Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease.

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, > 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.

Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes.

Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).

Infection of normal human epithelial cells by Epstein-Barr virus.

Primary cultures of epithelial cells were grown from the tonsils and adenoids of patients with diseases not related to Epstein-Barr virus. The cells could not be infected by Epstein-Barr virus. Fluorescein-labeled Epstein-Barr virus and a cytofluorograph were then used to show that the epithelial cells do not have detectable receptors for the virus. However, implantation with Epstein-Barr virus receptors gave the cells the ability to bind the labeled virus. One to 5 percent of receptor-implanted cells exposed to the transforming B95-8 substrain of the virus expressed Epstein-Barr nuclear antigen. The early and viral capsid Epstein-Barr virus-determined antigens were not detected in the virus-infected cultures. The results show that normal human epithelial cells from the nasopharynx become susceptible to infection by Epstein-Barr virus when the membrane barrier resulting from the lack of viral receptors is overcome by receptor implantation.

Immortalization of human T lymphocytes after transfection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, has the ability to transform human B lymphocytes. No other cell type has been experimentally transformed by EBV, either by intact virions or naked viral DNA and subgenomic fragments. Two immortalized human T-lymphoblastoid cell lines have now been established by transfecting cord blood lymphocytes with purified B95-8 viral DNA enclosed in fusogenic Sendai virus envelopes (RSVE) and then exposing the cells to EBV from a P3HR-1 cell subclone. One of these lines, which has been fully characterized, is termed HBD-1. This line is positive for EBV DNA and expresses surface OKT11, OKT4, and Tac receptors, but not M-1, mu immunoglobulin chains, EBV receptors, or B-1 surface markers. The cells contain fully rearranged T-cell receptor genes and germline immunoglobulin genes. The karyotype of the cells is normal, they do not require interleukin-2 for growth, and do not contain human T-lymphotropic virus type I. However, the HBD-1 cells contain incomplete EBV genomes and express several EBV-determined antigens, including the early antigen type D, membrane antigens, but not EBV-determined nuclear antigen (EBNA). This association of the EBV genome with permanently growing hematopoietic cells of non B-cell lineage should prove useful in studies on the mechanism of EBV-mediated cell transformation.

Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist.

The human immunodeficiency virus-1 (HIV-1) trans-activator Tat is an attractive target for the development of antiviral drugs because inhibition of Tat would arrest the virus at an early stage. The drug Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one], inhibited gene expression by HIV-1 at the level of transcriptional trans-activation by Tat. The compound did not inhibit the basal activity of the promoter. Both Tat and its target sequence TAR were required for the observed inhibitory activity. Ro 5-3335 reduced the amount of cell-associated viral RNA and antigen in acutely, as well as in chronically infected cells in vitro (median inhibition concentration 0.1 to 1 micromolar). Effective inhibition of viral replication was also observed 24 hours after cells were transfected with infectious recombinant HIV-1 DNA. The compound was active against both HIV-1 and HIV-2 and against 3'-azido-3'-deoxythymidine (AZT)-resistant clinical isolates.

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.

Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes.

Oxygen is vital for the development and survival of mammals. In response to hypoxia, the brain initiates numerous adaptive responses at the organ level as well as at the molecular and cellular levels, including the alteration of gene expression. Astrocytes play critical roles in the proper functioning of the brain; thus the manner in which astrocytes respond to hypoxia is likely important in determining the outcome of brain hypoxia. Here, we used microarray gene expression profiling and data-analysis algorithms to identify and analyze hypoxia-responsive genes in primary human astrocytes. We also compared gene expression patterns in astrocytes with those in human HeLa cells and pulmonary artery endothelial cells (ECs). Remarkably, in astrocytes, five times as many genes were induced as suppressed, whereas in HeLa and pulmonary ECs, as many as or more genes were suppressed than induced. More genes encoding hypoxia-inducible functions, such as glycolytic enzymes and angiogenic growth factors, were strongly induced in astrocytes compared with HeLa cells. Furthermore, gene ontology and computational algorithms revealed that many target genes of the EGF and insulin signaling pathways and the transcriptional regulators Myc, Jun, and p53 were selectively altered by hypoxia in astrocytes. Indeed, Western blot analysis confirmed that two major signal transducers mediating insulin and EGF action, Akt and MEK1/2, were activated by hypoxia in astrocytes. These results provide a global view of the signaling and regulatory network mediating oxygen regulation in human astrocytes.

Deficiency in Epstein-Barr virus receptors on B-lymphocytes of preleukemia patients.

Lymphocytes from eight preleukemia patients were exposed to Epstein-Barr virus (EBV) in vitro in an attempt to establish lymphoblastoid cell lines. No signs of viral infection were detected, and no cell lines were obtained. Studies using fluorescein-labeled EBV and flow cytometry revealed an unusual and consistent deficiency in EBV receptors in all patients examined. In control studies, about 15% of the unseparated lymphocytes from healthy donors bound fluorescein-labeled EBV. In spite of the lack of EBV receptors, B-lymphocytes amounted to 10 to 20% of the preleukemia lymphocyte populations, a proportion similar to that in healthy donors. When lymphocytes from preleukemic patients were first implanted with functional EBV receptors and then exposed to EBV, synthesis of EBV-determined nuclear, early, and viral capsid antigens was induced. Subsequently, several cell lines originating from preleukemic patients' lymphocytes were established. These lines are of a B-lymphocyte origin and carry EBV genome. They will provide experimental material for the molecular analysis of lymphocytic defects in preleukemia and their possible role in the transition to acute leukemia.

Inhibition of membrane fusion by suppression of lateral movement of membrane proteins.

Chicken erythrocytes were fused either by Sendai virus or by the combination of Ca2+ and ionophore A23187. Intramembrane particles and external anionic sites of cells undergoing fusion were found to acquire the ability to undergo a process of cold-induced clustering (thermotropic separation). Cationized ferritin (200 microgram/ml 5% (v/v) cell suspension) inhibited both the fusion process and the thermotropic separation of intramembrane particles and external anionic sites. The correlation between the mobility of membrane proteins and the fusion process is discussed. It is suggest that an increase in the lateral mobility of membrane proteins is a prerequisite for initiation of membrane fusion.

Epstein-Barr virus co-reconstituted with Sendai virus envelopes infects Epstein-Barr virus-receptor negative cells.

Epstein-Barr virus (EBV) was co-reconstituted with Sendai virus envelopes. The reconstituted "hybrid' virus could bind and penetrate into EBV-receptor negative cells. Using this approach, T-cell-derived human and mouse leukemia cells, human T-lymphocytes and mouse spleen cells were successfully infected as judged by the induction of EBV-determined antigens and stimulation of DNA synthesis. The T-cell-derived human leukemia line Molt-4, that can absorb EBV but without virus penetration, could be also infected by the reconstituted EBV.

Lateral mobility of beta-receptors involved in adenylate cyclase activation.

Cationized ferritin was found to inhibit the lateral mobility of intramembrane proteins in turkey erythrocyte membranes and the activation of adenylate cyclase by the (--)-epinephrine-bound beta-adrenergic receptor. It was observed that cationized ferritin has only a small direct effect on the beta-receptor and on the adenylate cyclase moiety. It is concluded that the cationized ferritin-induced inhibition of the hormone-dependent cyclase activity results from the inhibition of the lateral mobility of the receptor and therefore a decrease in the bimolecular rate of interaction between the receptor and the enzyme.

Uniform detection of HIV-1 in alveolar macrophages of pediatric but not adult AIDS patients.

Manifestations of pulmonary disease in pediatric AIDS patients differ from those in adults. To evaluate whether differences in the frequency of alveolar macrophage (AM) infection with human immunodeficiency virus type 1 (HIV-1) could account for these clinical distinctions, we undertook a comparative analysis of HIV-1 DNA in AMs from pediatric and adult AIDS patients by enzymatic amplification. A higher frequency of viral DNA detection in pediatric cases (100%) compared with adults (67%) was observed. The sensitivity of detection was 1 viral DNA copy per 4000 AMs; matched peripheral blood mononuclear cells from six of seven pediatric and eight of nine adult patients tested HIV-1 DNA positive. Adult but not pediatric patients exhibited a marked alveolar lymphocytosis, 32% mean lymphocyte count compared with 7.0%, respectively. These results suggest that the burden of HIV-1 in the lungs of pediatric AIDS patients is greater than that in adults.

Induction of dormant HIV-1 by sodium butyrate: involvement of the TATA box in the activation of the HIV-1 promoter.

Reactivation of latent HIV-1 is believed to play a major role in the pathogenesis of AIDS. Here we show that sodium butyrate (NaB), which can cause gene induction or cell differentiation, reactivates dormant HIV-1 in vitro in chronically infected cells of T-lymphoid and monocytoid origin. The effect of NaB on HIV-1 expression in T-lymphoid cells was apparent 3 h after addition of drug and peaked at 24 h. During this time the proportion of HIV-1 antigen expressing cells increased from less than 0.5 to greater than 90%, and virus production increased by three orders of magnitude. The virus released by the NaB-induced cells was infectious. The extent and kinetics of NaB effects were similar to effects of phorbol 12-myristate 13-acetate in T cells, but not monocytes. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that mutations which altered the nucleotide sequence in the TATA box significantly reduced the NaB effect. These data show that NaB is a potent inducer of dormant HIV-1 and suggest that the TATA motif is required for this activity.

The effect in vitro of 2'-deoxycytidine on the metabolism and cytotoxicity of 2',3'-dideoxycytidine.

We examined in vitro the effect of high, but clinically achievable and non-toxic, concentrations of 2'-deoxycytidine (dCyd) (greater than or equal to 100 mumols/l on the metabolism and cytotoxicity of 2',3'-dideoxycytidine (DDC) in normal human bone marrow mononuclear cells (BMMCs) and a cultured T-lymphocyte (HUT-102) cell line. Colony formation in semi-solid medium by bone marrow progenitor cells (CFU-GM and CFU-GEMM) was significantly protected by dCyd against the cytotoxic effects of high doses of DDC. In contrast, in HIV-infected HUT-102 cells, anti-HIV effect of DDC (10 mumols/l) was preserved in the presence of 100 mumols/l dCyd but partially reversed by higher levels of dCyd. dCyd reduced the generation of DDC triphosphate (DDC-TP) relative to dCyd triphosphate (dCTP) pools to a significantly greater extent in BMMCs versus HUT-102 cells. This might explain dCyd-mediated abrogation of DDC cytotoxicity against marrow progenitor cells with relative preservation of its anti-HIV-1 activity in HUT-102 cells.

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 DNA was confirmed by Southern blotting. HIV-1 gag DNA was 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 astrocytes and neurons by HIV-1 may contribute to the development of HIV-1 disease in the brain.

Cloning of differentially expressed genes in an HIV-1 resistant T cell clone by rapid subtraction hybridization, RaSH.

An HIV-1 resistant T cell clone R1c2 has been generated that carries mutant, latent HIV-1 in a minority of the cell population. Resistant cells express HIV-1 receptors CD4 and CXCR4 and display resistance to infection by wild type (wt) HIV-1 at the level of virus transcription. To begin to define the repertoire of genes modulated in R1c2 cells that correlate with and potentially control expression of the HIV-1 resistance phenotype we have employed a rapid subtraction hybridization (RaSH) technique. For this approach, cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified followed by incubation of tester and driver PCR fragments. The RaSH scheme resulted in the cloning of genes displaying differential expression between HIV-1 resistant (R1c2) and susceptible (SupT1) cells, including known genes and those not described in current DNA databases. Analysis of the pattern of expression of the differentially expressed genes documented eleven genes with enhanced (HR clones) and six genes with reduced (HS clones) expression in HIV-1 resistant versus HIV-1 susceptible T-cell clones.

Expression of the T4 molecule (AIDS virus receptor) by human brain-derived cells.

Three human cell lines of astrocytic origin were evaluated for expression of a human T-lymphocyte surface glycoprotein, T4, which also serves as a cellular receptor for the human immunodeficiency virus (AIDS virus, HIV). T4 antigen was detected on the cell surface of 2 of these cell lines using monoclonal OKT-4 antibody and flow cytometry. Gene transcripts encoding the T4 molecule were detected by a ribonuclease protection assay in surface T4-positive and -negative cells. Our results suggest that astrocytes may serve as targets for HIV infection in the brain.

Low-cytopathic infectious clone of human immunodeficiency virus type I (HIV-I).

Single genotypic variants of HIV-I, contained in a parental cytopathic HIV-I isolate, were isolated by molecular cloning and propagated in susceptible cells. Two such HIV-I clones, designated N1T-E and N1T-A, exhibited similar restriction endonuclease maps but strikingly different biological activities. Infection of T lymphocytes or monocytes by clone N1T-E was characterized by slow kinetics and lack of significant cytopathic effects, but high reverse transcriptase activity levels in culture supernatants of chronically-infected cells. Clone N1T-A, like the parental HIV-I isolate, exhibited fast kinetics of infection in T cells and monocytes and strong cytopathicity in these cells. Full characterization of the low-cytopathic virus in comparison to the structurally similar cytopathic clone may facilitate the elucidation of the molecular basis of HIV cytopathogenicity.

Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching.

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B-chloride (R-18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R-18-labeled HIV (R-18/HIV) with CD4-positive CEM and HUT-102 cells, but not with CD4-negative MLA-144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 20-25%. Similar level of DQ was observed upon incubation of CEM cells with R-18-labeled Sendai virus. DQ was observed when R-18/HIV was incubated with CD4+ cells at 37 degrees C, but not at 4 degrees C. Most of the increase in fluorescence occurred within 5 min of incubation at 37 degrees C and was independent of medium pH over the range of pH 5-8. Preincubation of cells with the lysosomotropic agent NH4Cl had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R-18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH-independent, receptor-mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.