3-nitrotyrosine and HIV-Infections

Advances in antiretroviral therapy have resulted in significantly decreased HIV-related mortality. HIV-associated neurocognitive disorders, however, continue to be a major problem in infected patients. The neuropathology underlying HIV-associated neurocognitive disorders has not been well characterized, and evidence suggests different contributing mechanisms. One potential mechanism is the induction of oxidative stress. Using the HIV-1 transgenic (Tg) rat model of HIV, we found increased striatal NADPH oxidase-4 and neuronal nitric oxide synthase expression in the adult (7- to 9-month-old) Tg rat compared with control rats but not in the young (1-month-old) Tg rats. This was accompanied by increased 3-nitrotyrosine (3-NT) immunostaining in the adult Tg rats, which worsened significantly in the old Tg rats (18 to 20 months old). There was, however, no concurrent induction of the antioxidant systems because there was no change in the expression of the nuclear factor-erythroid 2-related factor 2 and its downstream targets (thioredoxin and glutathione antioxidant systems). Colocalization of 3-NT staining with neurofilament proteins and evidence of decreased tyrosine hydroxylase and dopamine transporter expression in the old rats support dopaminergic involvement. We conclude that the HIV-1 Tg rat brain shows evidence of nitrosative stress without appropriate oxidation-reduction adaptation, whereas 3-NT modification of striatal neurofilament proteins likely points to the ensuing dopaminergic neuronal loss and dysfunction in the aging HIV-1 Tg rat.

Topics: Animals; Dopamine; Dopamine Plasma Membrane Transport Proteins; HIV Infections; HIV-1; Humans; Neurocognitive Disorders; Oxidative Stress; Rats; Rats, Transgenic; Tyrosine

HIV can infiltrate the brain and lead to HIV-associated neurocognitive disorders (HAND). The pathophysiology of HAND is poorly understood, and there are no diagnostic biomarkers for it. Previously, an increase in inducible nitric oxide synthase levels and protein tyrosine nitration in the brain were found to correlate with the severity of HAND.1,2 In this study, we analyzed human brains from individuals who had HIV infection without encephalitis and with encephalitis/HAND and compared them to the brains of healthy individuals. We identified the nitrated proteins and determined the sites of modification using affinity enrichment followed by high-resolution and high-mass-accuracy nanoLC-MS/MS. We found that nitrated proteins were predominantly present in the HIV-infected individuals with encephalitis, and, interestingly, the modifications were predominantly located on immunoglobulin variable regions. Our molecular model indicated potential interactions with HIV envelope proteins and changes on the heavy and light chain interface upon the nitration and nitrohydroxylation of these residues. Therefore, our findings suggest a role for these modifications in the immune response, which may have implications in disease pathogenesis.

Topics: Amino Acid Sequence; Antibodies; Brain; Brain Chemistry; Encephalitis, Viral; HIV Infections; HIV-1; Humans; Immunity, Innate; Immunoglobulin Variable Region; Models, Molecular; Molecular Sequence Data; Nitrates; Tyrosine

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.

Topics: Acetylcholine; Animals; Animals, Genetically Modified; Antioxidants; Aorta; Biopterins; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Endothelium, Vascular; Glutathione; HIV Infections; HIV-1; Human Immunodeficiency Virus Proteins; Male; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Nitroprusside; Oxidative Stress; Proviruses; Pyrrolidonecarboxylic Acid; Rats; Rats, Inbred F344; Superoxides; Thiazolidines; Tyrosine; Vasodilation; Vasodilator Agents; Xanthine Oxidase

Human immunodeficiency virus (HIV)-1 causes lung disease by increasing the host's susceptibility to pathogens. HIV-1 also causes an increase in systemic oxidative/nitrosative stress, perhaps enhancing the deleterious effects of secondary infections. Here we examined the ability of HIV-1 proteins to increase lung oxidative/nitrosative stress after lipopolysaccharide (LPS) (endotoxin) administration in an HIV-1 transgenic mouse model. Lung oxidative/nitrosative stress biomarkers studied 3 and 6 h after LPS administration were as follows: lung edema, tissue superoxide, NO metabolites, nitrotyrosine, hydrogen peroxide, and bronchoalveolar lavage fluid (BALF) glutathione (GSH). Blood serum cytokine levels were quantified to verify immune function of our nonimmunocompromised animal model. Results indicate that 3 h after LPS administration, HIV-1 transgenic mouse lung tissue has significantly greater edema and superoxide. Furthermore, NO metabolites are significantly elevated in HIV-1 transgenic mouse BALF, lung tissue, and blood plasma compared with those of wild-type mice. HIV-1 transgenic mice also produce significantly greater lung nitrotyrosine and hydrogen peroxide than wild-type mice. In addition, HIV-1 transgenic mice produce significantly less BALF GSH than wild-type mice 3 h after LPS treatment. Without treatment, serum cytokine levels are similar for HIV-1 transgenic and wild-type mice. After treatment, serum cytokine levels are significantly elevated in both HIV-1 transgenic and wild-type mice. Therefore, HIV-1 transgenic mice have significantly greater lung oxidative/nitrosative stress after endotoxin administration than wild-type mice, independent of immune function. These results indicate that HIV-1 proteins may increase pulmonary complications subsequent to a secondary infection by altering the lung redox potential.

Topics: Animals; Cytokines; Glutathione; HIV Infections; HIV-1; Hydrogen Peroxide; Lipopolysaccharides; Lung; Mice; Mice, Transgenic; Nitrates; Nitrites; Nitrosation; Oxidative Stress; Pulmonary Edema; Tyrosine

Oxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occurring 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).. 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.. Taken together, our results suggest that inhibition 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.

Topics: Apoptosis; Astrocytes; Astrocytoma; Catalysis; Culture Media, Conditioned; Ferric Compounds; Flow Cytometry; HIV Infections; HIV-1; Humans; Macrophages; Malondialdehyde; Metalloporphyrins; Peroxynitrous Acid; Tumor Cells, Cultured; Tyrosine

Balance and imbalance between oxidant stress and antioxidants contributes to the manifestations of human immunodeficiency virus (HIV) infection. Previously, we demonstrated a characteristic cytokine pattern in marrow supernatant from HIV patients who underwent diagnostic examinations.. In this report, we have determined the protein nitrotyrosine (pNT) concentration (an indicator of nitric oxide-superoxide interaction) as well as the concentration of the redox enzyme thioredoxin (Trx) in marrow supernatant from HIV patients, healthy controls, and other patients, and in serum from comparable subjects.. pNT concentrations were similar in serum and marrow supernatant and did not differ between subject subsets. Trx concentrations in both marrow supernatant and serum were higher for HIV patients than for other subjects; serum Trx concentrations were significantly higher than marrow Trx concentrations for the non-HIV patients and controls. The ratios of pNT/Trx concentrations in serum were similar in all subsets tested. In marrow aspirate, however, these ratios differed widely and significantly, with the highest values observed in non-HIV patients and the lowest in HIV patients. Only for HIV patients were serum and marrow supernatant pNT/Trx ratios similar. In HIV patients, marrow Trx concentrations correlated with CD4 count, CD4/CD8 ratio, and marrow colony-forming unit E (CFU-E) concentration; marrow pNT concentration correlated with the concentration of tumor necrosis factor in marrow supernatant, and with platelet count. No correlations were observed in other subject subsets.. These findings suggest that there is a degree of local regulation of the redox state of the marrow microenvironment that varies with the patients' clinical statuses, and which is associated with effects on hematopoiesis.

Topics: Adult; Blood Proteins; Bone Marrow; Case-Control Studies; Female; HIV Infections; Humans; Male; Middle Aged; Proteins; Thioredoxins; Tyrosine

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1beta, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.

Topics: Acquired Immunodeficiency Syndrome; Adult; Aged; AIDS Dementia Complex; Brain Chemistry; Female; HIV Infections; HIV-1; Humans; Immunohistochemistry; Interleukin-1; Interleukin-10; Macrophages; Male; Middle Aged; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; RNA, Messenger; Superoxide Dismutase; Superoxides; Tyrosine