Effect of chronic mucus hypersecretion on treatment responses to inhaled therapies in patients with chronic obstructive pulmonary disease: Post hoc analysis of the IMPACT trial.

BACKGROUND AND OBJECTIVE: Chronic mucus hypersecretion (CMH) is a clinical phenotype of COPD. This exploratory post hoc analysis assessed relationship between CMH status and treatment response in IMPACT. METHODS: Patients were randomized to once-daily fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100/62.5/25 µg, FF/VI 100/25 µg or UMEC/VI 62.5/25 µg and designated CMH+ if they scored 1/2 in St George's Respiratory Questionnaire (SGRQ) questions 1 and 2. Endpoints assessed by baseline CMH status included on-treatment exacerbation rates, change from baseline in trough forced expiratory volume in 1 second, SGRQ total score, COPD Assessment Test (CAT) score, proportion of SGRQ and CAT responders at Week 52 and safety. RESULTS: Of 10,355 patients in the intent-to-treat population, 10,250 reported baseline SGRQ data (CMH+: 62% [n = 6383]). FF/UMEC/VI significantly (p < 0.001) reduced on-treatment moderate/severe exacerbation rates versus FF/VI and UMEC/VI in CMH+ (rate ratio: 0.87 and 0.72) and CMH- patients (0.82 and 0.80). FF/UMEC/VI significantly (p < 0.05) reduced on-treatment severe exacerbation rates versus UMEC/VI in CMH+ (0.62) and CMH- (0.74) subgroups. Similar improvements in health status and lung function with FF/UMEC/VI were observed, regardless of CMH status. In CMH+ patients, FF/VI significantly (p < 0.001) reduced on-treatment moderate/severe and severe exacerbation rates versus UMEC/VI (0.83 and 0.70). CONCLUSION: FF/UMEC/VI had a favourable benefit: risk profile versus dual therapies irrespective of CMH status. The presence of CMH did not influence treatment response or exacerbations, lung function and/or health status. However, CMH did generate differences when dual therapies were compared and the impact of CMH should be considered in future trial design.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α).

OBJECTIVE: Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body's response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient (Hmox1-/-) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1-/- mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O2) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1-/- mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1-/- fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1-/- fibroblasts in response to hypoxia. CONCLUSIONS: Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1's protection against ischemic injury independent of neovascularization.

Inhibition of MPO (Myeloperoxidase) Attenuates Endothelial Dysfunction in Mouse Models of Vascular Inflammation and Atherosclerosis.

Objective- Inflammation-driven endothelial dysfunction initiates and contributes to the progression of atherosclerosis, and MPO (myeloperoxidase) has been implicated as a potential culprit. On release by circulating phagocytes, MPO is thought to contribute to endothelial dysfunction by limiting NO bioavailability via formation of reactive oxidants including hypochlorous acid. However, it remains largely untested whether specific pharmacological inhibition of MPO attenuates endothelial dysfunction. We, therefore, tested the ability of a mechanism-based MPO inhibitor, AZM198, to inhibit endothelial dysfunction in models of vascular inflammation. Approach and Results- Three models of inflammation were used: femoral cuff, the tandem stenosis model of plaque rupture in Apoe-/- mice, and C57BL/6J mice fed a high-fat, high-carbohydrate diet as a model of insulin resistance. Endothelial dysfunction was observed in all 3 models, and oral administration of AZM198 significantly improved endothelial function in the femoral cuff and tandem stenosis models only. Improvement in endothelial function was associated with decreased arterial MPO activity, determined by the in vivo conversion of hydroethidine to 2-chloroethidium, without affecting circulating inflammatory cytokines or arterial MPO content. Mechanistic studies in Mpo-/- mice confirmed the contribution of MPO to endothelial dysfunction and revealed oxidation of sGC (soluble guanylyl cyclase) as the underlying cause of the observed limited NO bioavailability. Conclusions- Pharmacological inhibition of MPO is a potential strategy to limit endothelial dysfunction in vascular inflammation. Visual Overview- An online visual overview is available for this article.

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Myeloperoxidase is a potential molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque.

Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque. Methods and results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene. Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

Endothelial indoleamine 2,3-dioxygenase-1 regulates the placental vascular tone and is deficient in intrauterine growth restriction and pre-eclampsia.

Indoleamine 2,3-dioxygenase-1 (IDO1) mediates the degradation of L-tryptophan (L-Trp) and is constitutively expressed in the chorionic vascular endothelium of the human placenta with highest levels in the microvasculature. Given that endothelial expression of IDO1 has been shown to regulate vascular tone and blood pressure in mice under the condition of systemic inflammation, we asked whether IDO1 is also involved in the regulation of placental blood flow and if yes, whether this function is potentially impaired in intrauterine growth restriction (IUGR) and pre-eclampsia (PE). In the large arteries of the chorionic plate L-Trp induced relaxation only after upregulation of IDO1 using interferon gamma and tumor necrosis factor alpha. However, ex vivo placental perfusion of pre-constricted cotyledonic vasculature with L-Trp decreases the vessel back pressure without prior IDO1 induction. Further to this finding, IDO1 protein expression and activity is reduced in IUGR and PE when compared to gestational age-matched control tissue. These data suggest that L-Trp catabolism plays a role in the regulation of placental vascular tone, a finding which is potentially linked to placental and fetal growth. In this context our data suggest that IDO1 deficiency is related to the pathogenesis of IUGR and PE.

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress per se can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 µm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking.

Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance.

Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered mitochondrial oxidants and insulin resistance while CoQ supplementation in either insulin-resistant cell models or mice restored normal insulin sensitivity. Specifically, lowering of mitochondrial CoQ caused insulin resistance in adipocytes as a result of increased superoxide/hydrogen peroxide production via complex II. These data suggest that mitochondrial CoQ is a proximal driver of mitochondrial oxidants and insulin resistance, and that mechanisms that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance.

Absence of the biliverdin reductase-a gene is associated with increased endogenous oxidative stress.

Bilirubin, a byproduct of heme catabolism, has been shown to be an effective lipid-soluble antioxidant in vitro. Bilirubin is able to inhibit free radical chain reactions and protects against oxidant-induced damage in vitro and ex vivo. However, direct evidence for bilirubin's antioxidant effects in vivo remains limited. As bilirubin is formed from biliverdin by biliverdin reductase, we generated global biliverdin reductase-a gene knockout (Bvra-/-) mice to assess the contribution of bilirubin as an endogenous antioxidant. Bvra-/- mice appear normal and are born at the expected Mendelian ratio from Bvra+/- x Bvra+/- matings. Compared with corresponding littermate Bvra+/+ and Bvra+/- animals, Bvra-/- mice have green gall bladders and their plasma concentrations of biliverdin and bilirubin are approximately 25-fold higher and 100-fold lower, respectively. Naïve Bvra-/- and Bvra+/+ mice have comparable plasma lipid profiles and low-molecular weight antioxidants, i.e., ascorbic acid, α-tocopherol and ubiquinol-9. Compared with wild-type littermates, however, plasma from Bvra-/- mice contains higher concentrations of cholesteryl ester hydroperoxides (CE-OOH), and their peroxiredoxin 2 (Prx2) in erythrocytes is more oxidized as assessed by the extent of Prx2 dimerization. These data show that Bvra-/- mice experience higher oxidative stress in blood, implying that plasma bilirubin attenuates endogenous oxidative stress.

Characterization of plasma labile heme in hemolytic conditions.

Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10-7 m and that 2-8% (~ 2-5 µm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10-7 m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Near-infrared autofluorescence induced by intraplaque hemorrhage and heme degradation as marker for high-risk atherosclerotic plaques.

Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology that allows identification of atherosclerotic plaques with intraplaque hemorrhage and ultimately holds promise for detection of high-risk plaques in patients.Atherosclerosis diagnosis relies primarily on imaging and early detection of high-risk atherosclerotic plaques is important for risk stratification of patients and stabilization therapies. Here Htun et al. demonstrate that vulnerable atherosclerotic plaques generate near-infrared autofluorescence that can be detected via emission computed tomography.

The roles of myeloperoxidase in coronary artery disease and its potential implication in plaque rupture.

Atherosclerosis is the main pathophysiological process underlying coronary artery disease (CAD). Acute complications of atherosclerosis, such as myocardial infarction, are caused by the rupture of vulnerable atherosclerotic plaques, which are characterized by thin, highly inflamed, and collagen-poor fibrous caps. Several lines of evidence mechanistically link the heme peroxidase myeloperoxidase (MPO), inflammation as well as acute and chronic manifestations of atherosclerosis. MPO and MPO-derived oxidants have been shown to contribute to the formation of foam cells, endothelial dysfunction and apoptosis, the activation of latent matrix metalloproteinases, and the expression of tissue factor that can promote the development of vulnerable plaque. As such, detection, quantification and imaging of MPO mass and activity have become useful in cardiac risk stratification, both for disease assessment and in the identification of patients at risk of plaque rupture. This review summarizes the current knowledge about the role of MPO in CAD with a focus on its possible roles in plaque rupture and recent advances to quantify and image MPO in plasma and atherosclerotic plaques.

Behavioral and cognitive data in mice with different tryptophan-metabolizing enzymes knocked out.

This article demonstrates behavioral changes in mice in response to free adaptation and drinking session adaptation modules implemented in their social home environment, the IntelliCage. These data complement the study "Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function" (Too LK, Li KM, Suarna C, Maghzal GJ, Stocker R, McGregor IS, et al., 2016) [1]. Prior to programmed drinking sessions, all mice were exposed to a home cage adaptation module during which there was no time limit on water access - the free adaptation module. The exploratory behaviors are here expressed as percentages of visits with nosepokes and of visits with licks. The measurements by percentage of exploratory activity showed minimal genotype effects. The number of nosepokes or licks per corner visit also was compared between WT and gene knockout (GKO) IDO1 mice, WT and GKO IDO2 mice and WT and GKO TDO2 mice and demonstrated unremarkable behavioral changes during the free adaptation module. Analysis of drinking session adaptation behavior showed no genotype effect between WT and GKO of IDO1, IDO2 or TDO2 background. Notwithstanding the absence of genotype differences, each IDO1, IDO2 or TDO2 animal group displayed a specific pattern of adaptation to the drinking session modules. Furthermore, IDO1 GKO mice showed a more rapid recovery of lick frequency to the baseline level compared to the WT equivalents in a simple patrolling task during the first complete testing cycle (R1). TDO2 GKO mice on the other hand did not differ from their WT equivalents in terms of lick frequency over the three test days of complex patrolling and discrimination reversal tasks. Lastly, IDO2 GKO mice reduced their visits to the permanently non-rewarding reference corners by the same degree as did the WT mice.

Absence of systemic oxidative stress and increased CSF prostaglandin F2α in progressive MS.

OBJECTIVE: We aimed to investigate the role of oxidative stress in the progression of multiple sclerosis (MS). METHODS: We determined by liquid chromatography-tandem mass spectrometry nonenzymatic (F2-isoprostanes) and enzymatic oxidation products of arachidonic acid (prostaglandin F2α [PGF2α]) in plasma and CSF of 45 controls (other neurologic disease [OND] with no signs of inflammation) and 62 patients with MS. Oxidation products were correlated with disease severity and validated biomarkers of inflammation (chemokine ligand 13; matrix metalloproteinase-9; osteopontin) and axonal damage (neurofilament light protein). RESULTS: Compared with OND controls, plasma concentrations of F2-isoprostanes and PGF2α were significantly lower in patients with progressive disease, and decreased with increasing disability score (Expanded Disability Status Scale). In contrast, CSF concentrations of PGF2α, but not F2-isoprostanes, were significantly higher in patients with progressive disease than OND controls (p < 0.01). The content of PGF2α in CSF increased with disease severity (p = 0.044) and patient age (p = 0.022), although this increase could not be explained by age. CSF PGF2α decreased with natalizumab and methylprednisolone treatment and was unaffected by the use of nonsteroidal anti-inflammatory drug in secondary progressive MS. CSF PGF2α did not associate with validated CSF markers of inflammation and axonal damage that themselves did not associate with the Expanded Disability Status Scale. CONCLUSIONS: Our data suggest that MS progression is associated with low systemic oxidative activity. This may contribute to immune dysregulation with CNS inflammation accompanied by increased local cyclooxygenase-dependent lipid oxidation.

Correction: AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation.

[This corrects the article DOI: 10.1371/journal.pone.0148213.].

Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function.

Tryptophan, an amino acid involved in routine energy metabolism, is a key modulator of sickness behaviors associated with inflammatory states and also plays roles in some psychiatric disorders. Tissue concentrations of tryptophan are regulated primarily by the enzymes indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO, encoded by TDO2). Altered IDO1 and TDO activities have been linked to the perturbed serotonergic neurotransmission that may underlie certain psychopathologies. Here we assessed mice genetically modified to be deficient in IDO1, IDO2 or TDO2 for their behavior and cognitive function using an automated home cage system, the IntelliCage™. A well-established behavioural and cognitive test battery was applied during two periods (Runs 1 and 2, "R1" and "R2") separated by one month. Various tryptophan-related neurochemicals also were measured in brain extracts. IDO1(-/-) mice displayed remarkable reductions of early diurnal exploration in the IntelliCage and this persisted in R2. In contrast, early diurnal hyperactivity was observed in IDO2(-/-) mice in both R1 and R2. TDO2(-/-) mice displayed increased diurnal and nocturnal exploration, but only in R2. Cognitive assessment suggested enhanced reference memory in IDO2(-/-) mice in a complex patrolling task, while TDO deficiency was associated with enhanced performance in complex patrolling and discrimination reversal tasks. Neurochemical measures showed attenuated brain serotonin levels in IDO1(-/-) mice and augmented tryptophan and serotonin levels in TDO2(-/-) animals, respectively. No neurochemical alterations were detected in IDO2(-/-) mice. Taken together, these findings reveal complex and dissimilar patterns of behavioral and cognitive changes induced by knockout of three different tryptophan-metabolizing enzymes.

Detailed protocol to assess in vivo and ex vivo myeloperoxidase activity in mouse models of vascular inflammation and disease using hydroethidine.

Myeloperoxidase (MPO) activity contributes to arterial inflammation, vascular dysfunction and disease, including atherosclerosis. Current assessment of MPO activity in biological systems in vivo utilizes 3-chlorotyrosine (3-Cl-Tyr) as a biomarker of hypochlorous acid (HOCl) and other chlorinating species. However, 3-Cl-Tyr is formed in low yield and is subject to further metabolism. Recently, we reported a method to selectively assess MPO-activity in vivo by measuring the conversion of hydroethidine to 2-chloroethidium (2-Cl-E(+)) by liquid chromatography with tandem mass spectrometry (LC-MS/MS) (J. Biol. Chem., 289, 2014, pp. 5580-5595). The hydroethidine-based method has greater sensitivity for MPO activity than measurement of 3-Cl-Tyr. The current methods paper provides a detailed protocol to determine in vivo and ex vivo MPO activity in arteries from mouse models of vascular inflammation and disease by utilizing the conversion of hydroethidine to 2-Cl-E(+). Procedures for the synthesis of standards, preparation of tissue homogenates and the generation of 2-Cl-E(+) are also provided in detail, as are the conditions for LC-MS/MS detection of 2-Cl-E(+).