HTLV-III infection in brains of children and adults with AIDS encephalopathy.

Unexplained debilitating dementia or encephalopathy occurs frequently in adults and children with the acquired immune deficiency syndrome (AIDS). Brains from 15 individuals with AIDS and encephalopathy were examined by Southern analysis and in situ hybridization for the presence of human T-cell leukemia (lymphotropic) virus type III (HTLV-III), the virus believed to be the causative agent of AIDS. HTLV-III DNA was detected in the brains of five patients, and viral-specific RNA was detected in four of these. In view of these findings and the recent demonstration of morphologic and genetic relatedness between HTLV-III and visna virus, a lentivirus that causes a chronic degenerative neurologic disease in sheep, HTLV-III should be evaluated further as a possible cause of AIDS encephalopathy.

Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA.

Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.

Diffusion alterations in corpus callosum of patients with HIV.

BACKGROUND AND PURPOSE: Diffusion alterations have been identified in the corpus callosum and frontal white matter of patients infected with human immunodeficiency virus (HIV), though the relevance of these findings to cognitive deterioration has not yet been determined. This study tested the hypothesis that diffusion tensor imaging can detect tissue status alterations in these regions in cognitively impaired patients infected with HIV and the acquired measurements correlate with the severity of cognitive impairment. METHODS: Fractional anisotropy (FA) and mean diffusivity (MD) were determined for corpus callosum (genu and splenium) and frontal white matter (FWM). The DTI measurements were compared in 11 HIV and 11 control participants. Patterns of relationship were examined with cognitive status measures from concurrent neurologic and neuropsychologic evaluations. RESULTS: FA values for the splenium were significantly reduced in the patients infected with HIV and correlated with dementia severity and deficits in motor speed. MD values for the splenium were significantly increased in the patients infected with HIV and correlated with deficits in motor speed. FA measurements were also significantly correlated with performance on visual memory (genu), visuoconstruction (FWM), and verbal memory (FWM) tasks. CONCLUSION: Diffusion abnormalities were identified in the splenium of the corpus callosum in patients infected with HIV, and these alterations were associated with dementia severity and motor speed losses. In vivo assessment of callosal integrity by using quantitative neuroimaging may have potential utility as a marker of brain injury in patients infected with HIV.

Bone marrow diffusion measures correlate with dementia severity in HIV patients.

BACKGROUND AND PURPOSE: Escalation in monocyte trafficking from the bone marrow into the brain may play a critical role in central nervous system injury and cognitive deterioration in patients with HIV infection. This study tested the hypothesis that the mean diffusivity is sensitive to marrow changes in HIV patients and that these quantitative imaging measurements correlate with the severity of dementia. METHODS: The mean diffusivity (MD), determined for clival and calvarial marrow regions, was compared in 11 HIV-infected patients and 9 control subjects. The imaging measurements were also evaluated for relationships with dementia severity and markers of disease progression (CD4 and viral load in plasma). RESULTS: The MD was significantly reduced in both clival and calvarial marrow in HIV-infected patients (P =.006). Diffusion measurements for clival (P =.02) and for calvarial (P =.03) regions were significantly correlated with the severity of dementia. CONCLUSION: The results of this investigation support the utility of diffusion strategies for monitoring the marrow and provide further evidence of a relationship between marrow status changes and neurologic progression in HIV patients.

HIV-1-induced neuronal injury in the developing brain.

HIV-1 infection of the nervous system causes neuronal injury and death, resulting in cognitive, motor, and behavioral dysfunction in both adults and children. In infants a characteristic feature of HIV-1 infection is impaired brain growth resulting in secondary microcephaly with onset between 2 and 4 months of age. This post-natal period of brain development is particularly vulnerable to excitotoxic neuronal injury due to the active synaptogenesis and pruning that takes place at this age associated with over-expression of excitatory amino acid (EAA) receptors. HIV-1 infection of brain microglia and perivascular macrophages results in chronic inflammation manifest pathologically as diffuse microglial activation and reactive astrogliosis. Several inflammatory products of activated microglia, including tumor necrosis factor alpha (TNF-alpha) and platelet-activating factor (PAF) have been shown to act as neuronal toxins. This toxic effect can be antagonized by blocking NMDA (or AMPA) glutamate receptors, suggesting that (weak) excitotoxicity leads to oxidative stress, neuronal injury, and apoptosis. HIV-1 infection and chronic inflammation may also contribute disruption of the blood-brain barrier and could result in further entry into the CNS of toxic viral or cellular products or additional HIV-1-infected cells. We hypothesize that prolonged microglial activation during HIV-1 infection underlies the neuronal injury and impaired brain growth in affected infants. Further investigation of the interaction between HIV-1-infected/activated microglia and developing neurons seems warranted. The current understanding of HIV neuropathogenesis implies that therapeutic strategies should target the sustained immune activation in microglia, attempt to repair the integrity of the blood-brain barrier, and provide "neuroprotection" from excitotoxic neuronal injury.

Human antibody response to a strain-specific HIV-1 gp120 epitope associated with cell fusion inhibition.

PEPSCAN analysis, performed using 536 overlapping nonapeptides derived from the HTLV-III B nucleotide sequence of the region encoding the external envelope protein of 120 kDa (gp120), identified in the V3 region of gp120 a major binding site for antibodies of HIV-1-infected humans. The minimal amino acid sequence of this antibody binding site was demonstrated by multiple length scanning to be five to eight amino acids in length: (G)PGRAF(VT), i.e. amino acids 312-319. A peptide (Neu 21) containing this binding site for human antibodies (KSIRIQRGPGRAFVTIG) was synthesized and shown to induce HTLV-III B cell fusion-inhibiting antibodies in rabbits and mice. Antibodies binding to this HTLV-III B/LAV-1-specific peptide were shown to be primarily of the IgG 1 subclass, appeared within 6 months after HIV-1 antibody seroconversion in six out of 14 men studied, and persisted throughout the follow-up period of 10-24 months. The other eight seroconverting men did not develop antibodies to Neu 21 during the observation period. The appearance of antibodies to Neu 21 paralleled the capacity of the serum to inhibit HTLV-III B in cell fusion. HIV-1-infected men with Kaposi's sarcoma exhibited a similar frequency of antibodies to the synthetic peptide Neu 21 (14 out of 39, 36%) as asymptomatic HIV-1-infected men (112 out of 319, 35%). Adults with Pneumocystis carinii pneumonia had a significantly lower frequency (11 out of 78, 14%) of antibodies to Neu 21. Similarly, a low prevalence of antibodies to Neu 21 (8 out of 43, 19%) was observed among symptomatic HIV-1-infected children.

Human fetal astrocytes as an ex vivo gene therapy vehicle for delivering biologically active nerve growth factor.

The therapeutic use of neurotrophic factors for neurodegenerative diseases is promising, however, optimal methods for continuous delivery of these substances to the human central nervous system (CNS) remains problematic. One approach would be to graft genetically engineered human cells that continuously secrete high levels of a biologically produced and processed neurotrophic factor. This ex vivo gene therapy approach has worked well in animal models of neurodegenerative diseases using a variety of nonneuronal cell types to deliver the transgene. In our studies, we have been investigating the potential of astrocytes, a cell type normally present in the CNS, as a vehicle for ex vivo gene therapy. Here, we demonstrate that astrocytes in the human fetal cortex can be isolated and efficiently infected with an amphotropic retrovirus harboring mouse beta-nerve growth factor (NGF). These transduced astrocytes express high levels of NGF mRNA and secrete bioactive NGF protein as demonstrated by stimulation of neurite outgrowth from adrenal chromaffin cells. NGF ELISA showed that these astrocytes secrete NGF protein at a rate of 41 ng/day per 10(5) cells after 2 weeks in vitro, whereas NGF is undetectable in medium conditioned by normal astrocytes. These data suggest that human fetal astrocytes can be used for delivering biologically produced neurotrophic factors to the human CNS.

Ultrastructural morphology and intracellular production of human immunodeficiency virus (HIV) in brain.

This is a comparative ultrastructural study of human immunodeficiency virus (HIV) particles in infected H9 lymphocyte cultures and in the brain of a six-year-old boy with acquired immunodeficiency syndrome (AIDS) encephalopathy. Viral particles in the cultures and the brain were of various sizes and shapes; particles ranged from 70 to over 160 nm in diameter, with a variable position of dense nucleoids and less dense core shells. In the brain, viral particles were located free in the cytoplasm of both multinucleated giant cells and mononuclear macrophage-like cells. There was intracellular budding of HIV particles from unidentified membranes, yielding intracellular immature or recently budded particles, with crescentic densities. By contrast, HIV particles in the infected H9 lymphocytes were not free in the cytoplasm but were instead located either extracellularly or in intracellular vacuoles. A small percentage of cells in the cultures were surrounded by immature particles only. Production (replication) of HIV occurred within infected macrophage-like cells in the brain of the child.

Excitotoxicity and dopaminergic dysfunction in the acquired immunodeficiency syndrome dementia complex. Therapeutic implications.

Human immunodeficiency virus infection is frequently complicated by a syndrome of central nervous system dysfunction known as the acquired immunodeficiency syndrome dementia complex (ADC). The ADC is characterized by abnormalities in cognition, motor performance, and behavior, and it produces serious morbidity in a significant number of patients with acquired immunodeficiency syndrome. The pathogenesis of ADC is unclear, but appears to be caused by the human immunodeficiency virus itself, rather than by a secondary opportunistic process. Herein, we review the data regarding the pathogenesis of ADC and hypothesize a mechanism involving excitotoxicity and dopaminergic dysfunction. N-methyl-D-aspartate receptor antagonists may be of therapeutic benefit, as these agents may both limit glutamate-mediated neuronal dysfunction and improve dopaminergic neuronal function.

Human immunodeficiency virus type 1 antigen in cerebrospinal fluid. Correlation with clinical neurologic status.

Human immunodeficiency virus type-1 (HIV-1) antigen was assayed in paired serum/cerebrospinal fluid (CSF) specimen from 85 adults and 58 children with acquired immunodeficiency syndrome and was compared with clinical neurological status. A quantitative comparison of HIV-1 antigen levels in matched serum and CSF specimens indicated that HIV-1 antigen expression in these compartments is independent and is correlated with acquired immunodeficiency syndrome dementia complex in adults and progressive encephalopathy in children. In a longitudinal study (n = 47), 16 patients tested positive for HIV-1 antigen in the CSF before (n = 2) or coincident (n = 14) with neurological deterioration. Six patients who tested positive for HIV-1 antigen in the CSF remained neurologically normal for a median duration of follow-up of 11 months. Six of 25 patients who tested negative for HIV-1 antigen in the CSF, subsequently showed neurological deterioration. These data indicate that HIV-1 antigen expression in the CSF is not useful in predicting neurological deterioration.

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.

Randomized trial of the platelet-activating factor antagonist lexipafant in HIV-associated cognitive impairment. Neurological AIDS Research Consortium.

OBJECTIVE: To assess the safety and tolerability of lexipafant in HIV-associated cognitive impairment. BACKGROUND: Cognitive impairment is the most common neurologic complication of advanced HIV-1 infection. There is evidence that a variety of inflammatory mediators, including platelet-activating factor (PAF), may contribute to neuronal injury. We hypothesized that lexipafant, a PAF antagonist, might improve cognitive dysfunction in HIV-infected people. METHODS: We conducted a randomized, double-blind, placebo-controlled clinical trial to assess the safety and tolerability of lexipafant 500 mg/day. The primary outcome measure for tolerability was the ability to complete the study on the originally assigned dosage of medication. Thirty patients with cognitive impairment were enrolled. RESULTS: Lexipafant was safe and well tolerated. Ninety-three percent in the placebo group and 88% in the lexipafant group completed the study at the originally assigned dosage. Trends toward improvement were seen in neuropsychological performance, especially verbal memory, in the lexipafant treatment group. CONCLUSIONS: This study shows that lexipafant, the first PAF antagonist used in HIV-associated cognitive impairment, is a safe and well tolerated compound. The observed trends toward improvement in neuropsychological test scores warrant the pursuit of a larger and longer efficacy trial to assess the impact of lexipafant on cognitive performance.

X-linked female band heterotopia-male lissencephaly syndrome.

We report a family with band heterotopia in a mother and daughter and lissencephaly in a son (X-linked inheritance pattern). Postmortem examination of the boy revealed classical lissencephaly and, among other findings, simplified and discontinuous inferior olives without inferior olivary heterotopia. The absence of inferior olivary heterotopia may distinguish X-linked lissencephaly from other conditions with classic lissencephaly such as Miller-Dieker syndrome.

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.

Proteasome blockers inhibit TNF-alpha release by lipopolysaccharide stimulated macrophages and microglia: implications for HIV-1 dementia.

HIV-1 infection of the central nervous system can cause severe neurologic disease although only microglial cells and brain macrophages are susceptible to productive viral infection. Substances secreted by infected cells are thought to cause disease indirectly. Tumor necrosis factor alpha (TNF-alpha) is one candidate neurotoxin and is upregulated during HIV-1 infection of the brain, likely via activation of the transcription factor NF-kappaB. We used the proteasome inhibitors, MG132 and ALLN (N-acetyl-Leu-Leu-Norleucinal), to inhibit NF-kappaB activation in primary human fetal microglia (PHFM) and primary monocyte derived-macrophages, and showed that they could block TNF-alpha release stimulated by lipopolysaccharide (LPS) or TNF-alpha. In addition, we performed electrophoretic mobility shift analysis and determined that in microglia, the p50/p65 heterodimer of NF-kappaB is activated by LPS stimulation, and is inhibited by MG132. Thus, blockade of NF-kappaB activation in microglia in vitro can decrease production of TNF-alpha and may prove to be a novel strategy for treating HIV-1 dementia.

Simultaneous in situ detection of apoptosis and necrosis in monolayer cultures by TUNEL and trypan blue staining.

A method for simultaneously detecting membrane permeability (characteristic of necrosis) and DNA fragmentation (characteristic of apoptosis) is described. By combining a common dye-exclusion method (Trypan Blue) with a commercially available terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) labeling kit, we have succeeded in developing a novel methodology for obtaining permanently mounted slides of monolayer cell cultures double-labeled for DNA fragmentation and cell lysis. This method should facilitate in situ studies of cell death by allowing for a more accurate quantification of total toxicity in monolayer cell cultures and perhaps further enhance our understanding of the different mechanisms of cell death as well.

Neurologic manifestations of human immunodeficiency virus infection in children.

This report describes the neurologic manifestations of 36 children with human immunodeficiency virus (HIV) infection. In this cohort, in 16 of 21 children with acquired immunodeficiency syndrome (AIDS), three of 12 children with AIDS-related complex, and one of three asymptomatic seropositive children, a progressive encephalopathy developed. Neurologic signs were often detected early but tended to worsen coincident with progression of the immunodeficiency. The presence of progressive encephalopathy correlated with the absence of serum neutralizing antibodies to HIV and with a poor, usually fatal, outcome. The incubation period from initial HIV infection in the perinatal period to the onset of progressive encephalopathy varied from 2 months to 5 years. Intrablood-brain barrier synthesis of HIV-specific antibodies was demonstrated in eight of 14 children with AIDS and AIDS-related complex, indicating active brain infection with HIV. In three cases this was unassociated with progressive neurologic signs. Unique neuropathologic findings in children who died with HIV infection further suggest that the progressive encephalopathy is the result of primary and persistent infection of the brain with this retrovirus. These findings broaden the spectrum of HIV infection in children and have important implications for the development of antiviral therapy.

Persistent human immunodeficiency virus type 1 antigenemia in children correlates with disease progression.

In a longitudinal study, human immunodeficiency virus type 1 (HIV-1) antigen (HIV-Ag) was measured in serum specimens from 54 children with HIV-1 infection followed for a median duration of 17 months. The persistent detection of free HIV-Ag in a group of 25 children was associated with clinical deterioration in 22 (88%) and a mortality of 52%, whereas the persistent nondetection of free HIV-Ag in a group of 18 children was associated with clinical deterioration in five (28%) and a mortality of 11% during the period of observation. Nine children had transient HIV-1 antigenemia and two children converted from HIV-Ag negative to positive during the study. Free HIV-Ag levels varied inversely with antibody reactivity to viral core proteins p24 and p17 determined by Western immunoblot, suggesting either the formation of immune complexes or a balance between viral expression and the host immune response. Five mother-infant pairs were studied for HIV-Ag expression in the perinatal period. In three of these pairs, both mother and infant were HIV-Ag negative, in one pair the mother had high levels of HIV-Ag and the infant was HIV-Ag negative. In the remaining mother-infant pair, the neonate became HIV-Ag positive but the mother was HIV-Ag negative prepartum and postpartum. These data suggest that HIV-Ag probably does not cross the placenta and that the detection of free HIV-Ag in the offspring of a HIV-1 infected mother most likely indicates viral infection.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Coreceptor ligand inhibition of fetal brain cell infection by HIV type 1.

The capacity of a panel of HIV-1 isolates to infect primary mixed fetal brain cell cultures was estimated and their sensitivity to inhibition by a range of coreceptor ligands assessed. Our results show that (1) HIV-1 strains that predominantly use CCR5 or only CXCR4 are able to infect microglia in primary brain cell cultures, and (2) ligands to these two coreceptors can inhibit brain cell infection. CCR5 ligands (including AOP-RANTES, a potent inhibitor of CCR5-dependent infection), however, blocked infection only weakly, raising the possibility that alternative unidentified coreceptors are also used. Interestingly, vMIP-II, a chemokine encoded by the Kaposi sarcoma-associated herpes virus (KSHV), reduced brain cell infection by all HIV-1 strains tested, including both R5 and X4 viruses. Our results therefore indicate that novel drugs targeted to the major HIV-1 coreceptors will influence HIV replication in the brain, if they cross the blood-brain barrier.