Tessa J. Mocatta

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-α), and interferon gamma (IFN-γ) to magnetic resonance–defined white matter injury in preterm infants. Relationships between CSF and plasma cytokine concentrations were also examined. Preterm infants (≤32 wk) and more mature infants from The Royal Womens Hospital, Melbourne, Australia, and Christchurch Womens Hospital, Christchurch, New Zealand, were eligible for study if they required a clinically indicated lumbar puncture. Plasma samples were obtained in a subgroup of Christchurch infants. Preterm infants underwent advanced quantitative volumetric magnetic resonance imaging using a 1.5-Tesla scanner at term equivalent. One hundred forty-six infants were enrolled and 190 CSF and 42 plasma samples obtained. There was no significant correlation between paired CSF and plasma concentrations for any cytokine. In comparing plasma and CSF concentrations, levels of IL-8 were significantly higher in CSF than plasma. Preterm infants with MRI-defined cerebral white matter injury had higher levels of IL-6, IL-10, and TNF-α in the CSF than infants without such injury. Plasma cytokine concentrations may not reflect CSF cytokine levels or inflammatory events within the brain. Elevated CSF levels of cytokines in infants with white matter injury suggest an altered inflammatory balance.

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.

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Background: Myeloperoxidase promotes oxidative stress during inflammation by producing hypochlorous acid. Results: Ceruloplasmin was a potent inhibitor of myeloperoxidase and slowed its activity in plasma from wild type mice compared with ceruloplasmin knock-out animals. Conclusion: Ceruloplasmin is a physiologically relevant inhibitor of myeloperoxidase. Significance: Ceruloplasmin will provide a protective shield against oxidant production by myeloperoxidase during inflammation. 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 FeV redox intermediate of myeloperoxidase, to Compound II, which has FeIV 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 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 (≤36 weeks gestational age) and 43 term infants. Protein carbonyls were significantly lower in pre-term (median for <32 weeks gestational age 0.048 nmol/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 μM 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.

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 κB 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 κB (NF-κB), 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-κB activation in lung neutrophils and macrophages on at least one occasion. Infants who had activated NF-κB showed elevated tumor necrosis factor-α 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-κB and elevated cytokines and were more likely to have Ureaplasma urealyticum colonization in their airways. Together, these observations suggest that NF-κB activation in pulmonary leukocytes may be involved in the lung inflammatory process in infants with HMD.

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.

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.

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 β epithelial sodium channel (βENaC) 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 βENaC 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 βENaC 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.