SummaryNitric oxide (NO)-related activity has been shown to be protective against Plasmodium fakiparum in vitro. It has been hypothesized, however, that excess NO production contributes to the pathogenesis of cerebral malaria. The purpose of this study was to compare markers of NO production [urinary and plasma nitrate + nitrite (NO~)], leukocyte-inducible nitric oxide synthase type 2 (NOS2), and plasma TNF-c~ and IL-10 levels with disease severity in 191 Tanzanian children with and without malaria. Urine NO• excretion and plasma NOx levels (corrected for renal impairment) were inversely related to disease severity, with levels highest in subclinical infection and lowest in fatal cerebral malaria. Results could not be explained by differences in dietary nitrate ingestion among the groups. Plasma levels of IL-10, a cytokine known to suppress NO synthesis, increased with disease severity. Leukocyte NOS2 antigen was detectable in all control children tested and in all those with subclinical infection, but was undetectable in all but one subject with cerebral malaria. This suppression of NO synthesis in cerebral malaria may contribute to pathogenesis. In contrast, high fasting NO x levels and leukocyte NOS2 in healthy controls and asymptomatic infection suggest that increased NO synthesis might protect against clinical disease. NO appears to have a protective rather than pathological role in African children with malaria.

Summary MRL-I/r/I/rHowever, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.

One of the phenotypes of mice with targeted disruption of the uncoupling protein-2 gene (Ucp2؊/؊) is greater macrophage phagocytic activity and free radical production, resulting in a striking resistance to infectious microorganisms. In this study, the molecular mechanisms of this enhanced immune response were investigated. We found that levels of nitric oxide measured in either plasma or isolated macrophages from Ucp2؊/؊ mice are significantly elevated in response to bacterial lipopolysaccharide challenge compared with similarly treated Ucp2؉/؉ mice. Likewise, expression of inducible nitric-oxide synthase and inflammatory cytokines is higher in Ucp2؊/؊ mice in vivo and in vitro. Key steps in the activation cascade of nuclear factor (NF)-B, including IB kinase and nuclear translocation of NF-B subunits, are all remarkably enhanced in Ucp2؊/؊ mice, most notably even under basal conditions. The elevated basal activity of IB kinase in macrophages from Ucp2؊/؊ mice can be blocked by cell-permeable inhibitors of superoxide and hydrogen peroxide generation, but not by a specific inhibitor for inducible nitric-oxide synthase. Isolated mitochondria from Ucp2؊/؊ cells produced more superoxide/hydrogen peroxide. We conclude that mitochrondrially derived reactive oxygen from Ucp2؊/؊ cells constitutively activates NF-B, resulting in a "primed" state to both potentiate and amplify the inflammatory response upon subsequent stimulation. Uncoupling protein (UCP)1 -2 is a mitochondrial inner membrane carrier protein that was discovered through its homology to the brown fat UCP1 (1). Whereas UCP1 has been clearly established as the molecular mediator of non-shivering thermogenesis (reviewed in Ref. 2), the function of UCP2 remains somewhat of an enigma. Several features of UCP2 led us to initially propose that it was a bona fide uncoupling protein involved in the dissipation of excess metabolic fuel as heat. These aspects of UCP2 included its structural resemblance to UCP1, its ability to uncouple respiration in model assay systems, and a chromosomal location to a region with genetic linkage to obesity and hyperinsulinemia (1, 3). However, although Ucp2 mRNA is expressed in a broad array of tissues in humans and rodent models (3, 4), including metabolically important organs, it exists in minute amounts compared with the level of UCP1 in brown fat. Moreover, it is present in cell types such as pancreatic ␤-cells, lymphocytes, and neurons that are not typically associated with thermogenesis (5-7). These and other features of UCP2 (8), together with the critical finding that targeted disruption of the Ucp2 gene did not result in obesity, cold sensitivity, or demonstrable differences in coupling efficiency in isolated mitochondria (9 -11), strongly suggested a functionally distinct role for this protein.One of the phenotypes of Ucp2Ϫ/Ϫ mice was a striking resistance to infectious microorganisms associated with greater macrophage phagocytic activity and free radical production (9). We showed that macrophages from Ucp2Ϫ/Ϫ mice produced...

Nitric oxide (NO) production and NO synthase (NOS) expression are increased in osteoarthritis and rheumatoid arthritis, suggesting that NO may play a role in the destruction of articular cartilage. To test the hypothesis that mechanical stress may increase NO production by chondrocytes, we measured the effects of physiological levels of static and intermittent compression on NOS activity, NO production, and NOS antigen expression by porcine articular cartilage explants. Static compression significantly increased NO production at 0.1 MPa stress for 24 h (P < 0.05). Intermittent compression at 0.5 Hz for 6 h followed by 18 h recovery also increased NO production and NOS activity at 1.0 MPa stress (P < 0.05). Intermittent compression at 0.5 Hz for 24 h at a magnitude of 0.1 or 0.5 MPa caused an increase in NO production and NOS activity (P < 0.05). Immunoblot analysis showed stress-induced upregulation of NOS2, but not NOS1 or NOS3. There was no loss in cell viability following any of the loading regimens. Addition of 2 mM 1400 W (a specific NOS2 inhibitor) reduced NO production by 51% with no loss of cell viability. These findings indicate that NO production by chondrocytes is influenced by mechanical compression in vitro and suggest that biomechanical factors may in part regulate NO production in vivo.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage- mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase- polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell- permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.