Antiinflammatory and Antimicrobial Effects of Thiocyanate in a Cystic Fibrosis Mouse Model.

Thiocyanate (SCN) is used by the innate immune system, but less is known about its impact on inflammation and oxidative stress. Granulocytes oxidize SCN to evolve the bactericidal hypothiocyanous acid, which we previously demonstrated is metabolized by mammalian, but not bacterial, thioredoxin reductase (TrxR). There is also evidence that SCN is dysregulated in cystic fibrosis (CF), a disease marked by chronic infection and airway inflammation. To investigate antiinflammatory effects of SCN, we administered nebulized SCN or saline to ß epithelial sodium channel (ßENaC) mice, a phenotypic CF model. SCN significantly decreased airway neutrophil infiltrate and restored the redox ratio of glutathione in lung tissue and airway epithelial lining fluid to levels comparable to wild type. Furthermore, in Pseudomonas aeruginosa-infected ßENaC and wild-type mice, SCN decreased inflammation, proinflammatory cytokines, and bacterial load. SCN also decreased airway neutrophil chemokine keratinocyte chemoattractant (also known as C-X-C motif chemokine ligand 1) and glutathione sulfonamide, a biomarker of granulocyte oxidative activity, in uninfected ßENaC mice. Lung tissue TrxR activity and expression increased in inflamed lung tissue, providing in vivo evidence for the link between hypothiocyanous acid metabolism by TrxR and the promotion of selective biocide of pathogens. SCN treatment both suppressed inflammation and improved host defense, suggesting that nebulized SCN may have important therapeutic utility in diseases of both chronic airway inflammation and persistent bacterial infection, such as CF.

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase.

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe(V) redox intermediate of myeloperoxidase, to Compound II, which has Fe(IV) in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.

The common G-866A polymorphism of the UCP2 gene and survival in diabetic patients following myocardial infarction.

BACKGROUND: A variant in the promoter of the human uncoupling protein 2 (UCP2) gene, the G-866A polymorphism, has been associated with future risk of coronary heart disease events, in those devoid of traditional risk factors and in those suffering from diabetes. We thus examined the impact of the G-866A polymorphism on 5-year survival in a cohort of 901 post-myocardial infarction patients, and the impact of type-2 diabetes on this relationship. The association of UCP2 with baseline biochemical and hormonal measurements, including levels of the inflammatory marker myeloperoxidase, was also examined. METHODS: UCP2 G-866A genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol. Myeloperoxidase levels were measured in plasma samples taken from 419 cohort patients 24-96 hours after admission. RESULTS: Genotypes were obtained for 901 patients with genotype frequencies AA 15.5%, GA 45.5%, and GG 39.0%. Genotype was not associated with survival in the overall cohort (mortality: AA 15.6%, GA 16.8%, GG 19.4%, p = 0.541). However, amongst diabetics, AA and GA genotype groups had significantly worse survival than GG diabetic patients (p < 0.05) with an attributable risk of 23.3% and 18.7% for those of AA and GA genotype respectively. Multivariate analysis using a Cox proportional hazards model confirmed that the interaction of diabetes with genotype was significantly predictive of survival (p = 0.031). In the cohort's diabetic subgroup AA/GA patients had higher myeloperoxidase levels than their GG counterparts (GA/AA, n = 51, 63.9 +/- 5.23; GG, n = 34, 49.1 +/- 3.72 ng/ml, p = 0.041). Further analysis showed that this phenomenon was confined to male patients (GA/AA, n = 36, 64.3 +/- 6.23; GG, n = 29, 44.9 +/- 3.72 ng/ml, p = 0.015). CONCLUSION: Diabetic patients in this post-myocardial infarction cohort with UCP2 -866 AA/GA genotype have poorer survival and higher myeloperoxidase levels than their GG counterparts.

Extracellular amino acids and lipid peroxidation products in periventricular white matter during and after cerebral ischemia in preterm fetal sheep.

It is widely hypothesized that accumulation of excitatory amino acids, and oxygen free radicals during or after exposure to hypoxia-ischemia play a pivotal role in preterm periventricular white matter injury; however, there is limited evidence in the intact brain. In preterm fetal sheep (0.65 gestation; term 147 days) we found no significant increase in extracellular levels of excitatory amino acids measured by microdialysis in the periventricular white matter during cerebral ischemia induced by bilateral carotid occlusion. There was no significant change in 8-isoprostane or malondialdehyde levels in the early phase of recovery after occlusion. In contrast, there was a significant delayed increase in most amino acids and in malondialdehyde (but not 8-isoprostane) that was maximal approximately 2-3 days after occlusion. The increase in glutamate was significantly correlated with a secondary increase in cortical impedance, an index of cytotoxic edema, and with white matter damage 3 days post-insult. In conclusion, no significant accumulation of cytotoxins was found within immature white matter during cerebral ischemia. Although a minority of fetuses showed a delayed increase in some cytotoxins, this occurred many days after ischemia, in association with secondary cytotoxic edema, strongly suggesting that these changes are mainly a consequence of evolving cell death.

A myeloperoxidase precursor, pro-myeloperoxidase, is present in human plasma and elevated in cardiovascular disease patients.

Myeloperoxidase (MPO)-derived oxidants have emerged as a key contributor to tissue damage in inflammatory conditions such as cardiovascular disease. Pro-myeloperoxidase (pro-MPO), an enzymatically active precursor of myeloperoxidase (MPO), is known to be secreted from cultured bone marrow and promyelocytic leukemia cells, but evidence for the presence of pro-MPO in circulation is lacking. In the present study, we used a LC-MS/MS in addition to immunoblot analyses to show that pro-MPO is present in human blood plasma. Furthermore, we found that pro-MPO was more frequently detected in plasma from patients with myocardial infarction compared to plasma from control donors. Our study suggests that in addition to mature MPO, circulating pro-MPO may cause oxidative modifications of proteins thereby contributing to cardiovascular disease.

The effect of gestational age and labour on markers of lipid and protein oxidation in cord plasma.

There are many potential sources of reactive oxidants around the time of birth and pre-term infants are considered to be particularly vulnerable to oxidative injury. To gain insight into these processes, we have measured biomarkers of lipid and protein oxidation in umbilical cord plasma and related concentrations to mode of delivery and gestational age. Protein carbonyls were measured by ELISA and malondialdehyde (MDA) by HPLC after reaction with thiobarbituric acid, for 54 pre-term (< or = 36 weeks gestational age) and 43 term infants. Protein carbonyls were significantly lower in pre-term (median for < 32 weeks gestational age 0.048nmol/mg protein) than in term infants (0.105 nmol/mg, p = 0.004), and were unrelated to mode of delivery. In contrast, MDA concentrations were higher in the very pre-term (<32 weeks gestation) group (2.47 compared with 1.83 microM for term infants, p < 0.0001). MDA concentrations were higher in infants who were born with labour compared with elective caesarean section. Pre-eclampsia in the mother was associated with higher cord blood MDA concentrations. The MDA results are consistent with other studies of this marker and could be interpreted as indicating increased oxidative stress associated with prematurity and labour. However, the lower protein carbonyls in pre-term infants would lead to an opposite interpretation. More information is needed on the source and fate of these and other biomarkers before drawing strong conclusions on how they reflect oxidative stress in this and other clinical situations.

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction.

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol.49:1993-2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized approximately 1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P<0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.

Factors influencing local and systemic levels of plasma myeloperoxidase in ST-segment elevation acute myocardial infarction.

Myeloperoxidase (MPO) is associated with risk in acute coronary syndromes. However, the precise role it plays in ST-elevation myocardial infarction (STEMI) remains unclear. In this study we tested the hypothesis that levels of MPO in plasma after a myocardial infarction are affected by its ability to bind to the endothelium and there is local release of the enzyme at the culprit lesion. We measured plasma MPO in systemic circulation and throughout the coronary circulation in patients with STEMI undergoing primary percutaneous coronary intervention (PCI). MPO levels at the femoral artery were higher (p <0.001) in patients with STEMI (n = 67, median 45 ng/ml, interquartile range 34 to 83) compared to control patients (n = 12, 25 ng/ml, 19 to 30) with chronic stable angina undergoing elective PCI. After administration of the anticoagulant bivalirudin in 13 patients with STEMI, plasma MPO was increased only at the culprit coronary artery lesion before PCI (178 ng/ml, 91 to 245) versus all other sites (femoral artery 86 ng/ml, 54 to 139, p = 0.019). Administration of heparin caused a marked increase of plasma MPO. Even so, it was still possible to detect an increase of plasma MPO at culprit lesion in patients with STEMI (n = 54, 171 ng/ml, 122 to 230) versus controls (n = 12, 136 ng/ml, 109 to 151, p <0.05) after heparin and before PCI. MPO levels were higher at the culprit lesion in patients with STEMI who presented early and in those with restricted flow (p <0.05). In conclusion, our results demonstrate that, in addition to a systemic increase of MPO in patients presenting early with STEMI, levels of this leukocyte enzyme are increased at the culprit coronary lesion before PCI.

Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction.

OBJECTIVES: This study investigated relationships between plasma myeloperoxidase (MPO), protein oxidation markers, and clinical outcome retrospectively in patients after acute myocardial infarction (MI). BACKGROUND: Reactive oxidants are implicated in cardiovascular disease, and elevated plasma MPO is reported to predict adverse outcome in acute coronary syndromes. METHODS: Detailed demographic information, radionuclide ventriculography, neurohormone measurements, and clinical history were obtained for 512 acute MI patients at hospital admission. Plasma levels of MPO and protein carbonyls were measured in patients and 156 heart-healthy control subjects. 3-chlorotyrosine was measured in selected patients. Patient mortality was followed for 5 years. RESULTS: Plasma MPO and protein carbonyl concentrations were higher in MI patients 24 h to 96 h after admission than in control subjects (medians: MPO 55 ng/ml vs. 39 ng/ml, and protein carbonyls 48 pmol/mg vs. 17 pmol/mg protein, p < 0.001 for each). Both markers were significantly correlated with each other and with cardiovascular hormone levels. Chlorotyrosine was not elevated in patients with high MPO or carbonyl levels. Above-median levels of MPO but not protein carbonyls were independently predictive of mortality (odds ratio 1.8, 95% confidence interval 1.0 to 3.0, p = 0.034). Patients with above-median MPO levels in combination with above-median plasma amino-terminal pro-brain natriuretic peptide (NT-proBNP) or below-median left ventricular ejection fraction (LVEF) had significantly greater mortality compared with other patients. CONCLUSIONS: Myeloperoxidase and protein carbonyl levels are elevated in plasma after acute MI, apparently via independent mechanisms. High MPO is a risk factor for long-term mortality and adds prognostic value to LVEF and plasma NT-proBNP measurements.

Angiotensin type-1 receptor A1166C gene polymorphism correlates with oxidative stress levels in human heart failure.

Oxidative stress plays a critical role in the pathogenesis of cardiovascular disease and diabetes. Studies in vascular cells and experimental animals have demonstrated that the angiotensin type-1 receptor (AT1R) contributes to formation of reactive oxygen species by activating nicotinamide-adenine dinucleotide phosphate oxidases, but the relevance of this pathway to human heart disease has not been established. Here we demonstrate that a polymorphism in the AT1R gene (A1166C), linked to increased receptor activity, is associated with elevated levels of oxidative stress markers in heart failure patients but not in healthy controls. Plasma protein carbonyls (PCs), a marker of oxidative protein modification, were 10-fold higher in heart-failure patients compared with controls [geometric means and 95% CIs for patients, 75 (57 to 100) pmol/mg; controls, 5 (4 to 7) pmol/mg; P<0.001]. Moreover, levels of PCs were 50-fold higher in patients homozygous for the polymorphism (CC) than in controls and significantly higher than the AA and AC genotype patient groups [CC: 273 (135-550); AC: 59 (35-98); AA: 65 (40-106) pmol/mg; P<0.001]. Levels of myeloperoxidase were also modestly increased in heart-failure patients [51 (46-57) ng/mL] compared with controls [37 (32-44) ng/mL; P<0.001], but were especially elevated in patients with a CC genotype [CC: 72 (58-89); AC: 52 (44-61); AA: 39 (34-46) ng/mL; P<0.001]. The AT1R genotype was demonstrated to be an independent predictor of both PCs and myeloperoxidase levels in heart-failure patients. These findings suggest that oxidative stress in human heart failure is regulated via angiotensin signaling and may involve the nicotinamide dinucleotide oxidase pathway.

Nuclear factor kappaB activation in pulmonary leukocytes from infants with hyaline membrane disease: associations with chorioamnionitis and Ureaplasma urealyticum colonization.

Unresolved pulmonary inflammation in hyaline membrane disease (HMD) may be a precursor to the development of chronic lung disease of early infancy. We investigated whether nuclear factor kappaB (NF-kappaB), a transcription factor that regulates the inflammatory process, is activated in pulmonary leukocytes in tracheal aspirates from premature infants with HMD. A total of 172 samples were obtained from 59 infants, two thirds of whom showed NF-kappaB activation in lung neutrophils and macrophages on at least one occasion. Infants who had activated NF-kappaB showed elevated tumor necrosis factor-alpha concentrations in their tracheal aspirates. These infants also required a longer period of mechanical ventilation support. Almost half of the infants with HMD had antenatal exposure to chorioamnionitis on the basis of placental histopathologic examination. These infants had evidence of activated NF-kappaB and elevated cytokines and were more likely to have Ureaplasma urealyticum colonization in their airways. Together, these observations suggest that NF-kappaB activation in pulmonary leukocytes may be involved in the lung inflammatory process in infants with HMD.

The relationship of CSF and plasma cytokine levels to cerebral white matter injury in the premature newborn.

Ischemia and systemic infection are implicated in the etiology of periventricular white matter injury, a major cause of adverse motor and cognitive outcome in preterm infants. Cytokines are signaling proteins that can be produced as part of the inflammatory response to both ischemia and infection. The aim of this study was to relate cerebrospinal fluid (CSF) concentrations of IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) to magnetic resonance-defined white matter injury in preterm infants. Relationships between CSF and plasma cytokine concentrations were also examined. Preterm infants (

Elevated free radical products in the cerebrospinal fluid of VLBW infants with cerebral white matter injury.

Free radical mediated cellular injury has been hypothesized to play a key role in the pathogenesis of white matter injury in the premature infant, although direct evidence is lacking. Between April 1999 and May 2001, 22 very low birthweight infants, 30 term infants, and 17 adults had samples of cerebrospinal fluid (CSF) collected for clinical indications. Only CSF samples without any evidence of meningeal inflammation were analyzed for the levels of the lipid peroxidation products, 8-isoprostane and malondialdehyde (MDA), and protein carbonyls as a measure of protein oxidation. Chlorotyrosine was monitored as a measure of neutrophil oxidative activity. In the premature infants with subsequent evidence of white matter injury on magnetic resonance imaging at term, there was a significant elevation in the CSF level of protein carbonyls in comparison with the level in healthy premature infants, term infants, and adult controls (all p < 0.001). A significant difference in the levels of the lipid peroxidation products, 8-isoprostane and MDA, was apparent between premature infants with white matter injury and adult controls (isoprostanes p = 0.02, MDA p = 0.014). There was a trend toward higher levels of 8-isoprostane in the premature infants with white matter injury in comparison with those without white matter injury (p = 0.08), with 5 of the 14 infants with white matter injury having levels that were more than 10-fold higher than the top of the adult range. There was no significant difference in the level of chlorotyrosines among any of the groups. These preliminary data provide evidence of an association of elevated oxidative products during the evolution of white matter injury in the human premature infant.

Markers of oxidative injury in the cerebrospinal fluid of a premature infant with meningitis and periventricular leukomalacia.

Free radicals have been hypothesized to play a key role in the evolution of periventricular leukomalacia, although direct evidence of oxidative injury in the human infant is lacking. This case report is the first to demonstrate a marked elevation in the levels of lipid and protein oxidative products in the cerebrospinal fluid during the evolution of periventricular leukomalacia in a premature infant with meningitis.

Myeloperoxidase and protein oxidation in the airways of young children with cystic fibrosis.

Cystic fibrosis (CF) is characterized by considerable oxidative stress. However, it is not known whether oxidative stress is an important feature early in this disease. We have investigated a group of infants and young children with CF to establish whether oxidants are produced in their airways. Bronchoalveolar lavage fluid (BALF) was assayed for myeloperoxidase as a measure of neutrophilic inflammation, and 3-chlorotyrosine as a biomarker of the potent oxidant hypochlorous acid, which is formed by myeloperoxidase. Protein carbonyls were also measured as a nonspecific indicator of reactive oxidant production. Myeloperoxidase and 3-chlorotyrosine levels in BALF from children with CF were 10- and fivefold higher, respectively, than in disease control subjects. There was a strong correlation between myeloperoxidase and 3-chlorotyrosine. Myeloperoxidase levels were fourfold higher in children with infections in their airways. Median protein carbonyls were elevated by only twofold compared with disease control subjects, but some children had extremely high levels of protein oxidation. We conclude that hypochlorous acid is produced early in CF and that it is a candidate for precipitating the fatal decline in lung function associated with this disease. Also, there must be other sourcesof oxidants because protein carbonyls were not related to either inflammation or infection.