Immunocytochemical quantitation of human immunodeficiency virus in the brain: Correlations with dementia

The pathogenesis of human immunodeficiency virus (HIV)-associated dementia is unclear, and the underlying pathological substrate has been a matter of debate. In a prospectively clinically characterized population of acquired immunodeficiency syndrome (AIDS) patients we investigated the relationship between the clinical syndrome of HIV-associated dementia and the presence and relative quantity of immunocytochemical Marchkers for HIV-1 (gp41 antibody), and for macrophages and microglia (HAM-56 antibody). Sections from the basal ganglia and frontal lobes from the brains of 51 patients were studied, and the data were stratified for severity of dementia (16 nondemented, 12 mildly demented, 23 severely demented), rate of dementia progression, duration of AIDS, use of antiretrovirals, and several other demographic features. We found a highly significant correlation between the degree of macrophage staining and the severity of dementia but only a borderline correlation between the presence and amount of gp41-positive cells and dementia. Several nondemented patients showed abundant gp41 immunoreactivity, and some severely demented showed little to no gp41 immunoreactivity. Other correlations with the immunostaining data, including antiretroviral use, were not significant. We conclude that the presence of macrophages and microglia is a better correlate with HIV-associated dementia than is the presence and amount of HIV-infected cells in the brain. These data support the concept that the pathogenesis of HIV-associated dementia is likely due to indirect effects of HIV-infection of the brain, possibly through the actions of macrophages and microglia.     

Cross talk between growth factors and viral and cellular factors alters neuronal signaling pathways: Implication for HIV-associated dementia

HIV-associated dementia (HAD) is a serious neurological disorder affecting about 7% of people with AIDS. In the brain, HIV-1 infects a restricted number of cell types, being primarily present in macrophages and microglial cells, less abundant in astrocytes, and rarely seen in oligodendrocytes and neurons. Lack of a productive HIV-1 infection of neuronal cells suggests the presence of an indirect pathway by which the virus may determine the brain pathology and neuronal dysfunction seen in AIDS patients. Among the participants in this event, viral proteins including gp120 and Tat, along with host factors including cytokines, chemokines, and several signaling pathways have received considerable attention. In this article, we discuss the most recent concepts pertaining to the mechanisms of HIV-1-induced neuronal dysfunction by highlighting the interplay between signal transduction pathways activated by viral and host factors and their consequences in neuronal cell function.     

Simultaneous detection of ferritin and HIV-1 in reactive microglia

Summary Using ferritin as a marker of reactive microglia, we demonstrated a close association between proliferation of reactive microglia and expression of human immunodeficiency virus type 1 (HIV-1) in brain tissue from autopsied cases of acquired immunodeficiency syndrome (AIDS). An increased number of ferritin-positive reactive microglia was observed in formalin-fixed paraffin-embedded brain sections from all 13 AIDS cases examined. Similar findings were observed in brain tissue from other neurological diseases (subacute sclerosing penencephalitis, herpes simplex encephalitis and multiple sclerosis). Multinucleated giant cells were found in 7 of the AIDS cases which were also intensely labeled for ferritin. Dual-label immunohistochemistry using anti-ferritin and cell-specific markers showed that ferritin-positive cells were distinct from astrocytes, neurons and endothelia using anti-glial fibrillary acidic protein (anti-GFAP), anti-neurofilament protein and Ulex europaeus agglutinin 1, respectively. In 5 AIDS brains, only ferritin-positive cells were shown to contain HIV-1 gp41 antigen using dual-label immunohistochemistry. In addition, HIV-1 RNA was localized in territin-positive reactive microglia but not in GFAP-positive astrocytes using immunohistochemistry combined with in situ hybridization. Ferritin-positive reactive microglia and multinucleated giant cells were colabeled with the microglial marker, Ricinus communis agglutinin 1 (RCA-1). Howerver, RCA-1 also extensively stained resting microglia only a few of which were colabeled for ferritin. The density of ferritin-positive cells was correlated with the presence of HIV-1 RNA-positive cells in AIDS brain. Thus, ferritin immunoreactivity can be used as an activation marker of microglia in archival paraffin sections and reflects the extent of inflammation in HIV-1-infected brain.Supported in part by NIH Grants RO1 DA04787, RO1 HD26621, PO1 NS25569, the Biopsychosocial Center for the Study of AIDS (NIMH P50 MH 43455), the Department of Veterans Affairs, the Mary Jane Crowe Foundation, the Swedish Society of Medicine (Stockholm, Sweden), and the Multiple Sclerosis Society of Göteborg (Göteborg, Sweden)     

Spectrum of human immunodeficiency virus-associated neocortical damage.

A spectrum of neurocognitive defects, termed human immunodeficiency virus type 1 (HIV-1)-associated cognitive/motor complex, has been described in patients with acquired immunodeficiency syndrome (AIDS). AIDS dementia complex (ADC) is a severe form of this disease seen in 20 to 30% of terminally ill patients. The etiology of this complex is distinct from commonly observed opportunistic infections seen in brains of patients with AIDS and has been attributed to HIV infection within the brain. At autopsy, the brains of patients with ADC contain numerous HIV-infected macrophages/microglia with prominent subcortical damage, together termed HIV encephalitis. We retrospectively analyzed all 107 brains from a three-year period (1988-1990) of AIDS autopsies using immunocytochemistry to detect HIV. Rather than breaking into distinct groups of HIV encephalitis versus non-HIV encephalitis, the specimens revealed a spectrum of severity of HIV infection. Although only 16% of the brains showed the histological hallmarks of HIV encephalitis, more than 50% of the autopsies showed moderate to severe HIV infection. In a subset of 23 AIDS autopsies during which short postmortem times and absence of significant opportunistic infection permitted quantitative analysis of dendritic and synaptic complexities, we identified a strong correlation between neocortical dendritic and presynaptic damage and abundance of HIV envelope protein in the neocortical gray and deep white matter. This correlation suggests that the presence of HIV-1 in the neocortex may be responsible by direct or indirect mechanisms for dendritic and synaptic damage.     

HIV-1 gp120 proteins and gp160 peptides are toxic to brain endothelial cells and neurons: possible pathway for HIV entry into the brain and HIV-associated dementia

Breakdown of the blood-brain barrier is commonly seen in patients with human immunodeficiency virus (HIV)-associated dementia, despite the lack of productive HIV-infection of the brain endothelium. Through this damaged blood-brain barrier, HIV and HIV-infected monocytes/macrophages infiltrate the brain and further infect microglia and brain macrophages. Neuronal cell death and dysfunction are the underlying cause of HIV-associated dementia, but no productive HIV-infection of neurons has been documented. It is likely that secreted viral products play a major role in blood-brain barrier damage and neuronal cell death. The aim of the present study was to examine the effect of HIV-1 gp160 peptides and gp120 proteins on brain microvascular endothelial cells and neurons from both human and rats. Four of the 7 gp160 peptides tested evoked significant neurotoxicity. Two different full-length recombinant HIV gp120 proteins (HIV-1CM235 gp120 and HIV-1MN gp120) also induced neuronal and brain endothelial cell death, and concentrations as little as 1 ng/ml evoked pronounced morphological changes in these cells and marked cytotoxicity. This study suggests that HIV proteins and peptides that are shed in vivo may be directly involved in blood-brain barrier damage and neuronal cell death in HIV-associated dementia.     

In vitro evidence for a dual role of tumor necrosis factor-α in human immunodeficiency virus type 1 encephalopathy

Microglial cell activation, myelin alteration, and abundant tumor necrosis factor (TNF)-α message have been observed in the brains of some human immunodeficiency virus type 1 (HIV - 1)vinfected and demented patients. We therefore used cultures of purified human microglia and oligodendrocytes derived from adult human brain to examine the role of TNF-α in HIV-1 encephalopathy. Human microglia synthesize TNF-α message and protein in vitro. When these cells were infected with HIV-1 JrFL and maintained in the presence of TNF-α antibodies, soluble TNF-α receptors, or the TNF-α inhibitor pentoxifylline, viral replication was delayed or strongly inhibited. Both human microglia and oligodendrocytes express the two TNF receptors, TNF-Rl, which has been implicated in cytotoxicity, and TNF-R2. While TNF-α may enhance HIV-1 replication in an autocrine manner, it is not toxic for microglia. In contrast, recombinant human TNF-α causes oligodendrocyte death in a dose-dependent manner. In situ detection of DNA fragmentation in some cells indicated that oligodendrocyte death may occur by apoptosis. Addition of live microglia or medium conditioned by these cells also resulted in 30 to 40% oligodendrocyte death, which was largely prevented by TNF-α inhibitors. We propose that TNF-α plays a dual role in HIV-1 encephalopathy, enhancing viral replication by activated microglia and damaging oligodendrocytes. Thus, TNF-α inhibitors may alleviate some ofthe neurological manifestations of acquired immunodeficiency syndrome.     

Expression of brain-derived neurotrophic factor protein in activated microglia of human immunodeficiency virus type 1 encephalitis

The role of neurotrophic factors and their therapeutic potential have been investigated in various neurodegenerative disorders. In neurodegeneration associated with human immunodeficiency virus (HIV) infection, neuronal function and survival may be affected by abnormal neurotrophic regulation involving HIV-infected microglia and reactive astrocytes. To characterize the cellular localization of brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor, trkB, proteins in HIV-1 encephalitis, we examined post-mortem brains from patients with acquired immunodeficiency syndrome and brains from non-HIV-infected controls. Using double immunofluorescent confocal microscopy, we found that BDNF immunoreactivity was distributed in neocortical neuronal perikarya and neuritic processes, while in the striatum only neurites were BDNF-immunoreactive. Additionally, the striatum with HIV infection was characterized by BDNF immunoreactivity in infiltrating activated microglia/macrophages and multinucleated giant cells. Catalytic trkB receptor immunoreactivity was observed in neuronal perikarya in the neocortex and striatum, as well as in reactive astrocytes within HIV-infected regions. Our findings suggest that expression of BDNF by activated microglia in HIV-1 encephalitis may affect neuronal survival and astroglial response through corresponding trkB receptors.     

AIDS dementia complex and HIV-1 brain infection: Clinical-virological correlations

To evaluate the presence and distribution of central nervous system infection by human immunodeficiency virus type 1 (HIV-1), we used immunohistochemical methods to map the HIV-1 p24 core protein in the brains of 55 autopsied patients with acquired immunodeficiency syndrome (AIDS). In a subset of 40 of these patients who had undergone antemortem neurological evaluation of the AIDS dementia complex (ADC), we analyzed the relation between the severities of the viral infection and clinical dysfunction. Viral antigen was detected in macrophages and cells with morphological and immunohistochemical characteristics of microglia as well as multinucleated cells. The distribution of antigen-positive cells preferentially involved certain deep brain structures, especially the globus pallidus, other basal ganglia nuclei, and the central white matter. Overall, the presence and frequency of infected cells were highly correlated with the histological findings of multinucleated-cell encephalitis and in general with the clinical ADC stage. However, infection was often more limited than might be “anticipated” from the severity of patients' clinical dysfunction: Only 61% of patients with at least ADC stage 1 had detectable antigen and of these only approximately 30% of the brain sections were antigen positive. These results suggest a pathogenetic model of ADC where virus- or cell-coded toxins amplify the effect of limited brain infection.     

Functional expression of the seven-transmembrane HIV-1 co-receptor APJ in neural cells

APJ is a recently described seven-transmembrane (7TM) receptor that is abundantly expressed in the central nervous system (CNS). This suggests an important role for APJ in neural development and/or function, but neither its cellular distribution nor its function have been defined. APJ can also serve as a co-receptor with CD4 for fusion and infection by some strains of human immunodeficiency virus (HIV-1) in vitro, suggesting a role in HIV neuropathogenesis if it were expressed on CD4-positive CNS cells. To address this, we examined APJ expression in cultured neurons, astrocytes, oligodendrocytes, microglia and monocyte-derived macrophages utilizing both immunocytochemical staining with a polyclonal anti-APJ antibody and RT - PCR. We also analyzed the ability of a recently identified APJ peptide ligand, apelin, to induce calcium elevations in cultured neural cells. APJ was expressed at a high level in neurons and oligodendrocytes, and at lower levels in astrocytes. In contrast, APJ was not expressed in either primary microglia or monocyte-derived macrophages. Several forms of the APJ peptide ligand induced calcium elevations in neurons. Thus, APJ is selectively expressed in certain CNS cell types and mediates intracellular signals in neurons, suggesting that APJ may normally play a role in signaling in the CNS. However, the absence of APJ expression in microglia and macrophages, the prinicpal CD4-positive cell types in the brain, indicates that APJ is unlikely to mediate HIV-1 infection in the CNS.     

Examination of brains of AIDS cases for human immunodeficiency virus and human cytomegalovirus nucleic acids.

The role of direct virus infection as a determining factor in acquired immunodeficiency syndrome (AIDS) dementia was investigated using in situ hybridisation for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV). Four of the five AIDS dementia patients in this series demonstrated HIV infected cells distributed in widely different parts of the brain, but only one case showed HCMV infected cells. The greater abundance of HIV was in subcortical white matter in nodular areas consisting of monocyte/macrophage infiltrates. The cells were occasionally arranged as a multinucleated syncitium. In two cases, a few large cells with the appearance of neurons were positive for HIV hybridisation. By appropriate treatment with ribonuclease, it was shown that hybridisation was primarily to HIV RNA. HCMV infected cells were observed in small numbers in only one of the positive cases, suggesting that HCMV is not a determining factor in AIDS dementia. HCMV positive cells were located in the grey matter, with an appearance suggestive of neurons. Cells expressing the MHC-class II antigen HLA-DR, a marker of reactive microglia and macrophages, were observed to be extensive in affected brain sections in the one case examined. These cells were present in greater number than HIV infected cells. In this case, extensive numbers of HIV infected cells were noticed along the peripheral margin of the substantia innominata. This could indicate infection in this case of a critical brain region from the cerebrospinal fluid.     

AIDS-associated progressive multifocal leukoencephalopathy (PML): comparison to non-AIDS PML with in situ hybridization and immunohistochemistry

We studied brain sections from 10 patients with the acquired immunodeficiency syndrome (AIDS) and progressive multifocal leukoencephalopathy (PML) by in situ hybridization with a biotin-labeled JC virus (JCV) DNA probe and by immunohistochemistry using antibody against the JCV capsid antigen. We compared the results with brain sections studied in the same fashion from 10 PML patients without AIDS. The pathology of JCV infection in AIDS was similar to non-AIDS PML except for minor differences in degree. AIDS-associated pathologic material showed a greater tendency toward necrosis and a higher density of JCV-infected cells. Replication of JCV was restricted to glial cells in all tissue studied. Bizarre astrocytes were less frequent in the AIDS patients, and perivascular inflammatory cells were more frequent. We could not demonstrate JCV in macrophages or microglial cells known to harbor HIV infection. In situ hybridization with nonradioactive probes serves as a useful technique for the confirmation of PML in AIDS.     

A novel action of minocycline: Inhibition of human immunodeficiency virus type 1 infection in microglia

Human immunodeficiency virus type 1 (HIV-1) infection of the brain produces a characteristic disease called acquired immunodeficiency syndrome (AIDS) dementia in which productive infection and inflammatory activation of microglia and macrophages play a central role. In this report, the authors demonstrate that minocycline (MC), a second-generation tetracycline with proven safety and penetration to the central nervous system, potently inhibited viral production from microglia. Inhibition of viral release was sustained through the entire course of infection and even when the drug exposure was limited to the first day of infection. Minocycline was effective even at low viral doses, and against R5- and X4R5-HIV, as well as in single-cycle reporter virus assays. Electrophoretic mobility shift analysis showed that minocycline inhibited nuclear factor (NF)-kappaB activation in microglia. HIV-1 long terminal repeat (LTR)-promoter activity in U38 cells was also inhibited. These results, combined with recently demonstrated in vivo anti-inflammatory effects of MC on microglia, suggest a potential utility for MC as an effective adjunct therapy for AIDS dementia.     

Oligodendrocyte reactions and cell proliferation markers in human demyelinating diseases

We have carried out immunocytochemical reactions using antibodies to markers of oligodendrocytes, astrocytes, microglia and proliferating cells (PCNA) in sections of human brain in a variety of demyelinating conditions and human immunodeficiency virus (HIV) infection. In the acute phases of demyelinating diseases we found marked reactive changes in oligodendrocytes with hyperplasia and an increased cytoplasmic reaction using antibodies to enzymes involved in myelin formation. Proliferative responses were implied by the hyperplasia and the common finding of clusters of two or three adjacent oligodendrocytes at sites of acute myelin dam age. This was borne out by studies using the PCNA antibody which gave negative reactions in normal brain but positive reactions in acute demyelination. Double staining for PCNA and cell markers showed that cells that had entered the cell proliferation cycle were to be found among astrocytes, microglia/macrophages and oligodendrocytes. In chronic demyelinating conditions, numbers of oligodendrocytes were reduced and cells in the proliferative cycle were not present, suggesting that the reactive potential of oligodendrocytes or their precursors and their capacity to respond to demyelination is limited.     

Human immunodeficiency virus type 1 infection of the nervous system: Pathogenetic mechanisms

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system is associated with characteristic virological, clinical, and neuropathological findings in adults and children. Productive infection in the brain and spinal cord occurs in blood-derived macrophages, resident microglia, and multinucleated giant cells. Previous work implicated indirect mechanisms for neurotoxicity by HIV-1 gene products or by factors secreted from HIV-1-infected macrophages. However, this cannot explain the paradox between the small numbers of infected cells and the widespread tissue pathology. Based on recent studies from our laboratories, we suggest that HIV-1-infected macrophages can initiate neurotoxicity, which is then amplified through cell-to-cell interactions with astrocytes. Macrophageastrocyte interactions produce cytokines tumor necrosis factor-α and interleukin-1b? and arachidonic metabolites that cause astroglial proliferation and neuronal injury. Inevitably, the astrogliosis serves to amplify these cellular processes while brain infection maintains itself in macrophage and microglia and possibly in astrocytes (by restricted infection). These findings, taken together, provide fresh insights into how low numbers of productively infected cells could elicit progressive and devastating neurological impairment during HIV-1 disease, and suggest therapeutic strategies to interrupt the pathological process.     

Stages of restricted HIV-1 infection in astrocyte cultures derived from human fetal brain tissue

The predominant cell types infected by HIV-1 in AIDS associated encephalopathy are cells of the macrophage/microglial lineage. There has been consistent evidence, however, that astrocytes also become infected although not at the same frequency or level of multiplication as microglial cells. HIV-1 antigens and/or nucleic acid have been identified in astrocytes in brain autopsy tissue from both adult and pediatric AIDS cases. In cell cultures, HIV-1 infection of astrocytes results in an initial productive but non-cytopathogenic infection that diminishes to a viral persistence or latent state. Understanding the nature of HIV-1 infection of astrocytes, which represents the largest population of cells in the brain, will contribute to the understanding of AIDS encephalopathy and the dementia that occurs in nearly one-quarter of all AIDS patients.