Decreased systemic bioavailability of L-arginine in patients with cystic fibrosis

BACKGROUND: L-arginine is the common substrate for nitric oxide synthases and arginases. Increased arginase levels in the blood of patients with cystic fibrosis may result in L-arginine deficiency and thereby contribute to low airway nitric oxide formation and impaired pulmonary function. METHODS: Plasma amino acid and arginase levels were studied in ten patients with cystic fibrosis before and after 14 days of antibiotic treatment for pulmonary exacerbation. Patients were compared to ten healthy non-smoking controls. RESULTS: Systemic arginase levels measured by ELISA were significantly increased in cystic fibrosis with exacerbation compared to controls (17.3 ± 12.0 vs. 4.3 ± 3.4 ng/ml, p < 0.02). Arginase levels normalized with antibiotic treatment. Plasma L-arginine was significantly reduced before (p < 0.05) but not after treatment. In contrast, L-ornithine, proline, and glutamic acid, all downstream products of arginase activity, were normal before, but significantly increased after antibiotic therapy. Bioavailability of L-arginine was significantly reduced in cystic fibrosis before and after exacerbation (p < 0.05, respectively). CONCLUSION: These observations provide further evidence for a disturbed balance between the L-arginine metabolic pathways in cystic fibrosis

Regulation of T Cell Priming by Lymphoid Stroma

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues

Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production

Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection

Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

Inactivation of [Fe-S] Metalloproteins Mediates Nitric Oxide-Dependent Killing of Burkholderia mallei

BACKGROUND: Much remains to be known about the mechanisms by which O(2)-dependent host defenses mediate broad antimicrobial activity. METHODOLOGY/PRINCIPAL FINDINGS: We show herein that reactive nitrogen species (RNS) generated by inducible nitric oxide (NO) synthase (iNOS) account for the anti-Burkholderia mallei activity of IFNgamma-primed macrophages. Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H(2)O(2), which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes. CONCLUSIONS/SIGNIFICANCE: Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners