Avindra Nath

The Tat Protein of HIV-1 Induces Tumor Necrosis Factor-α Production IMPLICATIONS FOR HIV-1-ASSOCIATED NEUROLOGICAL DISEASES

Human immunodeficiency virus (HIV) infection may cause a dementing illness. HIV-mediated dementia is clinically and pathologically correlated with the infiltration of activated macrophages and elevated levels of tumor necrosis factor (TNF)-α, both of which occur in an environment of small numbers of infected cells. We examined the possibility that HIV protein Tat, which is released extracellularly from infected cells, may induce the production of TNF-α. Tat induced TNF-α mRNA and protein production dose-dependently, primarily in macrophages but also in astrocytic cells. The TNF-α induction was NF-κB-dependent and could be eliminated by inhibiting protein kinase A or protein tyrosine kinase activity. In addition, Tat-induced TNF-α release was also linked to phospholipase C activation. However, Tat effects were independent of protein kinase C. These observations suggest that Tat may provide an important link between HIV and macrophage/glial cell activation and suggest new therapeutic approaches for HIV dementia.

Human immunodeficiency virus type 1 Tat protein induces death by apoptosis in primary human neuron cultures

Neuronal loss in HIV encephalopathy remains a mystery since HIV-1 productively infects macrophage and microglia and only rarely infects neurons in the central nervous system. Apoptosis is a mechanism which may account for the loss of neurons in HIV-1 infected brain. Putative toxic factors that result in neuronal cell death in HIV-1 infection include the regulatory protein Tat, since this protein is known to be released from HIV-1 infected cells. Here we show that Tat induces cell death by apoptosis in cultured human fetal neurons producing characteristic morphological and biochemical features associated with apoptosis. These findings suggest that Tat may play an important role as a secreted, soluble neurotoxin in HIV-1 associated dementia.

Neuronal excitatory properties of human immunodeficiency virus type 1 tat protein

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.

Infection of human fetal astrocytes with HIV-1: Viral tropism and the role of cell to cell contact in viral transmission

Astrocyte cultures from human fetal brain were infected with human immunodeficiency virus (HIV) either as free virus or with a chronically infected lymphoblastoid cell line and monitored for signs of infection. The lymphocytotropic strains HIV30 and HIVSF2(ARV-2) but not the monocytotropic strain HIVAda-M infected the human fetal astrocytes. The infected cells were monitored by immunocytochemistry, detection of p24 antigen in the supernatants and polymerase chain reaction amplification of the proviral DNA. No morphological or cytopathic effects were seen in these cells. Upon co-culture of astrocytes with a lymphoblastoid cell line chronically infected with HIVSF2(ARV-2), the lymphoblastoid cells readily adhered to the astrocytes as determined by a 51Cr adhesion assay and by light and electron microscopy. This cell to cell contact resulted in infection of increased numbers of astrocytes. Similar adhesion of lymphoblasts to microglia was not seen. Thus, astrocytes from human fetal brain can be infected in vitro directly by lymphocytotropic strains of HIV or by adherence to infected lymphoblastoid cells.

Genetic susceptibility to MS: a second stage analysis in Canadian MS families

Abstract. Four published genome screens have identified a number of markers with increased sharing in multiple sclerosis (MS) families, although none has reached statistical significance. One hundred and five markers previously identified as showing increased sharing in Canadian, British, Finnish, and American genome screens were genotyped in 219 sibling pairs ascertained from the database of the Canadian Collaborative Project on Genetic Susceptibility to MS (CCPGSMS). No markers examined met criteria for significant linkage. Markers located at 5p14 and 17q22 were analyzed in a total of 333 sibling pairs and attained mlod scores of 2.27 and 1.14, respectively. The known HLA Class II DRB1 association with MS was confirmed (P<0.0001). Significant transmission disequilibrium was also observed for D17S789xa0at 17q22 (P=0.0015). This study highlights the difficulty of searching for genes with only mild-to-moderate effects on susceptibility, although large effects of specific loci may still be present in individual families. Future progress in the genetics of this complex trait may be helped by (1) focussing on more ethnically homogeneous samples, (2) using an increased number of MS families, and (3) using transmission disequilibrium analysis in candidate regions rather than the affected relative pair linkage analysis.

Antioxidants and dipyridamole inhibit HIV-1 gp120-induced free radical- based oxidative damage to human monocytoid cells

Reactive oxygen species (ROS) may play an important role in HIV-1 pathogenesis and HIV-1 gp120-induced neurotoxicity. Our studies determined the extent to which gp120 increased ROS production in human monocytic U937 cells and the effectiveness of various agents, including dipyridamole (DPR), in blocking these responses. The thiobarbituric acid-reactive substances (TBARS) assay was used as a measure of recombinant gp120 (HIV-1[3B])-induced oxidative damage to U937 cells. As a control, TBARS production was measured using a hypoxanthine/xanthine superoxide generating system. There was gp120-induced oxidative damage in U937 cells with a concentration that produces 50% of maximal effect (apparent EC50 value) of 11 pM. Polyclonal antiserum to gp120 significantly (p < 0.05) inhibited gp120-induced oxidative damage. gp120-induced oxidative damage was significantly inhibited 81% (p < 0.01) by catalase/superoxide dismutase, 53% (p < 0.05) by (+/-)-alpha-tocopherol, 78% (p < 0.01) by desferrioxamine, and 82% (p < 0.01) by ethylene diamine tetraacetic acid (EDTA). These results indicate that gp120 is capable of promoting iron-based oxygen free radical damage to U937 cells. DPR potently (p < 0.05) inhibited both hypoxanthine/xanthine- and gp120-induced oxidative damage with concentrations that produce 50% inhibition (apparent IC50 values) of 1.3 microM for hypoxanthine/xanthine and 1.0 microM for gp120. Therapeutic intervention against ROS production may prevent HIV-1 neurotoxicity.

Primary headaches in HIV-infected patients

Headache in patients with human immunodeficiency virus (HIV) infection may indicate life‐threatening illnesses such as opportunistic infections or neoplasms. Alternatively, such patients may develop benign self‐limiting headaches. Hence, defining the various types of headache in these patients is essential for proper management. This study describes the clinical characteristics of primary headaches occurring in a group of HIV‐infected patients. Of 115 patients seen from 1990 to 1996, 44 (38%) had headaches. Primary headaches were present in 29 (66%) patients and secondary causes were identified in 15 (34%). Among those with primary headaches, migraine occurred in 22 (76%), tension‐type headache in 4 (14%), and cluster headache in 3 (10%) patients. Half of those with migraine (n=ll), 1 patient with tension‐type headache, and 1 patient with cluster headache developed chronic daily headaches which were severe and refractory to conventional headache or antiretroviral therapy. We conclude that primary headaches in patients with HIV infection are: (1) the commonest type of headache; (2) may present for the first time in individuals with severe immunosuppression; (3) usually bear no relationship to antiretroviral drug therapy; (4) polypharmacy, depression, anxiety, and insomnia are commonly associated comorbidities; (5) frequently do not respond to conventional management and carry a poor prognosis; and (6) do not require neuroradiological and/or cerebrospinal fluid evaluations.

HIV-1 coat protein gp120-induced increases in levels of intrasynaptosomal calcium

The effects of human immunodeficiency virus type-1 (HIV-1) coat protein gp120 on levels of intrasynaptosomal calcium ([Ca2+]i) were determined in rat cortical synaptosomes. gp120 at concentrations of > or = 400 pM, significantly (P < 0.05) increased levels of [Ca2+]i. Treatment with 20 mM KCl, reduced the concentrations of gp120 necessary to produce significant (P < 0.001) increases in [Ca2+]i. gp120-evoked increases in [Ca2+]i were prevented either by treatment with dantrolene or by removal of extracellular calcium with BAPTA. The peak levels of gp120-induced increases in [Ca2+]i were not affected by calcium channel blockers lanthanum and nicardipine, by glutamate receptor antagonists MK-801 and NBQX, or by removal of endogenous glutamate with glutamate dehydrogenase. gp120-induced [Ca2+]i increases in presynaptic terminals may play a role in HIV-mediated effects in the central nervous system.

Ocular motor dysfunction in HIV-1-infected subjects A quantitative oculographic analysis

We recorded eye and head movements in 13 human immunodeficiency virus type-1 (HIV-1)-infected patients with CD4 counts of less than or equal to 500 cells/mm3 using magnetic search coil oculography. Horizontal and vertical saccades, smooth pursuit, and vestibular smooth eye movements were recorded, as were horizontal antisaccades and vestibular memory-guided saccades. Rightward and leftward and upward and downward responses were analyzed separately. Compared to normal control subjects, HIV-1-infected patients performed the antisaccade test poorly, making the initial antisaccade in the correct direction (away from the target) in only 33% of trials. The mean final gaze position achieved during the vestibular memory-guided saccade task was less accurate for HIV-1-infected patients than for control subjects, and this correlated with inaccuracies on the antisaccade task. Horizontal saccades, horizontal and vertical smooth pursuit, and vestibular smooth eye movements were quantitatively normal. However, smooth pursuit showed directional asymmetries, vertically more than horizontally; horizontal and vertical unpredictable saccades were more inaccurate than predictable saccades; and vertical saccade latencies were prolonged. In patients with HIV-1 infection, abnormalities in vertical eye movements and relative asymmetries in smooth pursuit gains, both horizontally and vertically, are more sensitive and consistent indicators of CNS dysfunction than are horizontal eye movement abnormalities or measurements of absolute smooth pursuit gain and phase. NEUROLOGY 1996;46: 451-457

Lymphocyte Emperipolesis in Human Glial Cells

Astrocytes have been observed to contain intact, viable lymphocytes within their cytoplasm (emperipolesis) in multiple sclerosis plaques and some brain tumors. This study characterizes the adhesive, emperipoletic and phagocytic properties of glial cells in culture. Human fetal and adult astrocytes engaged in adhesion and emperipolesis of lymphocytes. Emperipolesis, and not adhesion, was temperature- and cation-dependent. The CD8 and MHC Class I antigens played a role in emperipolesis. Lymphocytes most often remained viable within the cytoplasm of astrocytes but occasionally underwent lysis or caused disruption of the astrocyte intermediate filaments. The phenomenon of emperipolesis is distinct from phagocytosis, since microglia showed prominent phagocytic properties but did not engage in emperipolesis. Conversely, astrocytes were efficient emperipolites and rarely demonstrated phagocytic properties.