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There is an urgent need to develop sensitive, non-invasive biomarkers that can track airway inflammatory activity for patients with cystic fibrosis (CF). Urinary glutathione sulfonamide (GSA) levels correlate well with GSA levels in BAL samples and other markers of neutrophilic inflammation, suggesting that this biomarker may be suitable for tracking disease activity in this population. We recruited 102 children (median 11.5 years-old) and 64 adults (median 32.5 years-old) who were admitted to hospital for management of an acute pulmonary exacerbation and/or eradication of infectious agents such as Pseudomonas aeruginosa or Staphylococcus aureus. Our aim was to explore how urinary GSA levels changed across admission timepoints. Urine samples were collected at admission and discharge, and GSA measured by liquid chromatography with mass spectrometry. Paired admission-discharge results were compared using Wilcoxon signed-rank test. Paired admission-discharge samples were available for 53 children and 60 adults. A statistically significant difference was observed between admission-discharge for children and adults. Spearman's correlation analysis identified a correlation between urinary GSA levels and sex and S. aureus infection for children only. Our preliminary findings suggest that urinary GSA is responsive to the resolution of an acute pulmonary exacerbation and therefore warrants further studies in this population.
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BACKGROUND AND AIMS: Inflammatory bowel disease [IBD], consisting of Crohn's disease [CD] and ulcerative colitis [UC], is a relapsing-remitting illness. Treat-to-target IBD management strategies require monitoring of gastrointestinal inflammation. This study aimed to investigate faecal myeloperoxidase [fMPO], a neutrophil granule enzyme, as a biomarker of IBD activity. METHODS: Prospectively recruited participants with IBD, undergoing ileocolonoscopy for disease assessment, provided biological samples and completed symptom questionnaires prior to endoscopy. fMPO, C-reactive protein [CRP], and faecal calprotectin [fCal] were compared with validated endoscopic indices [simple endoscopic score for CD and UC endoscopic index of severity]. Receiver operating characteristic [ROC] curves assessed the performance of fMPO, CRP, and fCal in predicting endoscopic disease activity. Baseline biomarkers were used to predict a composite endpoint of complicated disease at 12 months [need for escalation of biologic/immunomodulator due to relapse, steroid use, IBD-related hospitalisation, and surgery]. RESULTS: A total of 172 participants were recruited [91 female, 100 with CD]. fMPO was significantly correlated with endoscopic activity in both CD [râ =â 0.53, pâ <â 0.01] and UC [râ =â 0.63, pâ <â 0.01], and with fCal in all patients with IBD [râ =â 0.82, pâ <â 0.01]. fMPO was effective in predicting moderate-to-severely active CD [AUROC 0.86, pâ <â 0.01] and UC [AUROC 0.92, pâ <â 0.01]. Individuals with a baseline fMPOâ >â 26 µg/g were significantly more likely to reach the composite outcome at 12 months (hazard ratio [HR] 3.71, 95% confidence interval [CI] 2.07-6.64, pâ <â 0.01). CONCLUSIONS: Faecal myeloperoxidase is an accurate biomarker of endoscopic activity in IBD and predicted a more complicated IBD course during follow-up.
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Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Feminino , Humanos , Biomarcadores/metabolismo , Proteína C-Reativa/metabolismo , Doença Crônica , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/metabolismo , Doença de Crohn/diagnóstico , Doença de Crohn/metabolismo , Endoscopia Gastrointestinal , Fezes/química , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Peroxidase/metabolismo , Índice de Gravidade de Doença , MasculinoRESUMO
Targeting immune evasion tactics of pathogenic bacteria may hold the key to treating recalcitrant bacterial infections. Staphylococcus aureus produces bacillithiol (BSH), its major low-molecular-weight thiol, which is thought to protect this opportunistic human pathogen against the bombardment of oxidants inside neutrophil phagosomes. Here, we show that BSH was oxidized when human neutrophils phagocytosed S. aureus, but provided limited protection to the bacteria. We used mass spectrometry to measure the oxidation of BSH upon exposure of S. aureus USA300 to either a bolus of hypochlorous acid (HOCl) or a flux generated by the neutrophil enzyme myeloperoxidase. Oxidation of BSH and loss of bacterial viability were strongly correlated (r = 0.99, p < 0.001). BSH was fully oxidized after exposure of S. aureus to lethal doses of HOCl. However, there was no relationship between the initial BSH levels and the dose of HOCl required for bacterial killing. In contrast to the HOCl systems, only 50% of total BSH was oxidized when neutrophils killed the majority of phagocytosed bacteria. Oxidation of BSH was decreased upon inhibition of myeloperoxidase, implicating HOCl in phagosomal BSH oxidation. A BSH-deficient S. aureus USA300 mutant was slightly more susceptible to treatment with either HOCl or ammonia chloramine, or to killing within neutrophil phagosomes. Collectively, our data show that myeloperoxidase-derived oxidants react with S. aureus inside neutrophil phagosomes, leading to partial BSH oxidation, and contribute to bacterial killing. However, BSH offers only limited protection against the neutrophil's multifaceted killing mechanisms.
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Neutrófilos , Staphylococcus aureus , Cisteína/análogos & derivados , Cisteína/metabolismo , Glucosamina/análogos & derivados , Humanos , Ácido Hipocloroso/metabolismo , Ácido Hipocloroso/farmacologia , Neutrófilos/metabolismo , Oxidantes/metabolismo , Oxirredução , Peroxidase/metabolismo , Fagossomos/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.
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Líquido da Lavagem Broncoalveolar/imunologia , Fibrose Cística/imunologia , Inflamação/imunologia , Complexo Antígeno L1 Leucocitário/metabolismo , Neutrófilos/imunologia , Peptídeos/metabolismo , Sistema Respiratório/metabolismo , Criança , Pré-Escolar , Fibrose Cística/diagnóstico , Feminino , Humanos , Inflamação/diagnóstico , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/imunologia , Masculino , Ativação de Neutrófilo , Oxirredução , Peptídeos/genética , Peptídeos/imunologia , ProteóliseRESUMO
Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t 1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothiol-deficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system.
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Antibacterianos/metabolismo , Ácido Hipocloroso/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium smegmatis/fisiologia , Neutrófilos/metabolismo , Fagossomos/metabolismo , Células Cultivadas , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Humanos , Evasão da Resposta Imune , Imunidade Inata , Inositol/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Neutrófilos/imunologia , FagocitoseRESUMO
Bacillithiol is a major low-molecular-weight thiol in gram-positive firmicutes including the human pathogen Staphylococcus aureus. Bacillithiol is regarded as an important defence mechanism against oxidants produced by the immune system, especially myeloperoxidase-derived hypochlorous acid (HOCl). However, it is unknown how fast BSH reacts with HOCl and what products are formed in the reaction. In the present study, we used sensitive MRM-based LC-MS methods to characterize the reaction of BSH with HOCl in cell-free solutions and in S. aureus. In the cell-free system, BSH formed predominantly the disulfide dimer (BSSB) at low mole ratios of HOCl and the sulfinic and sulfonic acids at higher oxidant concentrations. HOCl also promoted the formation of bacillithiol sulfonamide. In S. aureus, the oxidation pattern was similar except that a small proportion of BSH also formed mixed disulfides with protein thiols. Using competition with methionine, we determined the second-order rate constant for the reaction of HOCl with BSH to be 6 × 107 M-1s-1, which indicated a fast, near diffusion-controlled reaction. Other reactive halogen species, including hypothiocyanous acid (HOSCN), also produced bacillithiol sulfonamide, albeit to a smaller extent than HOCl. The sulfonamide was not produced by hydrogen peroxide, which instead formed BSSB. This study helps our understanding of BSH redox biology and provides tools for gauging the exposure of BSH-producing bacteria to oxidative stress.
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Oxidantes , Peroxidase , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Humanos , Ácido Hipocloroso , Oxirredução , Peroxidase/metabolismo , Staphylococcus aureus/metabolismoRESUMO
BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by severe bacterial infections, excessive neutrophilic inflammation and oxidative stress. The neutrophil enzyme myeloperoxidase (MPO), which produces hypochlorous acid, is associated with worse disease outcomes. Therefore, pharmacological inhibition of MPO in the airways has therapeutic potential. We investigated whether treating mice with an MPO inhibitor during pulmonary infection decreases oxidative stress and improves infection outcomes in mice with CF-like lung inflammation without impacting on bacterial clearance. METHODS: Transgenic ß-epithelial sodium channel (ßENaC)-overexpressing mice (n = 10) were infected with Burkholderia multivorans and treated twice daily with the MPO inhibitor AZM198 (125 µmol/kg) or vehicle administered by oral gavage for two days. Bodyweight was recorded daily. MPO activity, markers of oxidative stress, inflammatory cytokines and leukocytes numbers were measured in bronchoalveolar lavage fluid (BALF). Bacterial burden was determined in lung tissue homogenates. RESULTS: During the course of infection, mice treated with AZM198 lost less weight than vehicle-treated mice (p < 0.01). MPO activity and glutathione sulfonamide, a hypochlorous acid-specific glutathione oxidation product, were significantly lower in BALF from AZM198-treated mice (p < 0.05). The inflammatory cytokines CXCL1 and TNF-α in BALF and bacterial burden in the lung were not significantly different between treated and control mice. CONCLUSIONS: Orally administered AZM198 inhibits MPO activity in epithelial lining fluid. Blocking hypochlorous acid production in epithelial lining fluid during pulmonary infections through inhibition of MPO improves morbidity in mice with CF-like lung inflammation without interfering with clearance of bacteria. Pharmacological inhibition of MPO is an approach to limit destructive oxidative stress in cystic fibrosis lung disease in humans.
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Fibrose Cística , Pneumonia , Animais , Líquido da Lavagem Broncoalveolar , Burkholderia , Fibrose Cística/tratamento farmacológico , Inflamação , Pulmão/metabolismo , Camundongos , Morbidade , Estresse Oxidativo , Peroxidase/metabolismo , Pneumonia/tratamento farmacológicoRESUMO
Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a nonredundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and hydrogen peroxide dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF-α that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent overactivation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.
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Comunicação Celular/imunologia , Retroalimentação Fisiológica , Imunidade nas Mucosas , Células T Invariantes Associadas à Mucosa/imunologia , Neutrófilos/imunologia , Movimento Celular , Técnicas de Cocultura , Escherichia coli/imunologia , Humanos , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , Contagem de Leucócitos , Fígado/citologia , Fígado/imunologia , Ativação Linfocitária , Células T Invariantes Associadas à Mucosa/citologia , Mucosa/citologia , Mucosa/imunologia , Neutrófilos/citologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Myeloperoxidase is a major neutrophil antimicrobial protein, but its role in immunity is often overlooked because individuals deficient in this enzyme are usually in good health. Within neutrophil phagosomes, myeloperoxidase uses superoxide generated by the NADPH oxidase to oxidize chloride to the potent bactericidal oxidant hypochlorous acid (HOCl). In this study, using phagocytosis assays and LC-MS analyses, we monitored GSH oxidation in Pseudomonas aeruginosa to gauge their exposure to HOCl inside phagosomes. Doses of reagent HOCl that killed most of the bacteria oxidized half the cells' GSH, producing mainly glutathione disulfide (GSSG) and other low-molecular-weight disulfides. Glutathione sulfonamide (GSA), a HOCl-specific product, was also formed. When neutrophils phagocytosed P. aeruginosa, half of the bacterial GSH was lost. Bacterial GSA production indicated that HOCl had reacted with the bacterial cells, oxidized their GSH, and was sufficient to be solely responsible for bacterial killing. Inhibition of NADPH oxidase and myeloperoxidase lowered GSA formation in the bacterial cells, but the bacteria were still killed, presumably by compensatory nonoxidative mechanisms. Of note, bacterial GSA formation in neutrophils from patients with cystic fibrosis (CF) was normal during early phagocytosis, but it was diminished at later time points, which was mirrored by a small decrease in bacterial killing. In conclusion, myeloperoxidase generates sufficient HOCl within neutrophil phagosomes to kill ingested bacteria. The unusual kinetics of phagosomal HOCl production in CF neutrophils confirm a role for the cystic fibrosis transmembrane conductance regulator in maintaining HOCl production in neutrophil phagosomes.
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Antibacterianos/farmacologia , Fibrose Cística/tratamento farmacológico , Ácido Hipocloroso/farmacologia , Neutrófilos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Fibrose Cística/microbiologia , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Dissulfeto de Glutationa/biossíntese , Humanos , Testes de Sensibilidade Microbiana , Neutrófilos/microbiologia , Pseudomonas aeruginosa/metabolismoRESUMO
Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10⯵M, cross-linked calprotectin (5⯵M) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10⯵M) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.
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Complexo Antígeno L1 Leucocitário/química , Complexo Antígeno L1 Leucocitário/metabolismo , Estresse Oxidativo , Cromatografia Líquida , Espectrometria de Massas , Modelos Moleculares , Peso Molecular , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Oxirredução , Peroxidase/metabolismo , Fagocitose , Conformação Proteica , Proteólise , Relação Estrutura-AtividadeRESUMO
The ability of helminths to manipulate the immune system of their hosts to ensure their own survival is often credited with affecting responses to other pathogens. We undertook co-infection experiments in mice to determine how infection with the intestinal helminth Heligmosomoides polygyrus affected the parasitological, immunological and physiological outcomes of a primary infection with a distinct species of helminth; the lung migratory parasite Nippostrongylus brasiliensis. We found that migrating N. brasiliensis larvae were killed in the lungs of H. polygyrus-infected mice by a process involving IL-33-activated CD4+ T cells that released IL-5 and recruited activated eosinophils. The lung pathology normally associated with N. brasiliensis larval migration was also reduced. Importantly, lung immunity remained intact in mice cleared of prior H. polygyrus infection and also occurred during infection with another entirely enteric helminth, Trichuris muris. This study identifies a cross-mucosal immune mechanism by which intestinal helminths may protect their hosts against co-infection by a different parasite at a distal site, via circulation of activated CD4+ T cells that can be triggered to release effector cytokines and mount inflammatory responses by tissue damage-associated alarmins, such as IL-33.
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Linfócitos T CD4-Positivos/imunologia , Coinfecção , Eosinófilos/imunologia , Interleucina-5/metabolismo , Pulmão/imunologia , Nematospiroides dubius/fisiologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Tricuríase/imunologia , Trichuris/fisiologia , Animais , Antígenos de Helmintos/imunologia , Movimento Celular , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Interações Hospedeiro-Parasita , Imunidade , Interleucina-33/metabolismo , Pulmão/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Urate is often viewed as an antioxidant. Here, we present an alternative perspective by showing that, when oxidized, urate propagates oxidative stress. Oxidation converts urate to the urate radical and the electrophilic products dehydrourate, 5-hydroxyisourate, and urate hydroperoxide, which eventually break down to allantoin. We investigated whether urate-derived electrophiles are intercepted by nucleophilic amino acid residues to form stable adducts on proteins. When urate was oxidized in the presence of various peptides and proteins, two adducts derived from urate (Mr 167 Da) were detected and had mass additions of 140 and 166 Da, occurring mainly on lysine residues and N-terminal amines. The adduct with a 140-Da mass addition was detected more frequently and was stable. Dehydrourate (Mr 166 Da) also formed transient adducts with cysteine residues. Urate-derived adducts were detected on human serum albumin in plasma of healthy donors. Basal adduct levels increased when neutrophils were added to plasma and stimulated, and relied on the NADPH oxidase, myeloperoxidase, hydrogen peroxide, and superoxide. Adducts of oxidized urate on serum albumin were elevated in plasma and synovial fluid from individuals with gout and rheumatoid arthritis. We propose that rather than acting as an antioxidant, urate's conversion to electrophiles contributes to oxidative stress. The addition of urate-derived electrophiles to nucleophilic amino acid residues, a process we call oxidative uratylation, will leave a footprint on proteins that could alter their function when critical sites are modified.
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Ácido Úrico/química , Aminas/química , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Inflamação/metabolismo , Modelos Moleculares , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Conformação Proteica , Albumina Sérica/química , Albumina Sérica/metabolismo , Ácido Úrico/metabolismo , Ácido Úrico/farmacologiaRESUMO
Since 1981, Gordon Research Conferences have been held on the topic of Oxygen Radicals on a biennial basis, to highlight and discuss the latest cutting edge research in this area. Since the first meeting, one special feature of this conference has been the awarding of the so-called Iron Bolt, an award that started in jest but has gained increasing reputation over the years. Since no written documentation exists for this Iron Bolt award, this perspective serves to overview the history of this unusual award, and highlights various experiences of previous winners of this "prestigious" award and other interesting anecdotes.
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Distinções e Prêmios , Radicais Livres , HumanosRESUMO
Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 µM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 µM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.
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Bromatos/química , Brometos/química , Proteínas da Matriz Extracelular/química , Matriz Extracelular/química , Peróxido de Hidrogênio/química , Peroxidase/química , Animais , Bromatos/análise , Linhagem Celular Tumoral , Colágeno Tipo IV/química , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Técnicas de Inativação de Genes , Iminas/química , Espectrometria de Massas , Camundongos , NAD/química , Peroxidase/antagonistas & inibidores , Peroxidase/genética , PeroxidasinaRESUMO
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.
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Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/enzimologia , Precursores Enzimáticos/sangue , Peroxidase/sangue , Sequência de Aminoácidos , Animais , Células CHO , Doenças Cardiovasculares/metabolismo , Cricetinae , Cricetulus , Células HL-60 , Halogenação , Humanos , Immunoblotting , Infarto do Miocárdio/sangue , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/metabolismo , Oxirredução , CoelhosRESUMO
Neutrophil-derived myeloperoxidase (MPO) is recognized as a major source of oxidative stress at the airway surface of a cystic fibrosis (CF) lung where, despite limited evidence, the antioxidant glutathione is widely considered to be low. The aims of this study were to establish whether oxidative stress or glutathione status are associated with bronchiectasis and whether glutathione deficiency is inherently linked to CF or a consequence of oxidative stress. MPO was measured by ELISA in 577 bronchoalveolar lavage samples from 205 clinically-phenotyped infants and children with CF and 58 children without CF (ages 0.2-6.92 years). Reduced glutathione (GSH), oxidized glutathione species (GSSG; glutathione attached to proteins, GSSP; glutathione sulfonamide, GSA) and allantoin, an oxidation product of uric acid, were measured by mass spectrometry. The odds of having bronchiectasis were associated with MPO and GSSP. GSH was low in children with CF irrespective of oxidation. Oxidized glutathione species were significantly elevated in CF children with pulmonary infections compared to uninfected CF children. In non-CF children, infections had no effect on glutathione levels. An inadequate antioxidant response to neutrophil-mediated oxidative stress during infections exists in CF due to an inherent glutathione deficiency. Effective delivery of glutathione and inhibition of MPO may slow the development of bronchiectasis.
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Bronquiectasia/metabolismo , Fibrose Cística/metabolismo , Glutationa/deficiência , Pulmão/metabolismo , Neutrófilos/enzimologia , Peroxidase/metabolismo , Idade de Início , Alantoína/metabolismo , Bronquiectasia/patologia , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Criança , Pré-Escolar , Fibrose Cística/patologia , Feminino , Glutationa/análogos & derivados , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Lactente , Inflamação , Pulmão/patologia , Masculino , Neutrófilos/metabolismo , Oxirredução , Estresse Oxidativo , Sulfonas/metabolismoRESUMO
BACKGROUND: In cystic fibrosis (CF) there is an urgent need for earlier diagnosis of pulmonary infections and inflammation using blood- and urine-based biomarkers. METHODS: Using mass spectrometry, oxidation products of glutathione and uric acid were measured in matched samples of bronchoalveolar lavage (BAL), serum and urine from 36 infants and children with CF, and related to markers of neutrophilic inflammation and infection in BAL. RESULTS: Oxidation products of glutathione (glutathione sulfonamide, GSA) and uric acid (allantoin), were elevated in BAL of children with pulmonary infections with Pseudomonas aeruginosa (PsA) compared to those without (p<0.05) and correlated with other markers of neutrophilic inflammation. Serum GSA was significantly elevated in children with PsA infections (p<0.01). Urinary GSA correlated with pulmonary GSA (r=0.42, p<0.05) and markers of neutrophilic inflammation. CONCLUSIONS: This proof-of-concept study demonstrates that urinary GSA but not allantoin shows promise as a non-invasive marker of neutrophilic inflammation in early CF lung disease.
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Fibrose Cística , Dissulfeto de Glutationa , Inflamação/diagnóstico , Infecções por Pseudomonas/diagnóstico , Infecções Respiratórias/diagnóstico , Ácido Úrico , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/urina , Líquido da Lavagem Broncoalveolar , Pré-Escolar , Fibrose Cística/diagnóstico , Fibrose Cística/microbiologia , Fibrose Cística/fisiopatologia , Diagnóstico Precoce , Feminino , Dissulfeto de Glutationa/análise , Dissulfeto de Glutationa/sangue , Dissulfeto de Glutationa/urina , Humanos , Lactente , Masculino , Neutrófilos/metabolismo , Reprodutibilidade dos Testes , Estatística como Assunto , Ácido Úrico/análise , Ácido Úrico/sangue , Ácido Úrico/urinaRESUMO
The modification of 5-hydroxyindoleacetic acid (5HIAA) by myeloperoxidase with a xanthine oxidase system was investigated by chromatographic analyses. Two major products were identified as a dimer and quinone (indoleacetate dione) of 5HIAA. The formation of a quinone moiety was also confirmed by chemical trapping with o-phenylenediamine. In the presence of N-acetyl-cysteine (NAC), a quinone-NAC adduct was formed. When glyceraldehyde 3-phosphate dehydrogenase was exposed to the myeloperoxidase system with 5HIAA, quinone adducts were formed on the protein molecule. A monoclonal antibody was prepared using a quinone-modified protein as an immunogen to immunochemically detect the quinone on a protein. The established antibody recognized the quinone-NAC adduct, quinone-modified poly-L-lysine, and quinone-modified low-density lipoprotein. Quinone-modified proteins in human atherosclerotic lesions were immunohistochemically observed using the established antibody to the quinone and also a monoclonal antibody to tryptamine dione-modified protein, suggesting an occurrence of in vivo oxidation of serotonin and 5HIAA, accompanied by covalent adduction to biomolecules.
Assuntos
Aterosclerose/sangue , Ácido Hidroxi-Indolacético/química , Quinonas/síntese química , Serotonina/química , Acetilcisteína/química , Idoso , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Aorta/química , Aorta/metabolismo , Aorta/patologia , Aterosclerose/patologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Ácido Hidroxi-Indolacético/metabolismo , Imuno-Histoquímica , Masculino , Microtomia , Peroxidase/química , Peroxidase/metabolismo , Fenilenodiaminas , Quinonas/metabolismo , Serotonina/metabolismo , Xantina Oxidase/química , Xantina Oxidase/metabolismoRESUMO
Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys.
Assuntos
Inflamação/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Estresse Oxidativo , Animais , Biomarcadores , Modelos Animais de Doenças , Cães , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Oxirredução , Peroxidase/metabolismo , Tirosina/metabolismoRESUMO
Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.