Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages

1471-2202-3-13 1471-2202 Research article Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages Muscoli Carolina muscoli@uniroma3.it Salvemini Daniela dsalvemini@metaphore.com Paolino Donatella fresta@unicz.it Iannone Michelangelo iannone@cs.cnr.it Palma Ernesto palma@unicz.it Cufari Antonio acufari@libero.it Rotiroti Domenicantonio rotiroti@unicz.it Perno Federico Carlo cf.perno@uniroma2.it Aquaro Stefano cf.perno@uniroma2.it Mollace Vincenzo mollace@libero.it

Faculty of Pharmacy, University of Catanzaro "Magna Graecia", Roccelletta di Borgia, Catanzaro, Italy

MetaPhore Pharmaceuticals. Inc., 1910 Innerbelt Business Center Drive, St Louis, MO 63114, USA

Department of Pharmaceutical Sciences, University of Catania, Italy

IBAF Centre, CNR, Roccelletta di Borgia, Catanzaro, Italy

Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", Rome, Italy

IRCCS "L. Spallanzani", Rome, Italy

BMC Neuroscience 1471-2202 2002 3 1 13 http://www.biomedcentral.com/1471-2202/3/13 10.1186/1471-2202-3-13 12234380 21 5 2002 16 9 2002 16 9 2002 2002 Muscoli et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Peroxynitrite astroglial cells therapy AIDS dementia complex FeTPMS

Abstract

Background

Oxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occuring in AIDS-dementia complex. Here we investigated the role of peroxynitrite in apoptosis occurring in astroglial cells incubated with supernatants of HIV-infected human primary macrophages (M/M).

Results

Flow cytometric analysis (FACS) of human cultured astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic cell death, an effect accompanied by pronounced staining for nitrotyrosine (footprint of peroxynitrite) and by abnormal formation of malondialdehyde (MDA). Pretreatment of astrocytes with the peroxynitrite decomposition catalyst FeTMPS antagonized HIV-related astrocytic apoptosis, MDA formation and nitrotyrosine staining.

Conclusions

Taken together, our results suggest that inibition of peroxynitrite leads to protection against peroxidative stress accompanying HIV-related apoptosis of astrocytes. Overall results support the role of peroxynitrite in HIV-related programmed death of astrocytes and suggest the use of peroxynitrite decomposition catalyst to counteract HIV-1-related neurological disorders.

Background

Evidence exists suggesting that HIV-1-infected patients are under chronic oxidative stress 12. Major causes of the increased concentration of free radicals are: i) a depletion of protective systems (glutathione peroxidase, superoxide dismutase, vitamin E, selenium), and an increased production of free radicals (superoxide anion, hydroxyl radical) associated with the activation of lymphocytes and M/M; ii) chronic inflammation; iii) increased polyunsaturated fatty acid concentration and lipid peroxidation; iv) the direct or indirect effects of several opportunistic pathogens 13. In addition, elevated serum levels of hydroperoxides and malondialdehyde, which are both indicative of oxidative stress, have also been found in asymptomatic HIV-1-infected patients early in the course of the disease 2. Despite the demonstrated role of free radicals in AIDS-dementia complex, the mechanism underlying HIV-related oxidative damage of CNS cells is still unknown. Recently, it has been shown that astroglial apoptosis play a key role in the neurological progression of HIV-1 associated dementia 4 and furthermore, incubation of astroglial cells with supernatants of HIV-infected macrophages leads to astrocytic oxidative stress and then to apoptotic cell death. This effect is attenuated by novel non peptidic superoxide dismutase mimetics, thus supporting that an abnormal generation of superoxide anions may contribute in HIV-related apoptosis 23. Evidence exists that HIV infetion is accompanied by simultaneous activation of free radical species in CNS cells other then superoxide anions, such as nitric oxide (NO). Since NO is able to combine with superoxide anions to generate peroxynitrite, a nitrogen free radical 5 accounted to produce relevant peroxidative HIV-related damage in CNS cells, it is likely that abnormal release of peroxynitrite may play a role in the apoptotic cell death which occurs when incubating astroglial cells with supernatants of HIV-infected M/M 6. The present experiments have been performed in order to evaluate the role of peroxynitrite in the apoptotic cell death of astroglial cells incubated with supernatants of HIV-1-infected M/M and ascertain the protective effect of FeTMPS, a novel peroxynitrite decomposition catalyst on HIV-1-related apoptosis of astroglial cells.

Results

Effect of HIV-1 infected macrophage supernatant on astroglial cells

Time-course studies revealed that astroglial cell apoptosis occurred between 6 and 8 days after addition of the supernatant from HIV-1 infected macrophages. Thus results will be shown at day 8. In particular, when astrocytes were incubated with supernatants of HIV-infected M/M, a dramatic reduction of cell viability was seen at day 8 by flow cytometric analysis (FACS). In sharp contrast, supernatants of mock-infected M/M only marginally affected astrocytes viability. The cytopathic effect observed in astrocytes exposed to HIV-1-infected M/M supernatants was mainly related to apoptosis. Indeed, FACS analysis showed apoptosis in 49% and 7% of astrocytes exposed to HIV-1-infected M/M or mock-infected cells, respectively (Fig. 1). Indeed, 121 nmol of MDA/mg protein of cell homogenate were found in astroglial cells at day 8 after exposure to HIV-1-infected M/M supernatants, with an increment of MDA production of about 9-fold compared to controls (Fig. 2). Neither apoptotic phenomena, nor MDA over production were generated after incubation of astroglial cells with supernatants from mock-infected M/M (Fig. 1 and 2).

Figure 1

FeTMPS prevents apoptosis in astroglial cells induced by HIV-1-infected M/M

FeTMPS prevents apoptosis in astroglial cells induced by HIV-1-infected M/M. Supernatants from HIV-1 infected M/M (M/M + HIV) produces apoptotic cell death of astroglial cells (c) greater than supernatants from mock-infected (M/M) (b) as evaluated by FACS analysis 8 days after exposure to supernatants. FeTMPS (d), but not H₂TMPS (e) or FeCl₃ (f), antagonized this effect.

Figure 2

Malondialdehyde increases within astroglial cells incubated with supernatants of HIV-infected M/M (M/M + HIV) but not after exposure to supernatants from mock-infected M/M (M/M)

Malondialdehyde increases within astroglial cells incubated with supernatants of HIV-infected M/M (M/M + HIV) but not after exposure to supernatants from mock-infected M/M (M/M). FeTMPS antagonized MDA overproduction, while H₂TMPS or FeCl₃ failed to antagonize lipid peroxydation. Values are the mean out of four independent experiments. Error bars represent standard deviations. ^* P < 0.05 FeTMPS-treated vs FeTMPS-untreated astroglial cells

When cells were treated with supernatants of HIV-1-infected M/M, a corresponding increase in the number of cells that stained with nitrotyrosine-specific mouse anti-serum was observed at day 2 (Fig. 3), indicating that an HIV-related peroxynitrite formation occurred in astroglial cells. No apoptosis was seen at day 2.

Figure 3

Nitrotyrosine staining in astroglial cells either untreated (a) or pre-treated with supernatants of HIV-1-infected macrophages (b,c,d)

Nitrotyrosine staining in astroglial cells either untreated (a) or pre-treated with supernatants of HIV-1-infected macrophages (b,c,d). In particular, the supernatant of HIV-infected macrophages enhanced the immunocytochemical expression of nitrotyrosine (b), indicating an increased production of peroxynitrite. Incubation of cells with FeTMPS (c) but not with the inactive catalyst H₂TMPS (d) inhibited HIV-related peroxynitrite formation in astrocytes. These are representative photomicrographs (optical microscopy 40×) out of four independent experiments.

Effect of FeTMPS on astroglial cells following incubation with supernatants from HIV-1 infected macrophages

Dose responses with FeTMPS were performed (1–30 μM) and maximal effects were obtained between 10 and 30 μM. Results at the highest dose used are shown. Pretreatment of astrocytes with peroxynitrite decomposition catalyst FeTMPS strongly antagonized apoptosis induced by HIV-1-infected M/M supernatants. FeTMPS showed a potent dose-dependent effect as assesed by FACS analysis at day 8, only 10% of astroglial cells exposed to supernatants from HIV-1-infected M/M, and treated with 30 μM FeTMPS, showed signs of apoptosis, compared to 49% of untreated astrocytes similarly exposed to HIV-1-infected M/M supernatants (Fig. 1). In addition, MDA overproduction was significantly decreased by FeTMPS: 121 and 38 nmol MDA/mg protein of cell homogenate were measured in not-treated and FeTMPS-treated astroglial cell cultures respectively, at day 8 after the exposure to HIV-1-infected M/M supernatants (Fig. 2). In sharp contrast, treatment of astrocytes with H₂TMPS (an inactive analogue compound of FeTMPS; 30 μM), or FeCl₃ (30 μM) failed to prevent both apoptosis and MDA formation (Fig. 1 and 2). On the other hand, nitrotyrosine staining was decreased using the active catalyst FeTMPS (Fig. 3). FeTMPS did not affect, when incubated in the absence of M/M supernatants, viability of astroglial cells at concentration up to 50 μM (data not shown).

Discussion

The alteration of the homeostasis induced by HIV-1 infection, with consequent production of toxic factors, is claimed to be the main cause of neuronal damage during AIDS. In particular, the release of some coating component of HIV-1, such as gp120 glycoprotein or Tat, by HIV-1-infected M/M produces both direct and indirect effects in the central nervous system (CNS) 789. Indeed, gp120, Tat and TNFα are capable to produce apoptotic cell death via cytokine release or caspase activation 10111213. In vitro incubation of glial cells and neurons with gp120 or Tat leads to release of large amount of free radicals and, in turn, to neurodegenerative effects by activation of both cytokine network and excitatory amino acid NMDA receptor sensitization 14151617.

Previous studies have shown that HIV-1-infected M/M are able to induce apoptosis of T-lymphocytes or astroglial cells via overproduction of several factors, including prostaglandins, CD95-ligand, and free-radicals 1819. Furthermore, arachidonic acid produced by infected M/M has been reported able to cause neuronal damage 14. Finally, it has been shown that HIV-1 infection induces a heavy perturbation of oxidative status of M/M, including increased production of MDA, and decreased synthesis of endogenous glutathione 20 thus indicating that the interaction of HIV-1 with macrophages/microglial cells and the release of HIV-1 components into CNS represents apoptotic cell death of brain cells in neuroAIDS, via ROS overproduction. This is also shown by our experiments where we found that the HIV-supernatants-mediated apoptosis of astroglial cells was accompanied by an increased generation of free radicals, in particular of peroxynitrite, the reaction product of nitric oxide and superoxide 5. The role of peroxynitrite in our study was assessed by the use of the peroxynitrite decomposition catalyst, FeTMPS which catalytically decompose peroxynitrite to nitrate 2122. In previous studies, FeTMPS but not H₂TMPS (inactive analog) 21 was found to be protective against peroxynitrite-mediated damage in vitro as well as in several animals models of acute and chronic inflammation 23242526. Importantly, FeTMPS does not interact with NO making it an important tool to assess peroxynitrite-mediated damage 232425.

Our results suggest that overproduction of peroxynitrite, possibly via the release of pro-inflammatory substances by HIV infected macrophages/microglial cells, may contribute in the pathophysiological mechanisms underlying astroglial apoptotic cell death in neuro-AIDS. In addition, due to their innovative pharmacological profile, the use of novel non peptidyl peroxynitrite decomposition catalyst, such as FeTMPS, may represent the basis for alternative and efficient strategies in the treatment of neuroAIDS.

Methods

Cell cultures

Human primary macrophages

Peripheral blood mononuclear cells (PBMCs) were obtained from the blood of healthy seronegative donors by separation over Ficoll-Hypaque gradient. After separation, PBMCs were seeded at a density of 6 × 10⁶ cells/ml in 25 cm² plastic flasks in RPMI 1640 with the addition of 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, and 20% heat-inactivated, mycoplasma- and endotoxin-free fetal calf serum (FCS) (hereinafter called complete medium). Cells were incubated at 37°C in humidified air containing 5% CO₂. After 5 days of culture, non-adherent cells were removed by repeated washing with warm medium. Macrophages obtained with this method resulted in >95% of purity analyzed by flow cytometry analysis.

Human astrocytoma cell line

The astroglial cell line Lipari was derived from a 51-year old male patient who presented a large right front-temporal mass (astrocytoma) 27, these cells are not permissive to HIV-1 infection 28. Cells were expanded and cultured by seeding them in 25 cm² plastic flasks at a density of 0.7 × 10⁶ cells/flask in complete medium, and incubated at 37°C in humidified air containing 5% CO₂.

HIV-1 strain and M/M infection

A monocytotropic strain of HIV-1, named HIV-1Ba-L, was used in all experiments. Characteristics and genomic sequence of this strain have been previously described 2930. The virus was expanded in M/M, collected, filtered and stored at -80°C before use. Its concentration was 2.1 × 10⁸ genomes, corresponding to 35 ng of p24 gag antigen (Ag), and 5,000 tissue culture infectious doses 50% per ml (TCID₅₀/ml) as assessed by virus titration in M/M. Macrophages were challenged for 2 hours with 300 TCID₅₀/ml of virus, then extensively washed with warm medium to remove the excess of virus, and finally cultured in complete medium at the same conditions as before. Macrophages were washed and fed every 7 days with fresh complete medium. Supernatants of HIV-1-infected and mock-infected M/M were collected at day 14 after virus challenge, spun to remove cells and cellular debris, and stored at -80°C until use. Virus production was determined by the antigen-capture assay using a commercially available p24 gag Ag kit (Abbott Pomezia, Italy).

Challenge of astrocytes

Astrocytes were incubated with supernatants from HIV-1-infected M/M or mock-infected M/M for 4 hours at 37°C in humidified incubator. FeTMPS (30 μM), H₂TMPS (30 μM) or FeCl₃ (30 μM) were added immediately before exposure to supernatants of HIV-1-infected or mock-infected M/M. Four hours later, cells were carefully and repeatedly washed (at least 5 times) to remove M/M supernatants, and cultured in complete medium with and without a daily treatment with all the compounds used in this study for the duration of the experiment (6–8 days).

Flow cytometric analysis (FACS)

Astrocytes treated or not treated with supernatants of HIV-infected or mock-infected M/M were gently detached from plastic 6–8 days after virus challenge. Aliquots of 5 × 10⁵ cells were centrifuged at 300 × g for 5 min; pellets were washed with PBS, placed on ice, and overlaid with 0.5 ml of a hypotonic fluorochrome solution containing 50 μg/ml propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. After gentle resuspension in this solution, astrocytes were left at 4°C for 30 min, in the absence of light, before analysis. Propidium iodide-stained cells were analyzed with a FACScan Flow Cytometer (Becton Dickinson), fluorescence was measured between 565 and 605 nm. The data were acquired and analysed by the Lysis II program.

Malondialdeyde (MDA) determinations

MDA has been used as a biochemical marker for lipid peroxidation and was measured by a method previously described 31. In particular, levels of MDA were measured in astroglial cell homogenates 8 days after cell exposure to supernatants of HIV-infected M/M as indicated above. In particular, astroglial cells were frozen in liquid nitrogen, and homogenized in potassium chloride (1.15%). Chloroform (2 ml) was then added to each homogenate and then spun for 30 min. The organic layer of the sample was removed and dried under nitrogen gas and reconstituted with 100 μl of saline. MDA generation was evaluated by the assay of thiobarbituric acid (TBA)-reacting compounds. In particular, the addition of a solution of 20 μl of sodium dodecyl sulphate (SDS; 8.1%), 150 μl of 20% acetic acid solution (pH3.5), 150 μl of 0.8% TBA and 400 μl of distilled water, produced a chromogenic product which was extracted in n-butanol and pyridine. The organic layer was removed and MDA was read at 532 nm and expressed as nmol MDA/mg protein of cell homogenate.

Immunocytochemical Staining

Immunocytochemical staining for nitrotyrosine was performed on treated or not treated astrocytes. Astrocytes were fixed with 4% paraformaldeyde dissolved in 0.1% phosphate buffer (pH 7.4). Nonspecific staining was blocked with 3% normal goat serum in 0.5 M Tris-HCl, pH 7.4 containing 0.2% Tween 20 for 1 h at room temperature. All subsequent incubations were carried out in this buffer. For detection of nitrotyrosine immunoreactivity, cells were incubated for 16 h at 4°C with an anti-nitrotyrosine monoclonal Ab (Cayman, 1:500) at 4°C. The primary Ab was then removed, and the cells were exposed to the secondary Ab, which was a biotinylated goat anti-mouse Ig (IgG), and avidin-biotin-horseradish peroxidase (VECTASTAIN Elite ABC Kit; Vector Laboratories), followed by diaminobenzedine.

Statistical analysis

Statistical significance and standards deviations were assessed using the Student t test.

Authors'contributions

Author 1 initials carried out the cytometric analysis, the immunocitochemistry, participated in the sequence alignment and drafted the manuscript. Author 2 partecipated in the design of the study and drafted the manuscript. Authors 3 initials carried out the biochemical analysis. Authors 4 and 5 initials partecipated in the sequence alignment. Author 6 performed the statistical analysis. Authors 7 and 8 conceived of the study. Author 9 initials carried out the virology study. Author 10 partecipated in the design of the study and in its coordination.

Acknowledgements

This work was supported by Ministry of University and Scientific Research (MURST; COFIN 2000 and COFIN 2001), Italian National Council for Research (CNR), by the European Found for Regional Development (POP 94/99), by grants from the AIDS Project (ISS), Italy, by the Ricerca Corrente of the Italian Ministry of Health, and by Biomed Project of European Comunity. Our thanks go to Mrs Tania Guenci, and Mr. Giovanni Politi (University of Rome "Tor Vergata", Rome, Italy) for their excellent technical support.

The redox pathway of S-<it>nitrosoglutathione</it>, glutathione and nitric oxide in cell to neuron communications. Chiueh CC Rauhala P Free Radic Res 1999 31 641 650 10630687 Oxidative stress and neuroAIDS: triggers, modulators and novel antioxidants. Mollace V Nottet HSLM Clayette P Turco C Muscoli C Salvemini D Perno CF Trends Neurosci 2001 24 411 416 10.1016/S0166-2236(00)01819-1 11410272 The contribution of oxidative stress in apoptosis of human-cultured astroglial cells induced by supernatants of HIV-1-infected macrophages. Mollace V Salvemini D Riley DP Muscoli C Granato T Masuelli L Modesti A Rotiroti D Nistico R Bertoli A Perno CF Aquaro S J Leukoc Biol 2002 71 1 65 72 11781381 Correlation between neurological progression and astrocyte apoptosis in HIV-associated dementia. Thompson KA McArthur JC Wesselingh SL Ann Neurol 2001 49 6 745 752 10.1002/ana.1011 11409426 Apparent hydroxyl radical production by peroxynitrite: implication for endothelial injury from nitric oxide and superoxide. Beckman JS Beckman TW Chen J Marshall PA Freeman BA Proc Natl Acad Sci USA 1990 87 1620 1624 53527 2154753 Increased peroxynitrite activity in AIDS dementia complex: implications for the neuropathogenesis of HIV-1 infection. Boven LA Gomes L Hery C Gray F Verhoef J Portegies P Tardieu M Nottet HS J Immunol 1999 162 4319 4327 10201964 Unraveling the neuroimmune mechanisms for the HIV-1-associated cognitive/motor complex. Nottet HS Gendelman HE Immunol Today 1995 16 441 4489 10.1016/0167-5699(95)80022-0 7546209 The cellular basis of central nervous system HIV-1 infection and the AIDS dementia complex. Price RW J Neuro-AIDS 1996 1 1 20 10.1300/J128v01n01_01 Neuronal apoptosis induced by HIV-1 Tat protein and TNF-alpha: potentiation of neurotoxicity mediated by oxidative stress and implications for HIV-1 dementia. Shi B Raina J Lorenzo A Busciglio J Gabuzda D J Neurovirol 1998 4 3 281 290 9639071 HIV-1 gp120-induced apoptosis in the rat neocortex involves enhanced expression of cyclo-oxygenase type 2 (COX-2). Bagetta G Corasaniti MT Paoletti AM Berliocchi L Nisticò R Giammarioli AM Finazzi-Agrò A Biochem Biophys Res Commun 1998 244 819 824 10.1006/bbrc.1998.8321 9535750 Involvement of interleukin-1β in the mechanism of human HIV-1 recombinant protein gp120-induced apoptosis in the neocortex of rat. Bagetta G Corasaniti MT Berliocchi L Nisticò R Giammarioli AM Malorni W Aloe L Finazzi Agrò A Neuroscience 1999 89 1051 1066 10.1016/S0306-4522(98)00363-7 10362294 The human immunodeficiency virus type 1 glycoprotein gp120 reduces the expression of neuronal nitric oxide synthase in the hippocampus but not in the cerebral cortex and medial septal nucleus of rat. Bagetta G Corasaniti MT Costa N Berliocchi L Finazzi-Agrò A Nisticò G Neurosci Lett 1997 224 75 78 10.1016/S0304-3940(97)13463-2 9086460 HIV-1 protein Tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress. Kruman II Nath A Mattson MP Exp Neurol 1998 154 2 276 288 10.1006/exnr.1998.6958 9878167 Release of nitric oxide from astroglial cells: a key mechanism in neuroimmune disorders. Mollace V Nisticò G Adv Neuroimmunol 1995 5 421 30 8746514 Human immunodeficiency virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures. Dawson VL Dawson TM Uhl GR Snyder SH Proc Natl Acad Sci USA 1993 90 3256 3259 46278 8097316 The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells. Mollace V Colasanti M Muscoli C Lauro GM Rotirorti D Nisticò G Br J Pharmacol 1998 124 742 746 9690866 Pro-inflammatory and pro-oxidant properties of the HIV protein Tat in a microglial cell line: attenuation by 17 beta-estradiol. Bruce-Keller AJ Barger SW Moss NI Pham JT Keller JN Nath A J Neurochem 2001 78 6 1315 1324 10.1046/j.1471-4159.2001.00511.x 11579140 Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes. Aquaro S Panti S Caroleo MC Balestra E Cenci A Forbici F Ippolito G Mastino A Testi R Mollace V Caliò R Perno CF J Leukoc Biol 2000 68 429 43 10985261 Correlation between induction of lymphocyte apoptosis and prostaglandin E2 production by macrophages infected with HIV. Mastino A Grelli S Piacentini M Oliverio S Favalli C Perno CF Garaci E Cell Immunol 1993 152 120 130 10.1006/cimm.1993.1272 8242755 Glutathione inhibits HIV replication by acting at late stages of the virus life cycle. Palamara AT Perno CF Aquaro S Bue MC Dini L Garaci E AIDS Res Hum Retroviruses 1996 12 1537 1541 8911579 Peroxynitrite decomposition catalysts. Stern MK Jensen MP Kramer K J Am Chem Soc 1996 118 8735 10.1021/ja961279f Mechanisms of peroxynitrite decomposition catalyzed by FeTMPS, a bioactive sulfonated iron porphyrin. Shimanovich R Groves JT Arch Biochem Biophys 2001 387 307 317 10.1006/abbi.2000.2247 11370855 Characterization of the cytoprotective action of peroxynitrite decomposition catalysts. Misko TP Highkin MK Veenhuizen AW Manning PT Stern MK Currie MG Salvemini D J Biol Chem 1998 273 15646 15653 10.1074/jbc.273.25.15646 9624158 Peroxynitrite decomposition catalyst: therapeutics for peroxynitrite-mediated pathology. Salvemini D Wang ZQ Stern MK Currie MG Misko TP Proc Natl Acad Sci 1998 95 2659 2663 19452 9482943 10.1073/pnas.95.5.2659 Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin-induced intestinal damage. Salvemini D Riley DP Lennon PJ Wang ZQ Currie MG Macarthur H Misko TP Br J Pharmacol 1999 127 685 692 10401559 Peroxynitrite is a major contributor to cytokine-induced myocardial contractile failure. Ferdinandy P Danial H Ambrus I Rothery RA Schulz R Circ Res 2000 87 241 247 10926876 Establishment, characterization and chemosensitivity of two human glioma derived cell lines. Zupi G Candiloro A Laudonio N Carapella C Benassi M Riccio A Bellocci M Greco C J Neurooncol 1988 6 169 177 3225639 Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes. Aquaro S Panti S Caroleo MC Balestra E Cenci A Forbici F Ippolito G Mastino A Testi R Mollace V Calio R Perno CF J Leukoc Biol 2000 68 429 435 10985261 Selected nucleotide sequence of the pol gene of the monocytotropic strain HIV type 1 BaL. Cenci A Perno CF Menzo S Clementi M Erba F Tavazzi B Di Pierro D Aquaro S Caliò R AIDS Res Hum Retroviruses 1997 13 629 632 9135882 Relative potency of protease inhibitors in monocytes/macrophages acutely and chronically infected with human immunodeficiency virus. Perno CF Newcomb FM Davis DA Aquaro S Humphrey RW Caliò R Yarchoan R J Infect Dis 1998 178 413 422 9697721 Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Ohkawa H Ohishi H Yagi K Anal Biochem 1979 95 351 358 36810