Glutathione and immunophenotypes of T and B lymphocytes in HIV-infected individuals.
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AIDS-Related Complex↗
Biomedical subjects
M Roederer
Publications and source records attributed to M Roederer.
Glutathione deficiency and human immunodeficiency virus infection.
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Glutathione↗
N-acetylcysteine: a new approach to anti-HIV therapy.
Several investigators have implicated depletion of glutathione (GSH) and production of reactive oxygen intermediates (ROIs) in the regulation of the human immunodeficiency virus (HIV). We have shown directly that N-acetylcysteine (NAC) blocks HIV expression in chronic and acute infection models, and HIV replication in normal peripheral blood mononuclear cells. NAC is a cysteine prodrug which maintains intracellular thiol levels during oxidative stress and replenishes depleted GSH. The observed antiviral effect of NAC is due to inhibition of viral stimulation by ROIs, which are produced in response to inflammatory cytokines. We have also shown that HIV-infected individuals have decreased intracellular GSH levels in their circulating T cells. Since GSH is the major protection against the production of ROIs, we hypothesize that the observed decrease is due to a chronic oxidative stress induced by continual exposure to elevated levels of inflammatory cytokines. Together, these results provide a rationale for clinical trials testing the efficacy of GSH-replenishing drugs such as NAC in the treatment of AIDS. NAC is different than many other antiviral drugs in that it inhibits host-mediated stimulation of viral replication arising in normal immune responses, and may thereby extend latency. In addition, it inhibits the action of inflammatory cytokines which may mediate cachexia, thereby raising the possibility that it may alleviate the deleterious wasting that accompanies late stage AIDS.
Acetylcysteine↗
Intracellular glutathione levels in T cell subsets decrease in HIV-infected individuals.
The authors have shown previously that intracellular glutathione (GSH) plays an important role in the regulation of human immunodeficiency virus (HIV) transcription and replication in vitro, through modulation of signal transduction by inflammatory cytokines. Moreover, intracellular GSH levels are known to regulate T-lymphocyte function. In multiparameter FACS studies presented here, we show that relative GSH levels in CD4+ and CD8+ T cells from HIV+ individuals are significantly lower than in corresponding subsets from uninfected controls. These studies define the relative intracellular glutathione (GSH) levels in CD4+ T cells, CD8+ T cells, B cells, and monocytes from 134 HIV-infected individuals and 31 uninfected controls. The greatest decreases in intracellular GSH occur in subsets of T cells in individuals in the later stages of the HIV infection. In AIDS patients, GSH levels are 63% of normal in CD4+ T cells (p less than 0.0001) and are 62% of normal in CD8+ T cells (p less than 0.0001). Similarly, in AIDS-related complex (ARC) patients, GSH levels are 66% of normal in CD4+ T cells (p less than 0.003) and are 69% of normal in CD8+ T cells (p less than 0.003). These findings suggest that low intracellular GSH levels may be an important factor in HIV infection and in the resulting immunodeficiency.
AIDS-Related Complex↗
CD20 expression is increased on B lymphocytes from HIV-infected individuals.
In studies presented here, we show that expression of the pan B cell marker CD20 is markedly increased on B lymphocytes from HIV-infected individuals and that this increase tends to be greater in individuals with more advanced disease. By using multiparameter FACS analyses to quantitate surface density of CD20 and intracellular glutathione (GSH) levels simultaneously, we further show that the distribution of intracellular glutathione (GSH) levels in B cells of HIV-infected individuals is more heterogeneous than in uninfected controls. Finally, we show that the intracellular GSH levels correlate with CD20 expression on a per-cell basis in all infected individuals. These findings suggest that CD20 expression, which can be precisely measured, may prove to be a useful surrogate marker for monitoring HIV infection.
Antigens, CD↗
Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.
The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.
Animals↗
N-acetylcysteine inhibits latent HIV expression in chronically infected cells.
The progression of the human immunodeficiency virus (HIV) infection from its early latent (asymptomatic) stage to active, late-stage acquired immunodeficiency syndrome (AIDS) apparently begins with the production of inflammatory cytokines that stimulate the expression and replication of the latent virus. We have shown that N-acetylcysteine, a cysteine precursor that is converted intracellularly into glutathione, blocks cytokine-stimulated HIV replication in an acutely infected T-cell line and in acutely infected peripheral blood mononuclear cells from normal individuals. In this report, we show that N-acetylcysteine also inhibits stimulated HIV expression in chronically infected monocyte and T-cell lines which are used as models for latent infection in AIDS. Furthermore, we show that N-acetylcysteine blocks viral production in monocyte cell lines more effectively than it blocks viral production in T cells. Since monocytes are a major reservoir for HIV in infected individuals, these results suggest that N-acetylcysteine may slow the change from latency to the later stages of AIDS in HIV-infected individuals.
Acetylcysteine↗
CD4 and CD8 T cells with high intracellular glutathione levels are selectively lost as the HIV infection progresses.
Maintenance of intracellular glutathione (GSH) levels has been implicated in blocking cytokine-stimulated HIV replication in vitro, in both acute and latent infection models. We demonstrate here that subsets of human peripheral blood mononuclear cells differ substantially in mean GSH levels, as measured on a cell-by-cell basis with the fluorescence-activated cell sorter (FACS): B cells have the lowest GSH levels; T cells are intermediate; and monocytes and macrophages have the highest levels. Furthermore, GSH levels subdivide the CD4 and CD8 T cell subsets into two classes each: high- and low-GSH cells, which cannot be distinguished by cell size or by currently known surface markers. Significantly, the high-GSH T cells are selectively depleted early during the HIV infection, and are effectively missing in all ARC and AIDS patients.
AIDS-Related Complex↗
Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine.
We show that the stimulation of human immunodeficiency virus (HIV) brought about by tumor necrosis factor alpha and phorbol 12-myristate 13-acetate can be inhibited by adding N-acetyl-L-cysteine (NAC). NAC, which replenishes intracellular glutathione, effectively inhibits the tumor necrosis factor alpha- or phorbol ester-stimulated replication of HIV in acutely infected cell cultures. NAC also inhibits the cytokine-enhanced HIV long terminal repeat-directed expression of beta-galactosidase in in vitro HIV model systems. These results show that intracellular thiol levels influence HIV production. Furthermore, because NAC reverses tumor necrosis factor alpha toxicity both in cells and in animals and is a well-known drug that can be administered orally without known toxicity in humans, these results suggest that NAC is a possible therapeutic agent in AIDS.
Acetylcysteine↗
Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus.
The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that intracellular thiol levels play a key role in regulating this process. That is, stimulation with tumor necrosis factor alpha and/or phorbol 12-myristate 13-acetate activates NF-kappa B and markedly decreases intracellular thiols; N-acetyl-L-cysteine, an efficient thiol source, prevents this thiol decrease and blocks the activation of NF-kappa B; and the lack of activated NF-kappa B prevents the activation of the HIV LTR and the transcription of genes under its control. These findings reveal a previously unrecognized genetic regulatory mechanism in which cytokine-induced shifts in intracellular thiol levels are crucial in the control of NF-kappa B activity and thereby influence the spectrum of genes expressed by cytokine-stimulated cells.
Base Sequence↗
Endosomes can undergo an ATP-dependent density increase in the absence of dense lysosomes.
On the basis of evidence that lysosomal enzymes and membrane proteins are present in endosomes, we have previously suggested that the production of lysosomes involves maturation rather than vesicle fusion (Roederer, M., R. Bowser, R. F. Murphy, J. Cell. Physiol. 131, 200-209 (1987)). Since the appearance of endocytosed material in lysosomes is associated with an increase in buoyant density from that of endosomes, a prediction of the model is that endosomes should be capable of undergoing such an increase in vitro. We observe that under appropriate conditions, isolated endosomes containing [125I]EGF can undergo an increase in density in vitro to that of dense lysosomes, mimicking the density change which occurs in vivo. This occurs in the absence of dense lysosomes with which to fuse. The density increase requires ATP and can be efficiently inhibited in vitro by the presence of benzylamine, suggesting that vesicular acidification is required. Since low pH has previously been shown to induce formation of a matrix by lysosomal enzymes in vitro (Buckmaster, M. J., A. L. Ferris, B. Storrie, Biochem. J. 249, 921-923 (1988)), we propose that a mechanism by which endosomes and/or lysosomes increase their density is a low pH induced aggregation of vesicle contents which decreases the osmotic pressure inside the vesicle. Together with previous data, the results provide highly suggestive evidence that the pathway to lysosomes includes a maturation of the postsorting compartment into what has classically been termed a lysosome.
Adenosine Triphosphate↗
Flow cytometry DNA ploidy analysis and catecholamine secretion profiles in neuroblastoma.
Previous studies have shown that catecholamine secretion patterns have been imperfect predictors of clinical behavior of neuroblastomas. Recently, studies of nuclear DNA content in neuroblastoma have shown that an aneuploid DNA content predicts favorable clinical behavior. To determine if a correlation exists between these tumor biologic indicators, the authors analyzed both in a series of 39 patients with neuroblastoma. Flow cytometric DNA analysis performed on paraffin blocks determined that 23 patients had tumors with aneuploid DNA content (aneuploid tumors) and 16 patients showed no demonstrable anomalies of tumor DNA content (nonaneuploid tumors). Comparison of catecholamine levels in urine and tumor homogenates with DNA content data indicate that nonaneuploid neuroblastomas include a significant number (P less than 0.02) of biochemically primitive tumors which secrete high levels of 3,4 dihydroxyphenylalanine (DOPA), dopamine and homovanillic acid (HVA). This suggests a dopamine-norepinephrine pathway block, which supports previous reports of deficiency of dopamine beta-hydroxylase activity in some neuroblastomas. The study shows that in contrast to aneuploid tumors, nonaneuploid neuroblastomas secrete higher levels of early pathway catecholamine metabolites and are more likely to present in higher (unfavorable) clinical stages of disease.
Aneuploidy↗
Effect of confluence on endocytosis by 3T3 fibroblasts: increased rate of pinocytosis and accumulation of residual bodies.
We have compared lysosomal enzyme distributions on density gradients and rates of transport of endocytic markers for actively-growing and confluent cells. While it has been previously established that mammalian cells accumulate lysosomal enzymes during quiescence, we show that this accumulation is predominantly in residual bodies (p greater than 1.12 g/ml) rather than in dense lysosomes (p = 1.08-1.10 g/ml) and does not represent a change in the endosomal and lysosomal enzyme content. The accumulation is not caused by a change in the rate of production of dense lysosomes, since the rate of transfer of epidermal growth factor (EGF) from light to dense compartments is the same between confluent and subconfluent cells. Confluent cultures have a higher rate of initial pinocytosis, and a higher rate of retroendocytosis and/or recycling, causing a net lower rate of accumulation of fluid-phase material. The accumulation of residual bodies in confluent cultures may be caused by a lower rate of exocytosis of their contents and/or a lack of dilution by cell division. The data indicate that the impact of culture confluence must be carefully assessed in experiments designed to analyze endocytic pathways.
Animals↗
Flow cytometric DNA analysis of neuroblastoma and ganglioneuroma. A 10-year retrospective study.
Retrospective quantitative DNA analysis was done on 147 samples from 89 patients with neuroblastoma and ganglioneuroma using flow cytometry. In the neuroblastoma patients, nuclear DNA content was found to be a stable tumor marker irrespective of site (primary versus metastatic) and despite changes with time in tumor progression, maturation, or therapy. The occurrence of DNA aneuploidy, which was detected in 60% of the neuroblastoma patients, paralleled other favorable indicators and was highly associated with survival (P less than 0.001). Of clinical stage, age, primary site, sex, and DNA content, only stage and DNA content correlated with survival. Those patients with favorable stage and DNA aneuploidy had higher survival rates. Further, favorable stage and the presence of DNA aneuploidy were independent prognostic indicators. Abnormal DNA content was also detected in samples from ganglioneuromas in which significant numbers of ganglion cell nuclei were recovered. These results indicate a striking difference between neuroblastoma and adult tumors in which DNA aneuploidy is generally a poor prognostic sign and provide a molecular link between ganglioneuromas and their malignant counterparts.
Adolescent↗
Flow cytometric DNA analysis of adrenocortical tumors in children.
Flow cytometric DNA analysis of isolated nuclei was performed on 14 lesions occurring in ten children with adrenocortical tumors. Unimodal DNA content distributions were obtained from seven tumors occurring in patients without metastases 2 to 18 years after diagnosis. Abnormal DNA contents were detected in all four primary lesions, which subsequently metastasized, and in the tumor of one patient who was followed for less than 2 years. Paraffin and frozen preparations were virtually identical, as were the analyses of the primary, recurrent, and metastatic disease occurring in one patient. These observations suggest that DNA content abnormalities detected by flow cytometry correlate with metastases, and may provide an objective measure of the biologic potential of these tumors.
Adrenal Gland Neoplasms↗
Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes.
The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.
Animals↗
Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts.
Autofluorescence of cells can be a major portion of the fluorescence signal in many systems, especially when fluorescent conjugates are used to study receptor-ligand systems for which there are less than 70,000 receptors per cell. We have devised a method for the cell-by-cell correction of autofluorescence for flow cytometric data by using an additional parameter to measure and correct for autofluorescence in the fluorescence channel. The principle has been extended to allow simultaneous correction for autofluorescence and dual-fluorescence spillover compensation in samples labeled with two different fluorochromes; all corrections were done in software, making them applicable to any flow cytometer. The autofluorescence correction method was used to analyze the acidification of epidermal growth factor (EGF) by Swiss 3T3 cells. EGF is acidified to pH 6.2 starting two min after labeling, with a half-time for acidification of 45 s.
Acids↗