Lung nitroxidative stress in mechanically-ventilated septic patients: A pilot study.

PURPOSE: During sepsis and mechanical ventilation oxidative stress is generated by endothelial and inflammatory lung cells. Our main objective was to study pulmonary NO (nitric oxide) production and nitroxidative stress in mechanically-ventilated septic patients. METHODS: We study 69 mechanically ventilated patients, 36 with sepsis and 33 without sepsis within the first 48 h of ICU admission compared with 33 mechanically ventilated patients without sepsis (MV) plus eight operating room patients without lung disease served as control healthy group (ORCG). Nitrite plus nitrate (NOx-), 3-nitrotyrosine and malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) were analyzed. RESULTS: BALF NOx-, BALF 3-nitrotyrosine, BALF MDA, and plasma NOx- were higher in the Sepsis than in MV patients (all p < .05). Both SG and MV patients had higher BALF NOx- than the healthy control group (p < .001). In the Sepsis patients, the ICU non-survivors had higher levels of BALF NOx- than ICU survivors 80(70-127) µM versus 31(15-47) µM, p < .001. CONCLUSIONS: We conclude that during early phases of sepsis there is an enhanced lung nitroxidative stress due to an increase of NO production leading to secondary NO-derived oxidants, which promote protein nitration and lipid peroxidation.

Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice

Nitric oxide (ÀNO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ÀNO and oxidants leads to the generation of nitrogen dioxide (ÀNO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ÀNO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ÀNO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ÀNO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ÀNO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ÀNO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice.

Nitric oxide (.NO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of .NO and oxidants leads to the generation of nitrogen dioxide (.NO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of .NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of .NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished .NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the .NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce .NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

Unraveling peroxynitrite formation in biological systems.

Peroxynitrite promotes oxidative damage and is implicated in the pathophysiology of various diseases that involve accelerated rates of nitric oxide and superoxide formation. The unambiguous detection of peroxynitrite in biological systems is, however, difficult due to the combination of a short biological half-life, limited diffusion, multiple target molecule reactions, and participation of alternative oxidation/nitration pathways. In this review, we provide the conceptual framework and a comprehensive analysis of the current experimental strategies that can serve to unequivocally define the existence and quantitation of peroxynitrite in biological systems of different levels of organization and complexity.

Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death.

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.

Low levels of interleukin-4 and high levels of transforming growth factor beta in rheumatoid synovitis.

Since interleukin-4 (IL-4) displays agonistic effects on both T and B cells, we studied whether this lymphokine is involved in rheumatoid synovitis, a disease characterized by intense T cell infiltration and B cell stimulation. Rheumatoid arthritis synovial fluids (RA SF) contained no (less than 15 pg/ml) or very low amounts (less than 25 pg/ml) of IL-4, as measured by a sensitive and specific enzyme-linked immunosorbent assay. No IL-4 was produced by unstimulated rheumatoid synovial membrane. RA SF were found to inhibit phorbol myristate acetate (PMA)-dependent proliferation of normal peripheral blood lymphocytes (PBL). An inhibitory fraction with an apparent molecular weight of 150 kd was isolated by gel filtration. The inhibitory fraction strongly blocked the proliferation of PBL induced by PMA, PMA + IL-2, or PMA + IL-4. However, this fraction was less effective in blocking the proliferation of PBL induced by PMA + IL-2 + IL-4. High levels of transforming growth factor beta (TGF beta) were found in these RA SF, and an anti-TGF beta antibody was able to partially reduce the inhibitory activity. RA SF were found to inhibit phytohemagglutinin-induced IL-4 production by PBL. These data indicate that IL-4, similar to other T cell lymphokines, cannot be detected in RA SF and that RA SF contains an inhibitory activity, related in part to TGF beta, which blocks mitogen-induced proliferation of PBL, at least in part through an inhibition of T cell-derived lymphokine release.