Potential environmental and host participants in the early white matter lesion of adreno-leukodystrophy: morphologic evidence for CD8 cytotoxic T cells, cytolysis of oligodendrocytes, and CD1-mediated lipid antigen presentation.

The 2 most common forms of X-linked adreno-leukodystrophy (ALD) are the juvenile or childhood cerebral form with inflammatory demyelination and the adult adrenomyeloneuropathy (AMN) involving spinal cord tracts without significant inflammation. Modifier genes or environmental factors may contribute to the phenotypic variability. We performed immunohistochemical, an in situ polymerase chain reaction, and TUNEL analyses to identify several viruses, lymphocyte subpopulations, apoptotic cells, and effector molecules, focusing on morphologically normal white matter, dysmyelinative and acute demyelinative lesions. No distinguishing viral antigens were detected. Most lymphocytes were CD8 cytotoxic T cells (CTLs) with the alpha/beta TCR, and they infiltrated morphologically unaffected white matter. Only a few oligodendrocytes were immunoreactive for caspase-3. MHC class II- and TGF-beta-positive microglia were present. CD44, which can mediate MHC-unrestricted target cell death, was seen on many lymphocytes and white matter elements. CD1 molecules, which play major roles in MHC-unrestricted lipid antigen presentation, were noted. Our data indicate that unconventional CD8 CTLs are operative in the early stages of dysmyelination/demyelination and that cytolysis of oligodendrocytes, rather than apoptosis, appears to be the major mode of oligodendrocytic death. The presentation of lipid antigens may be a key pathogenetic element in ALD and AMN-ALD.

Human herpesvirus 6-meningoencephalitis in an HIV patient with progressive multifocal leukoencephalopathy.

Human herpesvirus 6 (HHV6) has been reported as a rare cause of meningoencephalitis and leukoencephalitis. We present an HIV-infected patient with lesions of progressive multifocal leukoencephalopathy (PML), but also meningoencephalitis apparently due to HHV6. Immunohistochemistry for HHV6 antigens and in situ polymerase chain reaction for HHV6 genome showed many positive lymphocytes and microglia in the meningeal and cortical lesions. More importantly, dead and dying neurons were conspicuous; some were undergoing neuronophagia and some displayed evidence of HHV6 infection. A pathogenic role for this almost universal, and usually commensal, virus in inflammatory brain lesions and PML is briefly discussed.

The HHV6 paradox: ubiquitous commensal or insidious pathogen? A two-step in situ PCR approach.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) and multiple sclerosis (MS) are demyelinative diseases of the central nervous system (CNS). PML occurs mostly in individuals with AIDS-impaired immunity and is thought to be caused by JC polyoma virus (JCV). In MS a neurotrophic virus trigger is suspected, but the precise etiology remains unknown. Human herpesvirus 6 (HHV6) is a ubiquitous, commensal and usually benign beta-herpesvirus. Some researchers have found evidence for HHV6 infection in MS plaques and sera. We recently demonstrated a high frequency of cells containing HHV6 genome in PML lesions, as well as co-infection of oligodendrocytes by JCV and HHV6. This suggests that HHV6 may be a co-factor in the etiology of PML, and raises questions about its role in other demyelinative diseases. OBJECTIVES: To determine the prevalence and cellular localization of HHV6, JCV and HIV-1 infected cells in PML, MS, AIDS and control CNS tissues, and their potential relationship with disease. STUDY DESIGN: An unconventional, sensitive two-step in situ polymerase chain reaction (ISPCR) procedure was used to amplify and detect HHV6, JCV and HIV-1 genomic DNAs in formalin fixed, paraffin-embedded archival CNS tissues. HHV6, JCV and HIV-1 gene expression was detected by ICC for HHV6 p41 and gp101, JCV large T, and HIV-1 p24 gag and NEF proteins. RESULTS: A high frequency of HHV6 genome was consistently detected in both PML and MS white matter lesional cells; a peri-lesional concentration was notable. HHV6 was found mainly in oligodendrocytes, but neurons were also infected. HHV6 was present in larger amounts than JCV in PML lesions, while more HIV-1 than HHV6 was present in AIDS. Variable amounts of HHV6 genome were detected in normal, AIDS and other control brains; the frequency of infected cells tended to increase with patient age. CONCLUSIONS: High concentrations of HHV6 genome in association with PML and MS lesions, open the possibility that HHV6 activation may play a role in the pathogenesis of these demyelinative diseases.

Association of human herpesvirus 6 with the demyelinative lesions of progressive multifocal leukoencephalopathy.

Progressive Multifocal Leukoencephalopathy (PML) is a primary demyelinating disease of the central nervous system occurring almost exclusively in individuals with impaired cell-mediated immunity. The JC polyoma virus has been accepted as the etiologic agent ofPML. Using a two-step in-situ polymerase chain reaction procedure to amplify and detect genomic DNA of human herpesvirus-6 (HHV6) in formalin-fixed paraffin-embedded archival brain tissues, a high frequency of infected cells was consistently detected in PML white matter both within and surrounding demyelinative lesions and HHV6 genome was found mainly within oligodendrocytes. Lesser amounts of HHV6 genome were detected in most normal, AIDS, and other neurological disease control tissues. Immunocytochemistry for HHV6 antigens showed actively infected nuclei of swollen oligodendrocytic morphology only within the demyelinative lesions of PML but not in adjacent uninvolved tissue. In addition, no HHV6 antigens were detectable in control tissues including brains of individuals with HIV-1 encephalopathy but without PML. Double immunohistochemical staining for JC virus large T antigen and HHV6 antigens demonstrated co-labeling of many swollen intralesional oligodendrocytes in the PML cases. The evidence suggests that HHV6 activation in conjunction with JC virus infection is associated with the demyelinative lesions of PML.

Expression of pro- and anti-apoptosis gene products in brains from paediatric patients with HIV-1 encephalitis.

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV-1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene-directed neural cell death, we studied the in-situ expression of the Bcl-2 family of proteins, including the pro-apoptosis gene product Bax, the anti-apoptosis gene product Bcl-2, and Bcl-x. We demonstrate significantly elevated numbers of Bax-positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV-1 infected children without encephalitis or children who were seronegative for HIV-1. In contrast, patients with HIVE, but not HIV-1 without encephalitis, or seronegative controls, had increased expression of Bcl-2 and Bcl-x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up-regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl-2 in HIV-1 infected macrophages, and in LPS-activated macrophages, relative to levels in virus-negative unstimulated macrophages. These results suggest that productive HIV-1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain-resident macrophages and microglia in patients with HIV-1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV-1 infection in the CNS of children with HIV-1 encephalitis.

In situ amplification and detection of HIV-1 DNA in fixed pediatric AIDS brain tissue.

To examine whether latent infection by HIV-1 occurs in the central nervous system, we optimized a procedure for amplification and detection of HIV-1 DNA in situ, in formalin-fixed brain tissue from a child with severe HIV-1-associated progressive encephalopathy and severe HIV-1 encephalitis. By the use of a two-step technique, which involved polymerase chain reaction with incorporation of digoxigenin-labeled nucleotides followed by in situ hybridization with biotinylated probes, we found infection of numerous mononuclear cells and astrocytes in the cerebral white matter as well as of perineuronal satellite cells in basal ganglia, but not of neurons. Following PCR amplification, nuclear signal was found in 10 to 20 times as many cells as in parallel, control experiments using conventional, unamplified in situ hybridization.

Apoptotic neurons in brains from paediatric patients with HIV-1 encephalitis and progressive encephalopathy.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) associated dementia in adults involves neuronal loss from discrete areas of the neocortex and subcortical regions, but the mechanism for neuronal death is poorly understood. Gene-directed cell death resulting in apoptosis is thought to be a normal feature of neuronal development, but little is known about neuronal apoptosis in disease states. We investigated whether HIV-1 infection of the central nervous system is spatially associated with apoptosis of neurons. Using an in situ technique to identify newly cleaved 3'-OH ends of DNA as a marker for apoptosis, we demonstrate the presence of apoptotic neurons in cerebral cortex and basal ganglia of children that had HIV-1 encephalitis with progressive encephalopathy. Furthermore, an association was observed between the localization of apoptotic neurons and perivascular inflammatory cell infiltrates containing HIV-1 infected macrophages and multinucleated giant cells. Apoptotic neurons and p24-positive macrophages were observed infrequently in cerebral cortex and basal ganglia in children with HIV-1 infection without encephalitis or clinical encephalopathy. In nine control (HIV-1 negative) brains, ranging from the first post-natal month of life to 16.5 years of age, infrequent neuronal apoptosis was observed in three cases. These findings suggest that neuronal apoptosis is unlikely to be associated with post-natal development except in early post-natal germinal matrix, and that it may instead represent the end result of specific pathological processes, such as HIV-1 encephalitis.

Cellular localization of human herpesvirus-6 in the brains of children with AIDS encephalopathy.

Human herpesvirus-6, the etiologic agent of exanthem subitum, is a ubiquitous virus that infects almost all children by the age of 2 years and that has previously been shown to be neuroinvasive. These characteristics suggest that human herpesvirus-6 may be important in the neuropathogenesis of acquired immune deficiency syndrome (AIDS) in children. To address this hypothesis, we evaluated postmortem pediatric brain tissues for the presence of human herpesvirus-6 infection. Using in situ hybridization with a digoxigenin-labeled DNA probe for the large tegument protein gene of human herpesvirus-6, we detected nuclear signals in postmortem brain tissue from 4/5 children with human immunodeficiency virus-1 encephalitis. Human herpesvirus-6 DNA was found in numerous oligodendrocytes of the white matter and less frequently in astrocytes, macrophages, microglia and neurons. The human herpesvirus-6 positive cells detected by in situ hybridization were not immunoreactive either for human herpesvirus-6 early nuclear phosphoproteins or for surface glycoproteins associated with productive infection. Only rare human herpesvirus-6 infected cells were found in age-matched control brain tissues. No human herpesvirus-6 infected cells were found in human fetal brain tissue. These data suggest that human herpesvirus-6 is more extensively disseminated in neural cells in the presence of human immunodeficiency infection and immunodeficiency in pediatric AIDS patients, and it may contribute to the pathogenesis of AIDS encephalopathy.

Strand transfer mediated by human immunodeficiency virus reverse transcriptase in vitro is promoted by pausing and results in misincorporation.

Human immunodeficiency virus (HIV-1) is able to recombine by transfer of the growing DNA strand from internal regions of one genome to another. The strand transfer reaction, catalyzed by HIV-1 reverse transcriptase (RT), was conducted in vitro between donor and acceptor RNA templates that were derived from natural HIV-1 nef genes. The donor and acceptor templates shared a nearly homologous region where strand transfer could occur, differing only in that the acceptor had a 36-nucleotide insertion and 6 widely spaced base substitutions compared with the donor. We sequenced elongated primers that underwent transfer. The position of transfer was revealed by the change of sequence from that of the donor to that of the acceptor. Results showed a positive correlation between positions where the RT paused during synthesis and enhancement of strand transfer. Elimination of a pause site, with a minimal change in sequence, decreased the frequency of strand transfer in the immediate area. Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer. Remarkably, approximately 30% of the products contained mutations. Base substitutions, short additions and deletions were observed. Mutations did not appear in DNA products extended on the donor template without transfer. The identity of the mutations suggests that they were caused by a combination of slippage and non-template-directed nucleotide addition. These results indicated that the detected mutations were related to the process of strand transfer.

Interaction of HIV-1 and human salivary mucins.

Previous studies have suggested that salivary secretions may act as inhibitors of HIV-1 replication in vitro. This inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands, and subsequent electron micrographs revealed the association of viral particles with the salivary sediment. Fractionation of human submandibular-sublingual (HSMSL) saliva by size-exclusion chromatography was initiated, and resulting fractions were tested for their ability to modulate the replication of HIV-1 using a plaque assay on HeLa CD4+ cell monolayers. Results indicated that the filtration-sensitive inhibitory activity was primarily associated with the mucin-rich fractions, and the inhibitory activity was found to reduce the number of infectious units by 75%. To determine the identity of the salivary components involved, adsorption experiments involving the interaction of HIV particles with immobilized salivary components were performed. Immunological counter staining revealed an interaction of HIV particles as well as recombinant gp120 with the lower-molecular-weight mucin. Electron microscopic examination of the mucin-rich fractions-HIV incubates revealed the aggregation of virus particles by salivary components. These results suggest that human salivary mucins may have a role in modulating the infectivity of HIV-1.

HIV-1 infection of the developing nervous system: central role of astrocytes in pathogenesis.

Recent studies in our laboratory and that of Dr. Howard Gendelman have revealed two important pathways for neuronal damage during HIV-1 encephalopathy in children. First, substantial numbers of astrocytes are actively or latently infected with HIV-1. Astrocyte infection may lead to neuronal dysfunction through loss of supporting growth factors, excitotoxicity due to dysregulation of neurotransmitter reuptake, and loosening of the blood-brain barrier permitting further seeding of HIV-1 in the CNS. Significantly, infection of astrocytes is marked by near-exclusive synthesis of early regulatory gene products of HIV-1, while structural proteins characteristic of productive infection are found in macrophages, microglia and multinucleated giant cells. We propose the term 'restricted' to denote the non-productive infection found in astrocytes. Second, HIV-1-infected macrophages initiate inflammatory processes which are amplified through cell-cell interactions with astrocytes. Macrophage-astrocyte interactions produce arachidonic metabolites and potentially neurotoxic cytokines (TNF-alpha and IL-1 beta), leading to astroglial activation and proliferation which then amplifies these cellular processes. These new findings suggest that two major pathways leading to neurotoxicity in pediatric AIDS encephalopathy are linked to HIV-1 infection through astrocyte-mediated processes, and help explain how small numbers of productivity infected cells indirectly cause widespread tissue pathology and elicit profound neurological impairment.

Overexpression of nef as a marker for restricted HIV-1 infection of astrocytes in postmortem pediatric central nervous tissues.

In previous studies, using polymerase chain reaction amplification of HIV-1 genes directly from pathologic tissues of children who died with AIDS encephalopathy, we showed that the reading frame of the HIV-1 regulatory nef gene is open, suggesting that the nef protein was expressed. We now show, using immunocytochemistry and in situ hybridization with nef-specific probes in postmortem pediatric CNS tissues, that nef mRNA and protein are present in up to 20% of astrocytes in tissue sections selected for extensive histopathology. By contrast, HIV-1 structural proteins such as gag and their coding mRNAs are present in multinucleated giant cells that harbor productive infection and are the hallmark of HIV-1 infection in the CNS. These findings are consistent with the nonproductive infection of glial cells observed in vitro, and imply that HIV-1 infection of astrocytes is restricted to early regulatory gene products, of which nef is the best target as it is expressed at high levels and is membrane-anchored. In developing central nervous tissues of children, restricted and latent HIV-1 infection of astrocytes may be extensive and contribute significantly to HIV-1 neuropathogenesis.

Detection of HIV-1 DNA in pediatric AIDS brain tissue by two-step ISPCR.

In order to detect latent infection in neurons or other cell types in formalin-fixed brain tissue, we performed polymerase chain reaction amplification with incorporation of digoxigenin-conjugated deoxynucleotides, followed by in situ hybridization with biotinylated probes. The use of this two-step technique in brain tissue from a child with severe HIV-1 encephalitis revealed signal in both nuclear and perinuclear regions of cells identified as monocytes and astrocytes, and also in perineuronal satellite cells of glial morphology, but HIV-1 infection of neurons was not detected.

Aggregation of human immunodeficiency virus type 1 by human salivary secretions.

Human immunodeficiency virus (HIV-1) is generally transmitted by parenteral contact with infected body secretions. Although extensive epidemiological data and familial studies have failed to provide any conclusive data that saliva may act as a vehicle for transmission of AIDS, both professional and public anxieties remain. The present study, as well as others, suggests that salivary secretions may act as inhibitors of HIV-1 replication in vitro. In our study, the inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands. Human submandibular-sublingual (HSMSL) and parotid (HPS) salivas were collected and tested for their ability to modulate the replication of HIV-1, using a plaque assay on HeLa/CD4+ cell monolayers. Initial results examining freshly collected salivary samples from ten individuals confirmed the results previously obtained by Fox et al. (1988, 1989). An average plaque reduction of approximately 66% was obtained with HSMSL, in contrast to 34% reduction obtained with HPS. Titration of the inhibitory activity in HSMSL showed detectable levels at a 1:500 dilution. Comparison of inhibitory activity of dialyzed and lyophilized saliva to fresh saliva indicated little difference between the two samples when filtration occurred after the addition of HIV-1. However, the effect of filtration was significantly diminished in the lyophilized samples. Electron microscopic examination of the saliva-HIV incubates revealed the aggregation/entrapment of virus particles by salivary components. These results suggest that human salivary secretions (with HSMSL > HPS) may have a role in modulating the infectivity of HIV-1.

Human immunodeficiency virus type 1 nef quasispecies in pathological tissue.

The role of the nef gene in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. To provide a basis for studies on the role of nef in AIDS, we used targeted polymerase chain reaction amplification and DNA sequencing to determine the structure of nef genes in pathologic tissue from HIV-1-infected children and adults. We find that the nef reading frame is open in 92% of clones derived from both brain and lymphocytic tissue of children, suggesting that nef is expressed in these tissues. One HIV-1 clone, BRVA, obtained by coculture from the brain of an adult AIDS patient with progressive dementia, was previously shown to contain a duplicated region in nef. We show here that similar duplications are widespread in both adults and children with AIDS. However, coculture strongly selects against the broad spectrum of nef quasispecies found in tissue. These findings suggest functional selection for nef quasispecies in pathologic tissues during HIV-1 infection of the human host.

Human immunodeficiency virus type 1 infection of neural xenografts.

Human immunodeficiency virus type 1 (HIV-1) infection is highly specific for its human host. To study HIV-1 infection of the human nervous system, we have established a small animal model in which second-trimester (11 to 17.5 weeks) human fetal brain or neural retina is transplanted to the anterior chamber of the eye of immunosuppressed adult rats. The human xenografts vascularized, formed a blood-brain barrier, and differentiated, forming neurons and glia. The xenografts were infected with cell-free HIV-1 or with HIV-1-infected human monocytes. Analysis by polymerase chain reaction revealed HIV-1 sequences in DNA from xenograft tissue exposed to HIV-1 virions, and in situ hybridization demonstrated HIV-1 mRNA localized in macrophages and multinucleated giant cells. Pathological damage was observed only in neural xenografts containing HIV-1-infected human monocytes, supporting the hypothesis that these cells mediate neurotoxicity. This small animal model allows the study of direct and indirect effects of HIV-1 infection on developing human fetal neural tissues, and it should prove useful in evaluating antiviral therapies, which must ultimately target HIV-1 infection of the brain.

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.

HIV-1 V3 domain variation in brain and spleen of children with AIDS: tissue-specific evolution within host-determined quasispecies.

DNA coding for the principal neutralization epitope of HIV-1 (the V3 domain of the envelope glycoprotein gp120) was amplified by polymerase chain reaction from postmortem brain and spleen tissue of three perinatally infected children who died of AIDS with progressive encephalopathy. Sequences obtained directly (without cloning) from this DNA were compared with sequences of 52 molecular clones made from this DNA. Cluster analysis showed that V3 domain sequences from two of the three children were similar to sequences from the American MN/SC isolates, while those from one child were more closely similar to the Caribbean RF isolate. Comparison of sequences obtained directly with consensus sequences derived from cloned DNA showed that V3 sequences are characteristic for an individual host. In one child, the V3 sequence determined directly from brain DNA was very distant from the consensus brain clone sequence and from the spleen sequences, suggesting a diverging quasispecies distribution. Site-directed hybridization demonstrated that brain-specific sequences present in 33% of brain-derived clones were absent from clones derived from spleen. The evidence suggests that brain- and spleen-specific variants evolve independently within each host-delimited quasispecies.

Spinal cord disease in children with HIV-1 infection: a combined molecular biological and neuropathological study.

An autopsy study was performed on spinal cords from 18 children who died with HIV-1 infection, using standard histopathologic techniques as well as in situ hybridization and immunocytochemistry for HIV-1. Of 16 spinal cords examined by histology, nine had inflammatory cell infiltrates and six had multinucleated cells; both types of lesion are associated with the presence of HIV-1 in central nervous system tissue. HIV-1 type lesions were often present in the spinal cord and brain from the same patient. Pallor of myelin in corticospinal tracts in the cord was present in half of the cases; this change correlated with diffuse myelin pallor in the corresponding brains, but not with the HIV-1 associated changes in the cords. In situ hybridization for HIV-1 nucleic acid sequences gave positive results in seven of 18 spinal cords, with hybridizing signal usually localized to inflammatory cell infiltrates and multinucleated cells. Positive in situ hybridization, on frozen sections, correlated with the presence of HIV-1 associated changes on paraffin sections from the same cases. Immunocytochemistry for p25 core protein of HIV-1, using a monoclonal antibody on frozen sections, was positive in multinucleated cells, macrophages, and microglia. In this series there were two cases of vacuolar myelopathy, one a 30-month-old boy who had concomitant measles virus in the spinal cord grey matter, and the other nine-year-old girl who had severe HIV-1 infection of the cord. Other than the single case of measles virus, there were no opportunistic infections in the cords in this series. HIV-1 frequently involves the spinal cord in children with AIDS, while opportunistic infections are rare. Vacuolar myelopathy occurs in children with HIV-1 infection, although its occurrence is much less frequent than in adults with AIDS.

Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains.

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.