Leroy R. Sharer

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 immunocytoehemistry 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.

Immunopathology of secondary-progressive multiple sclerosis

Twenty‐three plaques obtained at early autopsy from 2 patients with secondary‐progressive multiple sclerosis were examined immunohistochemically for microglia/macrophages, and for immunoglobulins and components of activated complement. Most of the lesions examined in both cases exhibited evidence of low‐grade active demyelination of an unusual type (frustrated phagocytosis) in periplaque white matter. This included linear groups of microglia engaging short segments of disrupted myelin that were associated with deposits of C3d, an opsonin formed during complement activation. Similar microglia/C3d/myelin profiles were not observed in newly forming lesions in cases of acute multiple sclerosis or other central white matter diseases. As C3d coupling is known to increase the immunogenicity of potential antigens enormously, present findings point to disrupted myelin close to plaques as a possible source of the putative multiple sclerosis antigen. Ongoing myelin destruction found in a high proportion of old, established plaques was surprising. It suggests that slowly expanding lesions (progressive plaques), in which ongoing myelin breakdown occurs in the absence of florid perivascular cell cuffing or other histological signs of acute inflammation, contribute to disease progression in cases of secondary‐progressive multiple sclerosis.

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.

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.

Neuronal Fractalkine Expression in HIV-1 Encephalitis: Roles for Macrophage Recruitment and Neuroprotection in the Central Nervous System

HIV-1 infection of the brain results in chronic inflammation, contributing to the neuropathogenesis of HIV-1 associated neurologic disease. HIV-1-infected mononuclear phagocytes (MP) present in inflammatory infiltrates produce neurotoxins that mediate inflammation, dysfunction, and neuronal apoptosis. Neurologic disease is correlated with the relative number of MP in and around inflammatory infiltrates and not viral burden. It is unclear whether these cells also play a neuroprotective role. We show that the chemokine, fractalkine (FKN), is markedly up-regulated in neurons and neuropil in brain tissue from pediatric patients with HIV-1 encephalitis (HIVE) compared with those without HIVE, or that were HIV-1 seronegative. FKN receptors are expressed on both neurons and microglia in patients with HIVE. These receptors are localized to cytoplasmic structures which are characterized by a vesicular appearance in neurons which may be in cell-to-cell contact with MPs. FKN colocalizes with glutamate in these neurons. Similar findings are observed in brain tissue from an adult patient with HIVE. FKN is able to potently induce the migration of primary human monocytes across an endothelial cell/primary human fetal astrocyte trans-well bilayer, and is neuroprotective to cultured neurons when coadministered with either the HIV-1 neurotoxin platelet activating factor (PAF) or the regulatory HIV-1 gene product Tat. Thus focal inflammation in brain tissue with HIVE may up-regulate neuronal FKN levels, which in turn may be a neuroimmune modulator recruiting peripheral macrophages into the brain, and in a paracrine fashion protecting glutamatergic neurons.

Detection of HIV-1 DNA in Microglia/ Macrophages, Astrocytes and Neurons Isolated from Brain Tissue with HIV-1 Encephalitis by Laser Capture Microdissection

In HIV‐1 encephalitis, HIV‐1 replicates predominantly in macrophages and microglia. Astrocytes also carry HIV‐1, but the infection of oligodendrocytes and neurons is debated. In this study we examined the presence of HIV‐1 DNA in different brain cell types in 6 paraffin embedded, archival post‐mortem pediatric and adult brain tissues with HIV‐1 encephalitis by Laser Capture Microdissection (LCM). Sections from frontal cortex and basal ganglia were stained by immunohistochemistry for CD68 (microglia), GFAP (astrocytes), MAP2 (neurons), and p24 (HIV‐1 positive cells) and different cell types were microdissected by LCM. Individual cells or pools of same type of cells were lysed, the cell lysates were subjected to PCR using HIV‐1 gag SK38/SK39 primers, and presence of HIV‐1 DNAwas confirmed by Southern blotting. HIV‐1 gag DNAwas consistently detected by this procedure in the frontal cortex and basal ganglia in 1 to 20 p24 HIV‐1 capsid positive cells, and in pools of 50 to 100 microglia/macrophage cells, 100 to 200 astrocytes, and 100 to 200 neurons in HIV‐1 positive cases but not in HIV‐1 negative controls. These findings suggest that in addition to microglia, the infection of astro‐cytes and neurons by HIV‐1 may contribute to the development of HIV‐1 disease in the brain.

ARTERIOPATHY IN CHILDREN WITH ACQUIRED IMMUNE DEFICIENCY SYNDROME

Pathologic features of the arteries of different organs (heart, lungs, kidneys, spleen, intestine, brain) seen at autopsy in 6 children with acquired immune deficiency syndrome (AIDS) are described. Small and medium-sized arteries, which were the most commonly involved, showed intimal fibrosis with fragmentation of elastic tissue, fibrosis and calcification of media with variable luminal narrowing, and a vasculitis or perivasculitis that was seen only in the brain in association with AIDS encephalopathy. In 1 case aneurysms of the right coronary artery with thrombosis and myocardial infarction were seen. Vascular inflammation, seen only in the brain, may be related to the agent associated with AIDS encephalopathy. The fibrocalcific arterial lesions most closely resemble idiopathic arterial calcification of infancy, but because of differences in age incidence, clinicopathologic and immunologic features, and the size and distribution of the involved arteries, the arterial lesions of pediatric AIDS appear to constitute a distinctive arteriopathy. Infection, secondary to immunodeficiency and resulting in increased exposure to endogenous and exogenous elastases, may be the pathogenesis. Luminal narrowing caused by arterial lesions may play a contributory role in the pathogenesis of the atrophy, cell depletion, scarring, and necrosis or infarction found in organs of children with AIDS. Pediatricians should be alerted to the possibility of arterial involvement in pediatrics AIDS.

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: Potential role in neuropathogenesis

Neurodegeneration and dementia caused by human immunodeficiency virus type 1 (HIV-1) infection of the brain are common complications of acquired immunodeficiency syndrome (AIDS). Introduction of highly active antiretroviral therapy (HAART) reduced the incidence of HIV-1-associated dementia, but so far had no effect on the high frequency of milder neurological disorders caused by HIV-1. This indicates that some neuropathogenic processes persist during limited HIV-1 replication in the central nervous system (CNS). The authors are evaluating the hypothesis that interaction of HIV-1 with astrocytes, which bind HIV-1 but support limited productive HIV-1 infection, may contribute to these processes by disrupting astrocyte functions that are important for neuronal activity or survival. Using laser-capture microdissection on brain tissue samples from HIV-1-infected individuals, we found that HIV-1 DNA can be detected in up to 1% of cortical and basal ganglia astrocytes, thus confirming HIV-1 infection in astrocytes from symptomatic patients. Using rapid subtraction hybridization, the authors cloned and identified 25 messenger RNAs in primary human fetal astrocytes either up-regulated or down-regulated by native HIV-1 infection or exposure to gp120 in vitro. Extending this approach to gene microarray analysis using Affymetrix U133A/B gene chips, the authors determined that HIV-1 alters globally and significantly the overall program of gene expression in astrocytes, including changes in transcripts coding for cytokines, G-coupled protein receptors, transcription factors, and others. Focusing on a specific astrocyte function relevant to neuropathogenesis, the authors showed that exposure of astrocytes to HIV-1 or gp120 in vitro impairs the ability of the cells to transport l-glutamate and the authors related this defect to transcriptional inhibition of the EAAT2 glutamate transporter gene. These findings define new pathways through which HIV-1 may contribute to neuropathogenesis under conditions of limited virus replication in the brain.

Cytopathologic and Neurochemical Correlates of Progression to Motor/Cognitive Impairment in SIV-Infected Rhesus Monkeys

Neurochemical, pathologic, virologic, and histochemical correlates of simian immunodeficiency virus (SIV)- associated central nervous system (CNS) dysfunction were assessed serially or at necropsy in rhesus monkeys that exhibited motor and cognitive deficits after SIV infection. Some infected monkeys presented with signs of acquired immunodeficiency disease (AIDS) at the time of sacrifice. Seven of eight animals exhibited motor skill impairment which was associated with elevated quinolinic acid in cerebrospinal fluid (CSF). Examination of the brains revealed diffuse increases in glial fibrillary acidic protein immunoreacti vity in cerebral cortex in all animals, regardless of evidence of immunodeficiency disease. Reactive astrogliosis preceded or was coincident with the onset of neuropsychological impairments. Virus rescue from CSF of six of eight infected animals showed that one of three animals with AIDS and none of three animals without AIDS at necropsy had virus rescue-positive CSF. Multinucleated giant cells were seen in the brain of only one animal with end-stage AIDS and high systemic virus burden at death. Neither systemic nor CNS virus burden was associated with the onset of CNS dysfunction. SIV-associated motor/cognitive impairment is associated with subtle, widespread changes in CNS cytology and neurochemistry, rather than with large increases in brain virus burden or widespread virus-associated brain lesions.

A randomized clinical trial of CPI-1189 for HIV-associated cognitive-motor impairment.

Background: CPI-1189 is a compound with antioxidant properties that blocks tumor necrosis factor-α (TNFα) effects in animal models. It has neuroprotective properties in model systems for HIV-associated neurotoxicity and thus is a candidate for neuroprotective therapy in humans with HIV-associated CNS disease. Objective: To assess the tolerability and safety of CPI-1189 in treating HIV-associated cognitive–motor impairment. Methods: Sixty-four subjects with mild to moderate HIV-associated cognitive–motor impairment were randomized to receive either placebo or 50 or 100 mg daily of CPI-1189 in addition to optimal HIV therapy. Subjects were followed prospectively in a double-masked study for 10 weeks. The primary assessment was tolerability and safety of the compound. Secondary objectives examined neuropsychological and functional change associated with this treatment. Results: The study compound was well tolerated, with 91% of CPI-1189-treated subjects and 76% of placebo-treated subjects completing the trial. Skin rash was seen equally in placebo and active arms, but the only study withdrawals due to skin rash occurred in CPI-1189-treated subjects (n = 2). One subject developed a cataract on drug (100 mg/day). CD4 lymphocyte counts and plasma HIV viral load remained stable in all groups throughout the trial. No significant treatment effects were observed on the change in composite Z-scores for eight neuropsychologic measures (NPZ-8). The Grooved Pegboard Test (nondominant) showed improved performance with CPI-1189 at 100 mg/day (p = 0.01), but no other neuropsychometric or functional measures demonstrated significant improvement. Conclusions: CPI-1189 was well tolerated in HIV subjects with cognitive–motor disorder. This study was not powered to conclusively determine efficacy and showed no consistent treatment-associated improvement in cognitive or functional measures.