cases), and from the Manhattan Brain Bank (R24MH59724) at Mount Sinai Medical Center, New York, NY (6 cases). Ten samples were from HIV-1-infected patients, and 2 cases were of non-AIDS-related PML. Three normal brains from patients who died of nonneurologic conditions were used as negative controls.

Histologie and lmmunohistochemical Analysis

The formalin-fixed, paraffin-embedded tissue was sectioned at a 4-µm thickness and stained with hematoxylin-eosin for routine histologie diagnosis and characterization of the cases. A special staining for myelin (Luxol Fast Blue) was performed to evaluate demyelinated lesions of PML.

Immunohistochemistry was performed using the avidin-biotinperoxidase complex system according to the manufacturer's instructions (Vectastain Elite ABC Peroxidase Kit, Vector Laboratories Inc, Burlingame, Calif). Our modified protocol includes deparaffinization in xylenes and rehydration of the tissue through descending grades of alcohol up to water and nonenzymatic antigen retrieval in 0.01M sodium citrate buffer (pH 6.0) heated to 95°C for 40 minutes in a vacuum oven. After a cooling period of 30 minutes, the slides were rinsed in phosphate-buffered saline (PBS) and incubated in MeOH/3% H^sub 2^O^sub 2^ for 20 minutes to quench endogenous peroxidase. Sections were then rinsed with PBS and blocked with 5% normal horse or goat serum in 0.1% PBS/bovine serum albumin for 2 hours at room temperature. Primary antibodies were incubated overnight at room temperature in a humidifier chamber. The primary antibodies used in this study included the following: a rabbit polyclonal anti-Agnoprotein (1:500 dilution)31; a rabbit polyclonal antibody against the JCV capsid protein VP-1 (1:1000 dilution, kindly provided by Dr Walter Atwood, Brown University, Providence, RI); a mouse monoclonal antibody for the detection of SV-40 T-antigen, which cross-reacts with JCV T-antigen (clone pAb416, 1:100 dilution, Oncogene Science, Boston, Mass); a mouse monoclonal antibody against the HIV-1 capsid protein p24 (clone Kal-1, 1:10 dilution, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom); and a rabbit polyclonal anti-Tat (HIV-1BH10, 1:2000 dilution, a generous gift from Dr Avindra Nath, Department of Neurology, University of Kentucky, College of Medicine, Lexington). For the detection of cell cycle proteins, we used mouse monoclonal antibodies against TGF-[beta]1 (clone TGFB17, 1:200 dilution, Novocastra) and TGF-[beta] receptor 1 (clone 8A11, 1:100 dilution, Novocastra), a rabbit polyclonal antibody against Smad 3 (FL-425, 1:500 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, Calif), and a mouse monoclonal antibody against Smad 4 (clone B-8, 1:100 dilution, Santa Cruz Biotechnology). Biotinylated secondary anti-mouse or anti-rabbit antibodies were incubated for 1 hour at room temperature. Finally, sections were incubated with avidin-biotin complex (ABC kit, Vector Laboratories) for 1 hour at room temperature, rinsed with PBS, and developed with diaminobenzidine (Boehringer Mannheim, GmbH, Germany). Sections were counterstained with hematoxylin and mounted with Permount (Fisher Scientific, Pittsburgh, Pa).

Laser Capture Microdissection

Laser capture microdissection was performed on representative sections of 5-µm thickness. Immunohistochemistry to detect the JC capsid protein VP-I was performed as described above but, instead of mounting, sections were air dried on a slide heater overnight. Laser capture was performed under direct microscopic visualization to dissect VP-1-positive immunolabeled cell populations, specifically astrocytes and oligodenclrocytes, by melting a thermoplastic film mounted on laser capture microdissection caps onto the selected cells (Arcturus Engineering, Mountain View, Calif). The PixCell II LCM System (Arcturus) was set to the following parameters: 7.5-µm laser spot size, 80 mW of power, and 850 microseconds for JCV-infected oligodendrocytes and 15-µm laser spot size, 40 mW of power, and a 3.0-millisecond duration for JCV-infected astrocytes. A total of 20 VP-1 immunoreactive oligodendrocytes and 50 bizarre astrocytes were microdissected. Normal astrocytes and oligodendrocytes from the cortex of a patient who underwent a temporal lobectomy to control seizures were microdissected and used as a negative control for polymerase chain reaction amplification.

Polymerase Chain Reaction Amplification and Southern Blot Analysis

Polymerase chain reaction amplification of DNA sequences extracted from the microdissected cells was performed according to our previously described protocol.32 In this study, the following sets of primers were used: Pepl and Pep2 (nucleotides 4255-4274 and 4408-4427, respectively), which specifically amplify the amino-terminal region of the T-antigen gene; VP2 and VP3 (nucleotides 1818-1848 and 2019-2039, respectively), which amplify the VP-I late gene; and finally, Agnol and Agno2 (nucleotides 280-298 and 438-458, respectively), which amplify the accessory Agnoprotein gene region. Samples amplified in the absence of template DNA were used as negative controls, and plasmid pBJC, containing the JCV MAD-4 strain genome as a template, was used as a positive control. Southern blot hybridization was carried out according to the previously described protocol32 using JCV-specific probes: the JCV probe (nucleotides 1872-1891) for the T-antigen region-amplified sequences, the VP-1 probe (nucleotides 1872-1891) for the VP-1-amplified sequences, and the Agno probe (nucleotides 425-445) for detection of the sequences amplified with the Agno primers.

Cell Cultures and DNA Transfection

U-87MG cells were grown in D-MEM with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Transient transfections were carried out with the calcium phosphate method in Graham and Van der Eb33 with some variations. DNA concentrations were kept constant by adding empty vector DNA. After incubation of the DNA and calcium phosphate mixture for 4 to 5 hours, the cells were incubated with 10% glycerol in PBS; then, the precipitate was washed off, and the cells were fed with fresh complete media. JCV early and late plasmids containing the control sequence of the JCV MAD-1 strain placed at the 5' position from the reporter gene of chloramphenicol acetyl transferase were used to transfect cells. Smad 3 (pRK5-Smad3 Flag: BamHI-SalI into pRKSF) and Smad 4 (pRK5-Smad4 Flag: EcoKI-Sall into pRKSF) expressing plasmids were obtained from Dr Olay Batuman, SUNY Health Center, Brooklyn, NY.

Chloramphenicol Acetyl Transferase Assays

Cells were harvested 48 hours after transient transfection in TEN buffer (40mM Tris-HCl, pH 7.5, ImM EDTA, pH 8.0, and 150mM NaCl) and lysed in 250MM Tris, pH 7.8, by several freezes in a dry ice ethanol bath and subsequent thaws at 37°C. Equal amounts of extract protein were analyzed in each assay (5-20 µg). These extracts were incubated with 8 pmol of acetyl CoA and 0.1 µCi of ^sup 14^C-chloramphenicol at 37°C for 1 hour. After extraction with ethyl acetate, the samples were spotted onto thin-layer chromatography plates, and various forms migrated in a methanolchloroform (5:95) mixture. The percentage of conversion of ^sup 14^C-chloramphenicol was determined by liquid scintillation counting.

RESULTS

A total of 12 archival cases of PML were collected for this study. The patients ranged in age from 31 to 73 years. Of the total, 10 samples corresponded to HIV-1-infected patients, and 2 cases were of non-AIDS-related PML-one patient undergoing immunosuppressive chemotherapy for a renal transplant and the other suffering from chronic lymphocytic leukemia. Three samples of normal brains were used as negative controls. Table 1 summarizes the clinical data of the patients, including age and gender, country of origin, and other clinical-associated conditions.

PML was histologically characterized by multiple foci of demyelination in the subcortical white matter of the cerebral hemispheres, most frequently within the frontal lobe (Figure 1, A). The demyelinated plaques were highlighted with Luxol Fast Blue staining (Figure 1, E). Other areas affected included the parietal lobe, the temporal lobe, and, in one case, the white matter tracts of the cerebellum, midbrain, and pons. Within the margins of the demyelination plaques, the presence of multiple enlarged oligodendrocytes harboring intranuclear eosinophilic inclusion bodies was observed (Figure 1, B). The demyelinated plaques also contained bizarre transformed astrocytes (Figure 1, F) and multiple activated foamy macrophages, which are presumably in charge of phagocytizing the myelin released by the lyrically infected oligodendrocytes.

To determine the localization of JCV and HIV-1 viral proteins and cellular cytokines such as TGF-[beta]1 and Smads 3 and 4 in the clinical samples of patients with PML, immunohistochemical analysis was performed. The JCV capsid protein VP-1 was found in the nuclei of enlarged, infected oligodendrocytes (Figure 1, C) and, in some instances, to a lesser degree of intensity in the cytoplasm of the same oligodendrocytes (Figure 1, C insert, arrow), as well as in the nuclei and cytoplasm of bizarre transformed astrocytes within the demyelinated plaques (Figure 1, G), indicating the presence and active replication of JCV within these cells. The late gene product Agnoprotein showed robust immunoreactivity in the cytoplasm of both the enlarged, infected oligodendrocytes (Figure 1, D) and the bizarre astrocytes (Figure 1, H). The early gene product of JCV, T-antigen, was not detectable by immunohistochemistry in any of the cells studied (data not shown). Although T-antigen is important for the early stages of viral replication, the lack of immunoreactivity in PML samples is not an uncommon finding because of the low expressed levels of the protein.

To determine the presence of JCV DNA sequences in different types of cells within demyelinated plaques, we performed laser capture microdissection on tissue sections immunohistochemically labeled for the JCV capsid protein VP-1 specifically dissecting astrocytes (Figure 2, A1) and oligodendrocytes (Figure 2, B1). Once the cells were microdissected (Figure 2, A2, A3, B2, and B3), DNA was extracted, and polymerase chain reaction analysis was performed. Results from DNA amplification showed JCV DNA sequences from the early genes as well as DNA from the late capsid VP-1 region and the late region encoding the accessory Agnoprotein (Figure 2, D) in both types of microdissected cells, bizarre astrocytes, and oligodendrocytes.

Next, immunohistochemistry to detect HIV-1 viral proteins showed the presence of the HIV-1 capsid protein p24 in the cytoplasm of reactive astrocytes (Figure 3, B); however, the JCV-infected oligodendrocytes, harboring intranuclear inclusion bodies, remained negative (Figure 3, A), indicating that HIV-1 is actively infecting reactive astrocytes but not oligodendrocytes. HIV-1 Tat was detected in the cytoplasm of bizarre reactive astrocytes (Figure 3, D) but was also strongly immunoreactive in the nuclei of JCV-infected oligodendrocytes (Figure 3, C), suggesting that HIV-1 Tat is produced and secreted by HIV-1-infected cells-in this case, bizarre astrocytes-and then absorbed by neighboring uninfected cells, including oligodendrocytes harboring JCV, as have been suggested by previous data from our laboratory and other laboratories.22,23 As anticipated, in non-AIDS-related cases, the p24 capsid protein and Tat were not detected by immunohistochemistry (data not shown).

In the next series of experiments, we investigated the involvement of the TGF-[beta]/Smad pathway as an indirect mechanism of JCV promoter activation. For this purpose, we performed immunohistochemistry using specific antibodies for TGF-[beta] and TGF-[beta] receptor 1, which demonstrated robust immunolabeling of infected oligodendrocytes (Figure 4, A and B, respectively), suggesting that TGF-[beta]1 and its receptor are up-regulated in these cells, which also contain Tat and VP-1 in their nuclei. Bizarre astrocytes also show cytoplasmic immunoreactivity (Figure 4, E and F, respectively). Immunohistochemistry for the TGF-[beta]1 downstream factor, Smads 3 and 4, demonstrated cytoplasmic reactivity in astrocytes (Figure 4, G and H, respectively) and, most importantly, in the nuclei of JCV-infected oligodendrocytes (Figure 4, C and D). Normal, noninfected oligodendrocytes in adjacent white matter showed nondetectable levels of Smad antibodies (data not shown). Smads 3 and 4 were detected in the nuclei of oligodendrocytes and in the cytoplasm of bizarre astrocytes, a pattern similar to that observed with the Tat protein. To further characterize the phenotype of the cells containing JCV viral inclusions and expressing TGF-[beta] and Smad proteins, we performed double-labeling immunohistochemistry. Bizarre astrocytes were labeled with a fluorescein-tagged glial fibrillary acidic protein antibody and robustly expressed the cytoplasmic marker, and a second antibody for VP-1 showed the presence of nuclear viral replication in the same cells (Figure 4, M). The same glial fibrillary acidic protein-positive bizarre astrocytes (Figure 4, N) were labeled with a rhodamin TGF-[beta] (Figure 4, O) and demonstrated the presence of both proteins in the cytoplasm (Figure 4, P). Oligodendrocytes demonstrated the presence of myelin basic protein in the cytoplasm and JCV capsid protein VP-1 in their nuclei (Figure 4, I). Fluorescein-tagged myelin basic protein was found in the cytoplasm of enlarged oligodendrocytes (Figure 4, J), and Smads 3 and 4 were found in the nuclei of the same cells (Figure 4, K). Superimposition of the 2 images demonstrates the oligodendroglial nature of the cells expressing Smad 3 (Figure 4, L).

Table 2 summarizes the immunohistochemical data observed in bizarre astrocytes, and Table 3 shows the results of immunohistochemistry in JCV-infected oligodendrocytes in the PML clinical samples.

To corroborate the hypothesis of JCV promoter activation by Smads in an in vitro system, we performed chloramphenicol acetyl transferase assays on lysates from U-87-MG cell cultures transiently transfected with JCV early and late promoters and Smads 3 and 4. Results from these experiments show that Smads 3 and 4, both alone and together, activated the JCV early and late promoters, respectively (Figure 5, A). Smad 3 activated the JCV early promoter by 8.38 and the late promoter by 10.02-fold, while Smad 4 caused a 6.76-fold activation of the early promoter and a 14.6-fold activation of the late promoter. Cotransfection with Smads 3 and 4 activated the JCV early promoter by 12.53-fold and the late promoter by 27.5-fold, respectively, suggesting cooperativity between these 2 proteins (Figure 5, B).

COMMENT

PML is a subacute and fatal demyelinating disease of the central nervous system, very frequently seen in patients with AIDS. Although the human neurotropic virus JCV is the well-established opportunistic agent of this disease, very little is known about the mechanisms of JCV promoter activation, which result in the viral replication of oligodendrocytes and in the development of PML. That PML is more frequently seen in HIV-1-infected patients than in any other immunosuppressive conditions suggests that the molecular interaction between these 2 viruses is responsible for the activation of JCV replication in the setting of neuro-AIDS.

Previous studies have demonstrated the ability of the HIV-1-encoded Tat protein to be secreted by HIV-1-infected cells in the central nervous system, particularly microglial cells and astrocytes, and taken up by neighboring HIV-1-negative cells, including oligodendrocytes.22,23 In addition, in this study, we demonstrate the presence of JCV DNA sequences in both bizarre reactive astrocytes and infected oligodendrocytes and the expression of JCV proteins (the capsid protein VP-1 and the accessory product Agnoprotein) in the nuclei and cytoplasm of astrocytes and oligodendrocytes, indicating active viral replication in these cells. However, when the samples were analyzed for the presence of HIV-1 proteins, we found a complete absence of HIV-1 p24 in enlarged oligodendrocytes, but the transactivator protein Tat was present, corroborating the notion of the absence of infection and the uptake of soluble Tat secreted by neighboring HIV-1-infected cells.

Although a direct interaction between Tat and the JCV late promoter has been shown in different models,13,71,18 it is possible that indirect mechanisms for JCV activation also occur. Numerous reports have shown that the HIV-1 transactivator protein Tat can stimulate TGF-[beta]1 production.20,34,35 TGF-[beta]1 binding to a type II receptor starts the signaling cascade, which leads to the phosphorylation of Smad 3 and the subsequent nuclear import and localization of Smad 3 and its partner Smad 4, which in turn can activate targeted gene transcription.36,37 In the present study, we demonstrate that the TGF-[beta]1 receptor and TGF-[beta]1 are detected in the cytoplasm of astrocytes and oligodendrocytes and are overexpressed when compared to normal oligodendrocytes and astrocytes in the control brain. We have also found that where Smads 3 and 4 are present in the cytoplasm of bizarre astrocytes, in JCV-infected oligodendrocytes, these proteins are imported into the nucleus where, presumably, they interact and activate the JCV promoter, leading to viral replication. To corroborate this notion, we performed in vitro studies, which demonstrate that Smads 3 and 4, alone and together, are capable of activating both JCV early and late promoters in transfected glial cell cultures.

We suggest 2 alternate pathways for JCV reactivation in the brain of HIV-1-infected individuals. In the first, the HIV-1 transactivator protein Tat, produced by HIV-1-infected astrocytes, is secreted into the extracellular matrix and taken up by neighboring JCV-infected oligodendrocytes. The HIV-1-encoded transregulatory protein Tat has been shown to be a potent activator of JCV gene expression, particularly by its ability to increase the rate of transcription from the JCV late promoter. Tat binds to the JCV promoter, either directly or by its interaction with other cellular proteins, to result in increased transcription of the JCV genes, which in turn results in viral replication, capsid protein formation, and new viral particle assembly. The target sequence for Tat induction on the HIV-1 promoter is located in the leader of the viral transcripts between nucleotides + 19 and +42 in the R region of the long terminal repeat. This transacting responsive region has the capacity to form a stem-loop structure within the RNA molecule, important for initiating viral RNA synthesis and hence activation of the viral promoter. Figure 6 depicts a schematic representation of the transacting responsive-like sequences in the JCV control region. In the second, but not mutually exclusive, model, Tat stimulates the production of several cytokines, including TGF-[beta]1, which binds to the TGF-[beta]1 receptor of JCV-infected oligodendrocytes, which in turn results in the transactivation of the JCV promoter by Smads 3 and 4. A scheme for both of the proposed pathways is shown in Figure 7.

In summary, the present study introduces data that support a model in which the HIV-1 transactivator protein Tat, in the absence of HIV infection of oligodendrocytes, may participate, either directly or indirectly through activation of the TGF-[beta] and its downstream proteins, Smads, in the activation of the JCV promoter and therefore in the development of the fatal demyelinating disease PML.

We thank Walter Atwood, MD, Brown University, Providence, RI, and Avindra Nath, MD, Kentucky University, Lexington, for kindly sharing antibodies. PML tissue was obtained from the Manhattan Brain Bank (R24 MH59724) at Mount Sinai Medical Center, under the direction of Susan Morgello, MD, and from the University Institute of Pathology in Laussane, Switzerland, generously provided by Judith Miklossy, MD. We also wish to acknowledge the present and past members of the Center for Neurovirology and Cancer Biology for their support, insightful discussion, and sharing of ideas and reagents. Finally, we thank Cynthia Schriver for editorial assistance and preparation of this manuscript. This work was made possible by grants awarded by the National Institutes of Health to Drs Amini, Khalili, and Del Valle.

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Sahnila Enam, BS; Thersa M. Sweet, PhD; Shohreh Amini, PhD; Kamel Khalili, PhD; Luis Del Valle, MD

Accepted for publication November 10, 2003.

From the Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pa.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Luis Del Valle, MD, Center for Neurovirology and Cancer Biology, Temple University, 1900 N 12th St, Suite 240, Philadelphia, PA 19122 (e-mail: lvalle@temple.edu).

Copyright College of American Pathologists Mar 2004
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