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Peroxynitrite1 is a powerful oxidant produced in biological systems from superoxide (O 2˙-) and nitric oxide (˙NO). Peroxynitrite has been implicated in a host of disease states, including neurodegenerative disorders [1], chronic inflammation and autoimmune diseases [2,3], ischemia-reperfusion injury [4] and septic shock [5].The chemistry of peroxynitrite is complex and highly pHdependent [6,7]. Not only peroxynitrite anion (ONOO -) and its conjugated acid, peroxynitrous acid (ONOOH) (pK a = 6.8) are potent oxidants by themselves [8,9] but also they can lead to the production of secondary reactive species. Moreover, peroxynitrite is capable of nitrating aromatics [10] in a process that can be enhanced by metal centres and CO 2 [11].Despite of being a reactive and short-lived species, which precludes its direct isolation or measurement, peroxynitrite detection and quantitation can be achieved with the use of detector molecules as well as molecular footprints that take advantage of oxidation and nitration reactions. A good detector molecule should be sensitive and outcompete the multiple reactions that peroxynitrite can undergo in biological systems. Moreover, both detector molecules and footprints should be specific, in order to discriminate between the reactions of peroxynitrite from those of other species, including its precursors ˙NO and O2 -, and other ˙NO or O˙2 --derived oxidants. None of the detection systems used until now fulfil all these requisites, and a subtle knowledge of the biological chemistry of peroxynitrite is required in order to choose and utilise the best approach in different biochemical or biological systems. Peroxynitrite formationAlthough several possible routes lead to peroxynitrite formation, the main one in biology arises from the radicalradical combination reaction of ˙NO[12] and O˙2 - [13,14] NOThis is a diffusion-controlled reaction with a second order rate constant (k 2 ) that has been independently determined as 4. 1 The term peroxynitrite refers to the sum of peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH). IUPAC recommended names for peroxynitrite anion, peroxynitrous acid, nitroso-peroxocarboxylate (ONOOCO 2 -) and nitric oxide are oxoperoxynitrate (1-), hydrogen oxoperoxynitrate, 1-carboxylato-2-nitrosodioxidane and nitrogen monoxide respectively.

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 (<15 pg/ml) or very low amounts (C25 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 (PMAWependent 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 p (TGFP) were found in these RA SF, and an anti-TGFP antibody was able to Interleukin-4 (IL-4), a T cell-derived lymphokine first described in terms of its ability to activate B cells, was later shown to act on T cells (6,7). Human IL-4 can induce the proliferation of activated T and B cells (%lo), the expression of the low-affinity receptor for IgE (Fc, R2) on B cells (1 l), the production of IgG and IgM by activated B cells (12). and the expression of CD8 on CD4+ T cell clones (13). Thus, the large number of activated T and B cells in rheumatoid synovitis (1-3). the presence of CD4+, CD8+ T cells (14). and the production of immunoglobulins by the synovium (2) led us to consider a role for IL-4 in rheumatoid synovitis.We studied synovial fluids (SF) from rheuma-

Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO 2 ) 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 -nitroarginine methyl ester treatment abolished • NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanidoethyl 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.

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.