AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p

in this review, current understanding of the prevention and treatment of Incontinence Associated Dermatitis (IAD) is discussed. The need for preventative measures which target specific faecal/urinary irritants is highlighted, including the role of urease inhibitors. There is no existing internationally and clinically accepted method to diagnose and categorise the severity of IAD. Diagnosis currently relies on visual inspection; non-invasive techniques to assess skin barrier function could remove subjectiveness, particularly in darker skin tones. Impedance spectroscopy is a non-invasive technique which can be used to monitor skin barrier function, supporting visual assessments. Six studies (2003–2021) which used impedance to assess dermatitis were reviewed; inflamed skin was distinguishable from healthy skin in each case. This suggests that impedance spectroscopy could be useful in diagnosis early-stage IAD, potentially enabling earlier intervention. Finally, the authors present their initial findings on the role of urease in skin breakdown in an in vivo IAD model, using impedance spectroscopy.

This study tested the hypothesis that the increases in salivary and plasma [NO2−] after dietary NO3− supplementation would be greater when oral temperature and pH were independently elevated, and increased further when oral temperature and pH were elevated concurrently. Seven healthy males (mean ± SD, age 23 ± 4 years) ingested 70 mL of beetroot juice concentrate (BR, which provided ~6.2 mmol NO3−) during six separate laboratory visits. In a randomised crossover experimental design, salivary and plasma [NO3−] and [NO2−] were assessed at a neutral oral pH with a low (TLo-pHNorm), intermediate (TMid-pHNorm), and high (THi-pHNorm) oral temperature, and when the oral pH was increased at a low (TLo-pHHi), intermediate (TMid-pHHi), and high (THi-pHHi) oral temperature. Compared with the TMid-pHNorm condition (976 ± 388 µM), the mean salivary [NO2−] 1–3 h post BR ingestion was higher in the TMid-pHHi (1855 ± 423 µM), THi-pHNorm (1371 ± 653 µM), THi-pHHi (1792 ± 741 µM), TLo-pHNorm (1495 ± 502 µM), and TLo-pHHi (2013 ± 662 µM) conditions, with salivary [NO2−] also higher at a given oral temperature when the oral pH was increased (p < 0.05). Plasma [NO2−] was higher 3 h post BR ingestion in the TMid-pHHi, THi-pHHi, and TLo-pHHi conditions, but not the TLo-pHNorm and THi-pHNorm conditions, compared with TMid-pHNorm (p < 0.05). Therefore, despite ingesting the same NO3− dose, the increases in salivary [NO2−] varied depending on the temperature and pH of the oral cavity, while the plasma [NO2−] increased independently of oral temperature, but to a greater extent at a higher oral pH.

Dietary nitrate (NO3-) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2-]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3-. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3- ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3-, ∼11 mg NO3-/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3-, NO2-, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2-] and reduced BP, but increased RBC [NO2-] was correlated with decreased systolic BP (rs = -0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = -0.68, P = 0.001), diastolic (rs = -0.59, P = 0.008) and mean arterial pressure (rs = -0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2-] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3- supplementation.

Watercress is an enriched source of phenethyl isothiocyanate (PEITC), among other phyto-chemicals, with an antioxidant capacity. The aim of this study was to (i) chemically characterize and (ii) biologically evaluate the profile of the main health-promoting compounds contained in edible (i.e. mixture of leaves and lateral buds) and non-edible (i.e. stems) parts of watercress in an in vitro model of malignant melanoma consisting of human malignant melanoma (A375), non-melanoma (A431) and keratinocyte (HaCaT) cells. The extraction of the main constituents of watercress was performed by subjecting the freeze-dried edible and non-edible samples through different extraction protocols, whereas their concentration was obtained utilizing analytical methodologies. In addition, cell viability was evaluated by the Alamar Blue assay, whereas levels of oxidative stress and apoptosis were determined by commercially available kits. The edible watercress sample contained a higher amount of various nutrients and phytochemicals in the hexane fraction compared to the non-edible one, as evidenced by the presence of PEITC, phenolics, flavonoids, pigments, ascorbic acid, etc. The cytotoxicity potential of the edible watercress sample in the hexane fraction was considerably higher than the non-edible one in A375 cells, whereas A431 and HaCaT cells appeared to be either more resistant or minimally affected, respectively. Finally, levels of oxidative stress and apoptotic induction were increased in both watercress samples, but the magnitude of the induction was much higher in the edible than the non-edible watercress samples. Herein, we provide further evidence document-ing the potential development of watercress extracts (including watercress waste by-products) as promising anti-cancer agent(s) against malignant melanoma cells.

Objectives: This is a protocol for a Cochrane Review (diagnostic). The objectives are as follows:. To determine the diagnostic accuracy of biomarkers for detecting acute appendicitis in hospitalised adults with suspected acute appendicitis. Secondary objectives to determine (if possible) the diagnostic accuracy of biomarkers for detecting: uncomplicated and complicated (gangrenous or perforated) acute appendicitis; acute appendicitis in males and females; acute appendicitis in women of reproductive age; acute appendicitis in pregnancy; acute appendicitis at different time intervals from onset of pain.

BACKGROUND: Early identification and treatment of kidney transplant rejection episodes is vital to limit loss of function and prolong the life of the transplanted kidney and recipient. Current practice depends on detecting a creatinine rise. A biomarker to diagnose transplant rejection at an earlier time point than current practice, or to inform earlier decision making to biopsy, could be transformative. It has previously been shown that urinary nitrate concentration is elevated in renal transplant rejection. Nitrate is a nitric oxide (NO) oxidation product. Transplant rejection upregulates NO synthesis via inducible nitric oxide synthase leading to elevations in urinary nitrate concentration. We have recently validated a urinary nitrate concentration assay which could provide results in a clinically relevant timeframe. Our aim was to determine whether urinary nitrate concentration is a useful tool to predict renal transplant rejection in the context of contemporary clinical practice. METHODS: We conducted a prospective observational study, recruiting renal transplant participants over an 18-month period. We made no alterations to the patients' clinical care including medications, immunosuppression, diet and frequency of visits. We collected urine samples from every clinical attendance. We assessed the urinary nitrate to creatinine ratio (uNCR) between patient groups: routine attendances, biopsy proven rejection, biopsy proven no rejection and other call backs. uNCR was examined over time for those with biopsy proven transplant rejection. These four groups were compared using an ANOVA test. RESULTS: a total of 2656 samples were collected. uNCR during biopsy proven rejection, n = 15 (median 49 μmol/mmol, IQR 23-61) was not significantly different from that of routine samples, n = 164 (median 55 μmol/mmol, IQR 37-82) (p = 0.55), or biopsy proven no rejection, n = 12 (median 39 μmol/mmol, IQR 21-89) (P = 0.77). Overall uNCR was highly variable with no diagnostic threshold for kidney transplant rejection. Furthermore, within-patient uNCR was highly variable over time, and thus it was not possible to produce individualised patient thresholds to identify rejection. The total taking Tacrolimus was 204 patients, with no statistical difference between the uNCR of all those on Tacrolimus, against those not, p = 0.18. CONCLUSION: the urinary nitrate to creatinine ratio is not a useful biomarker for renal transplant rejection.

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00–1.34) and 0.97 (0.00–1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

In vivo, mammalian cells reside in an environment of 0.5–10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

The authors regret that they have to correct the acknowledgement of the above mentioned publication as follows: This article/publication is based upon work from COST Action BM1203 (EU-ROS), supported by COST (European Cooperation in Science and Technology) which is funded by the Horizon 2020 Framework Programme of the European Union. COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks. Our Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. For further information see www.cost.eu. The authors would like to apologise for any inconvenience caused.

Graphene has a promising future in applications such as disease diagnosis, cancer therapy, drug/gene delivery, bio-imaging and antibacterial approaches owing to graphene's unique physical, chemical and mechanical properties alongside minimal toxicity to normal cells, and photo-stability. However, these unique features and bioavailability of graphene are fraught with uncertainties and concerns for environmental and occupational exposure. Changes in the physicochemical properties of graphene affect biological responses including reactive oxygen species (ROS) production. Lower production of ROS by currently available theranostic agents, e.g. magnetic nanoparticles, carbon nanotubes, gold nanostructures or polymeric nanoparticles, restricts their clinical application in cancer therapy. Oxidative stress induced by graphene accumulated in living organs is due to acellular factors which may affect physiological interactions between graphene and target tissues and cells. Acellular factors include particle size, shape, surface charge, surface containing functional groups, and light activation. Cellular responses such as mitochondrial respiration, graphene-cell interactions and pH of the medium are also determinants of ROS production. The mechanisms of ROS production by graphene and the role of ROS for cancer treatment, are poorly understood. The aim of this review is to set the theoretical basis for further research in developing graphene-based theranostic platforms.

Biomolecular fractions affect the fate and behaviour of quantum dots (QDs) in living systems but how the interactions between biomolecules and QDs affect the bioavailability of QDs is a major knowledge gap in risk assessment analysis. The transport of QDs after release into a living organism is a complex process. The majority accumulate in the lungs where they can directly affect the inhalation process and lung architecture. Here, we investigate the bioavailability of graphene quantum dots (GQDs) to the lungs of rats by measuring the alterations in macromolecular fractions via Fourier transform infrared spectroscopy (FTIR). GQDs were intravenously injected into the rats in a dose-dependent manner (low (5 mg kg21) and high (15 mg kg21) doses of GQDs per body weight of rat) for 7 days. The lung tissues were isolated, processed and haematoxylin–eosin stained for histological analysis to identify cell death. Key biochemical differences were identified by spectral signatures: pronounced changes in cholesterol were found in two cases of low and high doses; a change in phosphorylation profile of substrate proteins in the tissues was observed in low dose at 24 h. This is the first time biomolecules have been measured in biological tissue using FTIR to investigate the biocompatibility of foreign material. We found that highly accurate toxicological changes can be investigated with FTIR measurements of tissue sections. As a result, FTIR could form the basis of a non-invasive pre-diagnostic tool for predicting the toxicity of GQDs.

As an inexpensive monolayer archetypal member of the carbon family, graphene has triggered a new ‘gold rush’ in nanotechnology for achieving unique properties that were not available in many traditional materials. Owing to these unique features, graphene-related materials are finding new uses in nanomedicine and synthetic biology in addition to their diverse applications in electronics, optoelectronics, photonics and environmental clean-up. The increased production of graphene nanostructures and increased likelihood of exposures to these substances in environmental and occupational settings has raised concerns about adverse health outcomes. In particular, the biological effects of these materials need to be assessed to ensure risk free, sustainable development of graphene for widespread applications. In this work, for the first time, we studied the in vitro and in vivo interactions of a relatively new derivative of graphene, graphene nanopores (GNPs) in mammalian systems, to systematically elucidate the possible mechanism of their toxicity over time. This study showed that GNPs induced early apoptosis in both SKMES-1 and A549 lung cancer cells. However, late apoptosis is only induced at concentrations higher than 250 μg/ml, suggesting that, although GNPs at lower concentrations induce upregulation of phosphatidylserine on the cell surface membrane (i.e. early apoptotic event), GNPs do not significantly disintegrate the cell membrane. We also showed that rats intraperitoneally injected with GNPs suffered sub-chronic toxicity in a period of 27 days when tested at single and multiple doses of GNPs (5 and 15 mg/kg) as evidenced by blood biochemistry, organo-somatic index, liver and kidney enzymes functions analysis, oxidative stress biomarkers and histological examinations. In sum, our results show that GNPs are likely to have a low bioavailability in SKMES-1 and A549 lung cancer cells and rats. Nevertheless, this must be considered against the context of a wider lack of knowledge regarding the bioavailability, fate and behaviour of this type of new porous framework of graphene in natural systems. Therefore, a more long-term GNPs exposure regime, more relevant to real-life environmental consequences, is needed to fully determine the transport capacities of GNPs in living systems.

A diet rich in vegetables is known to provide cardioprotection. However, it is unclear how the consumption of different vegetables might interact to influence vascular health. This study tested the hypothesis that nitrate-rich vegetable consumption would lower systolic blood pressure but that this effect would be abolished when nitrate-rich and thiocyanate-rich vegetables are co-ingested. On four separate occasions, and in a randomized cross-over design, eleven healthy males reported to the laboratory and consumed a 750 mL vegetable smoothie that was either: low in nitrate (∼0.3 mmol) and thiocyanate (∼5 μmol), low in nitrate and high in thiocyanate (∼72 μmol), high in nitrate (∼4 mmol) and low in thiocyanate and high in nitrate and thiocyanate. Blood pressure as well as plasma and salivary [thiocyanate], [nitrate] and [nitrite] were assessed before and 3 h after smoothie consumption. Plasma [nitrate] and [nitrite] and salivary [nitrate] were not different after consuming the two high-nitrate smoothies, but salivary [nitrite] was higher after consuming the high-nitrate low-thiocyanate smoothie (1183 ± 625 μM) compared to the high-nitrate high-thiocyanate smoothie (941 ± 532 μM; P

Imbalances in the oral microbial community have been associated with reduced cardiovascular and metabolic health. A possible mechanism linking the oral microbiota to health is the nitrate (NO3-)-nitrite (NO2-)-nitric oxide (NO) pathway, which relies on oral bacteria to reduce NO3- to NO2-. NO (generated from both NO2- and L-arginine) regulates vascular endothelial function and therefore blood pressure (BP). By sequencing bacterial 16S rRNA genes we examined the relationships between the oral microbiome and physiological indices of NO bioavailability and possible changes in these variables following 10 days of NO3- (12 mmol/d) and placebo supplementation in young (18–22 yrs) and old (70–79 yrs) normotensive humans (n = 18). NO3- supplementation altered the salivary microbiome compared to placebo by increasing the relative abundance of Proteobacteria (+225%) and decreasing the relative abundance of Bacteroidetes (−46%; P < 0.05). After NO3-supplementation the relative abundances of Rothia (+127%) and Neisseria (+351%) were greater, and Prevotella (−60%) and Veillonella (−65%) were lower than in the placebo condition (all P < 0.05). NO3- supplementation increased plasma concentration of NO2- and reduced systemic blood pressure in old (70–79 yrs), but not young (18–22 yrs), participants. High abundances of Rothia and Neisseria and low abundances of Prevotella and Veillonella were correlated with greater increases in plasma [NO2-] in response to NO3- supplementation. The current findings indicate that the oral microbiome is malleable to change with increased dietary intake of inorganic NO3-, and that diet-induced changes in the oral microbial community are related to indices of NO homeostasis and vascular health in vivo.

The management of patients with autoimmune rheumatic diseases such as rheumatoid arthritis (RA) remains a significant challenge. Often the rheumatologist is restricted to treating and relieving the symptoms and consequences and not the underlying cause of the disease. Oxidative stress occurs in many autoimmune diseases, along with the excess production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The sources of such reactive species include NADPH oxidases (NOXs), the mitochondrial electron transport chain, nitric oxide synthases, nitrite reductases, and the hydrogen sulfide producing enzymes cystathionine-β synthase and cystathionine-γ lyase. Superoxide undergoes a dismutation reaction to generate hydrogen peroxide which, in the presence of transition metal ions (e.g. ferrous ions), forms the hydroxyl radical. The enzyme myeloperoxidase, present in inflammatory cells, produces hypochlorous acid, and in healthy individuals ROS and RNS production by phagocytic cells is important in microbial killing. Both low molecular weight antioxidant molecules and antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and peroxiredoxin remove ROS. However, when ROS production exceeds the antioxidant protection, oxidative stress occurs. Oxidative post-translational modifications of proteins then occur. Sometimes protein modifications may give rise to neoepitopes that are recognized by the immune system as ‘non-self’ and result in the formation of autoantibodies. The detection of autoantibodies against specific antigens, might improve both early diagnosis and monitoring of disease activity. Promising diagnostic autoantibodies include anti-carbamylated proteins and anti-oxidized type II collagen antibodies. Some of the most promising future strategies for redox-based therapeutic compounds are the activation of endogenous cellular antioxidant systems (e.g. Nrf2-dependent pathways), inhibition of disease-relevant sources of ROS/RNS (e.g. isoform-specific NOX inhibitors), or perhaps specifically scavenging disease-related ROS/RNS via site-specific antioxidants.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Background and Purpose: Exposure to nanoparticulate pollution has been implicated in platelet-driven thrombotic events such as myocardial infarction. Inflammation and impairment of NO bioavailability have been proposed as potential causative mechanisms. It is unclear, however, whether airways exposure to combustion-derived nanoparticles such as diesel exhaust particles (DEP) or carbon black (CB) can augment platelet aggregation in vivo and the underlying mechanisms remain undefined. We aimed to investigate the effects of acute lung exposure to DEP and CB on platelet activation and the associated role of inflammation and endothelial-derived NO. Experimental Approach: DEP and CB were intratracheally instilled into wild-type (WT) and eNOS−/− mice and platelet aggregation was assessed in vivo using an established model of radio-labelled platelet thromboembolism. The underlying mechanisms were investigated by measuring inflammatory markers, NO metabolites and light transmission aggregometry. Key Results: Platelet aggregation in vivo was significantly enhanced in WT and eNOS−/− mice following acute airways exposure to DEP but not CB. CB exposure, but not DEP, was associated with significant increases in pulmonary neutrophils and IL-6 levels in the bronchoalveolar lavage fluid and plasma of WT mice. Neither DEP nor CB affected plasma nitrate/nitrite concentration and DEP-induced human platelet aggregation was inhibited by an NO donor. Conclusions and Implications: Pulmonary exposure to DEP and subsequent platelet activation may contribute to the reports of increased cardiovascular risk, associated with exposure to airborne pollution, independent of its effects on inflammation or NO bioavailability.

Uptake of inorganic nitrate (NO3-) into the salivary circulation is a rate-limiting step for dietary NO3- metabolism in mammals. It has been suggested that salivary NO3- uptake occurs in competition with inorganic iodide (I-). Therefore, this study tested the hypothesis that I- supplementation would interfere with NO3- metabolism and blunt blood pressure reductions after dietary NO3- supplementation. Nine healthy adults (4 male, mean ± SD, age 20 ± 1 yr) reported to the laboratory for initial baseline assessment (control) and following six day supplementation periods with 140 mL·day-1 NO3--rich beetroot juice (8.4 mmol NO3-·day-1) and 198 mg potassium gluconate·day-1 (nitrate), and 140 mL·day-1 NO3--rich beetroot juice and 450 μg potassium iodide·day-1 (nitrate + iodide) in a randomized, cross-over experiment. Salivary [I-] was higher in the nitrate + iodide compared to the control and NIT trials (P 

The redox state of cellular exofacial molecules is reflected by the amount of available thiols. Furthermore, surface thiols can be considered as indicators of immune cell activation. One group of thiol containing proteins, peroxiredoxins, in particular, have been associated with inflammation. In this study, we assessed surface thiols of the U937 and Thp1 monocyte cell lines and primary monocytes in vitro upon inflammatory stimulation by irreversibly labelling the cells with a fluorescent derivative of maleimide. We also investigated exofacial thiols on circulating blood mononuclear cells in patients with rheumatoid arthritis and healthy controls. When analysing extracellular vesicles, we combined thiol labelling with the use of antibodies to specific CD markers to exclude extracellular vesicle mimicking signals from thiol containing protein aggregates. Furthermore, differential detergent lysis was applied to confirm the vesicular nature of the detected extracellular events in blood plasma. We found an increase in exofacial thiols on monocytes upon in vitro stimulation by LPS or TNF, both in primary monocytes and monocytic cell lines (p

BACKGROUND Inorganic nitrate from the oxidation of endogenously synthesized nitric oxide (NO) or consumed in the diet can be reduced to NO via a complex enterosalivary circulation pathway. The relationship between total nitrate exposure by measured urinary nitrate excretion and blood pressure in a large population sample has not been assessed previously. METHODS for this cross-sectional study, 24-hour urinary nitrate excretion was measured by spectrophotometry in the 919 participants from the InChianti cohort at baseline and blood pressure measured with a mercury sphygmomanometer. RESULTS After adjusting for age and sex only, diastolic blood pressure was 1.9 mm Hg lower in subjects with ≥2 mmol urinary nitrate excretion compared with those excreting

Nitric oxide alters gastric blood flow, improves vascular function, and mediates glucose uptake within the intestines and skeletal muscle. Dietary nitrate, acting as a source of nitric oxide, appears to be a potential low-cost therapy that may help maintain glucose homeostasis. In a randomized, double-blind, placebo-controlled crossover study, 31 young and older adult participants had a standardized breakfast, supplemented with either nitrate-rich beetroot juice (11.91 mmol nitrate) or nitrate-depleted beetroot juice as placebo (0.01 mmol nitrate). MRI was used to assess apparent diffusion coefficient (ADC), portal vein flux, and velocity. Plasma glucose, incretin, and C-peptide concentrations and blood pressure were assessed. Outcome variables were measured at baseline and hourly for 3 h. Compared with a placebo, beetroot juice resulted in a significant elevation in plasma nitrate and plasma nitrite concentration. No differences were seen for the young or older adult cohorts between placebo and beetroot juice for ADC, or portal vein flux. There was an interaction effect in the young adults between visits for portal vein velocity. Nitrate supplementation did not reduce plasma glucose, active GLP-1, total GLP-1, or plasma C-peptide concentrations for the young or older adult cohorts. Despite a significant elevation in plasma nitrite concentration following an acute dose of (11.91 mmol) nitrate, there was no effect on hepatic blood flow, plasma glucose, C-peptide, or incretin concentration in healthy adults.

Dietary supplementation with inorganic nitrate (NO3-) has been reported to improve cardiovascular health indices in healthy adults. Cigarette smoking increases circulating thiocyanate (SCN-), which has been suggested to competitively inhibit salivary nitrate (NO3-) uptake, a rate-limiting step in dietary NO3- metabolism. Therefore, this study tested the hypothesis that dietary NO3- supplementation would be less effective at increasing the circulating plasma nitrite concentration ([NO2-]) and lowering blood pressure in smokers (S) compared to non-smokers (NS). Nine healthy smokers and eight healthy non-smoking controls reported to the laboratory at baseline (CON) and following six day supplementation periods with 140 mL day-1 NO3--rich (8.4 mmol NO3- day-1; NIT) and NO3--depleted (0.08 mmol NO3- day-1; PLA) beetroot juice in a cross-over experiment. Plasma and salivary [SCN-] were elevated in smokers compared to non-smokers in all experimental conditions (P 

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

OBJECTIVES: Abnormal circadian oscillations of blood pressure (BP) and nocturnal-diurnal BP differences (i.e. dipping) increase cardiovascular risk. Whether inorganic nitrate supplementation influences 24-hr BP variability is currently unknown. We studied the effects of high-nitrate beetroot juice supplementation on BP variability measured by 24-hr ambulatory BP monitoring (24-hr ABPM) in older subjects. METHODS: Data from four independent randomised clinical trials were collated. Eighty-five older participants (age range: 55-76 years) were included in the final database. Two trials had an open-label, parallel design and two trials had a cross-over, double-blind design. Participants were randomised to either beetroot juice or placebo. Changes in 24-hr ABPM (daily, diurnal, nocturnal), variability (weighted-SDs), night-dipping, morning surge for systolic and diastolic BP were measured. Meta-analysis was conducted to obtain pooled estimates of the effect size for each BP outcome. Sub-group analyses were conducted to evaluate the influence of age, BMI, gender, BP status and changes in nitrite concentrations on the effect size. RESULTS: the pooled effect of beetroot juice on all BP outcomes was not significant. Beetroot juice ingestion determined a significant decrease in nocturnal systolic BP variability in subjects aged less than 65 y (2.8 mmHg, -4.5 -1.0, p = 0.002) compared to the older group (≥ 65 y; 1.0 mmHg, -2.2 4.2, p = 0.54). A greater change in NO2(-) concentrations after beetroot supplementation was associated with significant differences for nocturnal mean (-3.4 mmHg, -0.6 -2.4, p = 0.02) and variability (-0.8 mmHg, -1.5 -0.06, p = 0.03) of systolic BP. CONCLUSIONS: the vascular responsiveness to inorganic nitrate may be modified by mechanisms of vascular ageing influencing the reducing capacity to convert inorganic nitrate into nitrite and tissue-specific responses to dietary nitrate supplementation.

There is currently no biochemical test for detection of early-stage osteoarthritis (eOA). Tests for early-stage rheumatoid arthritis (eRA) such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibodies require refinement to improve clinical utility. We developed robust mass spectrometric methods to quantify citrullinated protein (CP) and free hydroxyproline in body fluids. We detected CP in the plasma of healthy subjects and surprisingly found that CP was increased in both patients with eOA and eRA whereas anti-CCP antibodies were predominantly present in eRA. A 4-class diagnostic algorithm combining plasma/serum CP, anti-CCP antibody and hydroxyproline applied to a cohort gave specific and sensitive detection and discrimination of eOA, eRA, other non-RA inflammatory joint diseases and good skeletal health. This provides a first-in-class plasma/serum-based biochemical assay for diagnosis and type discrimination of early-stage arthritis to facilitate improved treatment and patient outcomes, exploiting citrullinated protein and related differential autoimmunity.

A BDD-BDD dual-plate microtrench electrode with 6μm inter-electrode spacing is investigated using generator-collector electrochemistry and shown to give microtrench depth-dependent sulfide detection down to the μM levels. The effect of the microtrench depth is compared for a "shallow" 44 μm and a "deep" 180μm microtrench and linked to the reduction of oxygen to hydrogen peroxide which interferes with sulfide redox cycling. With a deeper microtrench and a fixed collector potential at -1.4V vs. SCE, two distinct redox cycling potential domains are observed at 0.0V vs. SCE (2-electron) and at 1.1V vs. SCE (6-electron).

SIGNIFICANCE: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. RECENT ADVANCES: an increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. CRITICAL ISSUES: the literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. FUTURE DIRECTIONS: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker.

Nitrate derived from vegetables is consumed as part of a normal diet and is reduced endogenously via nitrite to nitric oxide. It has been shown to improve endothelial function, reduce blood pressure and the oxygen cost of sub-maximal exercise, and increase regional perfusion in the brain. The current study assessed the effects of dietary nitrate on cognitive performance and prefrontal cortex cerebral blood-flow (CBF) parameters in healthy adults. In this randomised, double-blind, placebo-controlled, parallel-groups study, 40 healthy adults received either placebo or 450 ml beetroot juice (~5.5 mmol nitrate). Following a 90 minute drink/absorption period, participants performed a selection of cognitive tasks that activate the frontal cortex for 54 min. Near-Infrared Spectroscopy (NIRS) was used to monitor CBF and hemodynamics, as indexed by concentration changes in oxygenated and deoxygenated-haemoglobin, in the frontal cortex throughout. The bioconversion of nitrate to nitrite was confirmed in plasma by ozone-based chemi-luminescence. Dietary nitrate modulated the hemodynamic response to task performance, with an initial increase in CBF at the start of the task period, followed by consistent reductions during the least demanding of the three tasks utilised. Cognitive performance was improved on the serial 3s subtraction task. These results show that single doses of dietary nitrate can modulate the CBF response to task performance and potentially improve cognitive performance, and suggest one possible mechanism by which vegetable consumption may have beneficial effects on brain function.

Dietary nitrate supplementation has been shown to reduce the oxygen (O2) cost of exercise and enhance exercise tolerance in healthy individuals. This study assessed whether similar effects could be observed in individuals with type 2 diabetes (T2DM). In a randomized, double-blind, placebo-controlled crossover study, 48 participants with T2DM supplemented their diet for 4 days with either nitrate-rich beetroot juice (70ml/day, 6.43mmol nitrate/day) or nitrate-depleted beetroot juice as placebo (70ml/day, 0.07mmol nitrate/day). After each intervention period, resting plasma nitrate and nitrite concentrations were measured subsequent to participants completing moderate-paced walking. Pulmonary gas exchange was measured to assess the O2 cost of walking. After a rest period, participants performed the 6-min walk test (6MWT). Relative to placebo, beetroot juice resulted in a significant increase in plasma nitrate (placebo, 57±66 vs beetroot, 319±110µM; P < 0.001) and plasma nitrite concentration (placebo, 680±256 vs beetroot, 1065±607nM; P < 0.001). There were no differences between placebo juice and beetroot juice for the O2 cost of walking (946±221 vs 939±223ml/min, respectively; P = 0.59) and distance covered in the 6MWT (550±83 vs 554±90m, respectively; P = 0.17). Nitrate supplementation did not affect the O2 cost of moderate-paced walking or improve performance in the 6MWT. These findings indicate that dietary nitrate supplementation does not modulate the response to exercise in individuals with T2DM.

The purpose of this study was to compare the effects of L-citrulline (Cit) and L-arginine (Arg) supplementation on nitric oxide (NO) biomarkers, pulmonary O2 uptake (Vo2) kinetics, and exercise performance. In a randomized, placebo (Pla)-controlled, crossover study, 10 healthy adult men completed moderate- and severe-intensity cycling exercise on days 6 and 7 of a 7-day supplementation period with Pla, Arg (6 g/day), and Cit (6 g/day). Compared with Pla, plasma Arg concentration was increased by a similar magnitude with Arg and Cit supplementation, but plasma Cit concentration was only increased (P < 0.001) with Cit supplementation. Plasma nitrite (NO-2) concentration was increased with Arg supplementation (P < 0.05) and tended to increase with Cit supplementation (P = 0.08) compared with Pla (83 ±) 25, 106 ± 41, and 100 ± 38 nM with Pla, Arg, and Cit, respectively); however, mean arterial blood pressure was only lower (P < 0.05) after Cit supplementation. The steady-state Vo2 amplitude during moderateintensity cycle exercise was not significantly different between supplements, but Cit lowered the Vo2 mean response time (59 ± 8 and 53 ± 5 s with Pla and Cit, respectively, P < 0.05) during severe-intensity exercise, improved tolerance to severe-intensity exercise (589 ± 101 and 661 ± 107 s with Pla and Cit, respectively), and increased the total amount of work completed in the exercise performance test (123 ± 18 and 125 ± 19 kJ with Pla and Cit, respectively, P < 0.05). These variables were not altered by Arg supplementation (P > 0.05). In conclusion, these results suggest that short-term Cit, but not Arg, supplementation can improve blood pressure, Vo2 kinetics, and exercise performance in healthy adults.

It is now recognised that administration of oral nitrate (NO3(-)), in its various forms, increases the level of nitric oxide (NO) metabolites in the circulation of humans. Its application to modulate physiology and alleviate cardiovascular dysfunction in some patients is now recorded and shows particular promise in hypertension, in modifying platelet activation/aggregation, and in conditions where tissue ischaemia prevails. The potential of oral NO3(-) to modify exercise/performance via elevation of plasma nitrite concentration ([NO2(-)]) has been applied across a range of human test systems. Herein we discuss how the choice of NO3(-) source, route of administration and resulting pharmacokinetics might influence the outcome of physiological measures and potentially contribute to discrepancies in performance trials. There are but a few examples of detailed pharmacokinetic data on which the majority of researchers base their test protocols in different cohorts/settings. We compare and contrast the results of key publications with the aim of highlighting a consensus of our current understanding and critical considerations for those entering the field.

A dual-electrode sensor is developed for rapid detection of nitrite/nitrate at micromolar levels in phosphate buffer media and in dilute horse serum without additional sample pre-treatment. A generator-collector configuration is employed so that on one electrode nitrate is reduced to nitrite and on the second electrode nitrite is oxidised back to nitrate. The resulting redox cycle gives rise to a specific and enhanced current signal which is exploited for sensitive and reliable measurement of nitrite/nitrate in the presence of oxygen. The electrode design is based on a dual-plate microtrench (approximately 15 μm inter-electrode gap) fabricated from gold-coated glass and with a nano-silver catalyst for the reduction of nitrate. Fine tuning of the phosphate buffer pH is crucial for maximising collector current signals whilst minimising unwanted gold surface oxidation. A limit of detection of 24 μM nitrate and a linear concentration range of 200-1400 μM is reported for the microtrench sensor in phosphate buffer and dilute horse serum. Relative standard deviations for repeat measurements were in the range 1.8-6.9% (n=3) indicating good repeatability in both aqueous and biological media. Preliminary method validation against the standard chemiluminescence method used in medical laboratories is reported for nitrate analysis in serum. © 2014 Elsevier B.V.

Inorganic nitrate is present at high levels in beetroot and celery, and in green leafy vegetables such as spinach and lettuce. Though long believed inert, nitrate can be reduced to nitrite in the human mouth and, further, under hypoxia and/or low pH, to nitric oxide. Dietary nitrate has thus been associated favorably with nitric-oxide-regulated processes including blood flow and energy metabolism. Indeed, the therapeutic potential of dietary nitrate in cardiovascular disease and metabolic syndrome-both aging-related medical disorders-has attracted considerable recent research interest. We and others have shown that dietary nitrate supplementation lowers the oxygen cost of human exercise, as less respiratory activity appears to be required for a set rate of skeletal muscle work. This striking observation predicts that nitrate benefits the energy metabolism of human muscle, increasing the efficiency of either mitochondrial ATP synthesis and/or of cellular ATP-consuming processes. In this mini-review, we evaluate experimental support for the dietary nitrate effects on muscle bioenergetics and we critically discuss the likelihood of nitric oxide as the molecular mediator of such effects.

Advanced oxidation protein products (AOPP) are reportedly elevated in the plasma of patients with a number of diseases, including diabetes mellitus, that involve oxidative stress. However, the accurate measurement of AOPP in human plasma is hampered by the formation of a precipitate following the addition of potassium iodide and glacial acetic acid according to the published assay procedure. Here we describe a modification of the AOPP assay which eliminates interference by precipitation and provides a robust, reliable, and reproducible protocol for the measurement of iodide oxidising capacity in plasma samples (intra-assay CV 1.7-5.3%, interassay CV 5.3-10.5%). The improved method revealed a significant association of AOPP levels with age (p

Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

Chlorhexidine-containing mouthwash (STRONG), which disturbs oral microflora, has been shown to diminish the rise in plasma nitrite concentration ([NO2-]) and attenuate the reduction in resting blood pressure (BP) typically seen after acute nitrate (NO3-) ingestion. We aimed to determine whether STRONG and weaker antiseptic agents attenuate the physiological effects of chronic NO3- supplementation using beetroot juice (BR). 12 healthy volunteers mouth-rinsed with STRONG, non-chlorhexidine mouthwash (WEAK) and deionised water (CON) 3 times a day, and ingested 70 mL BR (6.2 mmol NO3-), twice a day, for 6 days. BP (at rest and during 10 min of treadmill walking) and plasma and salivary [NO3-] and [NO2-] were measured prior to and on day 6 of supplementation. The change in salivary [NO3-] 4 h post final ingestion was higher (P

BACKGROUND: Chronic obstructive pulmonary disease (COPD) results in exercise intolerance. Dietary nitrate supplementation has been shown to lower blood pressure (BP), reduce the oxygen cost of exercise, and enhance exercise tolerance in healthy volunteers. This study assessed the effects of dietary nitrate on the oxygen cost of cycling, walking performance and BP in individuals with mild-moderate COPD. METHODS: Thirteen patients with mild-moderate COPD were recruited. Participants consumed 70 ml of either nitrate-rich (6.77 mmol nitrate; beetroot juice) or nitrate-depleted beetroot juice (0.002 mmol nitrate; placebo) twice a day for 2.5 days, with the final supplement ~3 hours before testing. BP was measured before completing two bouts of moderate-intensity cycling, where pulmonary gas exchange was measured throughout. The six-minute walk test (6 MWT) was completed 30 minutes subsequent to the second cycling bout. RESULTS: Plasma nitrate concentration was significantly elevated following beetroot juice vs. placebo (placebo; 48 ± 86 vs. beetroot juice; 215 ± 84 µM, P = 0.002). No significant differences were observed between placebo vs. beetroot juice for oxygen cost of exercise (933 ± 323 vs. 939 ± 302 ml: min(-1); P = 0.88), distance covered in the 6 MWT (456 ± 86 vs. 449 ± 79 m; P = 0.37), systolic BP (123 ± 14 vs. 123 ± 14 mmHg; P = 0.91), or diastolic BP (77 ± 9 vs. 79 ± 9 mmHg; P = 0.27). CONCLUSION: Despite a large rise in plasma nitrate concentration, two days of nitrate supplementation did not reduce the oxygen cost of moderate intensity cycling, increase distance covered in the 6 MWT, or lower BP.

Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell development in vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential.

Thiols and disulfides are ubiquitous and important analytical targets. However, their redox properties, in particular on gold sensor electrodes, are complex and obscured by strong adsorption. Here, a gold-gold dual-plate microtrench dual-electrode sensor with feedback signal amplification is demonstrated to give well-defined (but kinetically limited) steady-state voltammetric current responses for the cysteine-cystine redox cycle in nondegassed aqueous buffer media at pH 7 down to micromolar concentration levels.

We tested the hypothesis that the time constants (τ) of postexercise T2. MRI signal intensity (an index of O2 delivery) and muscle [PCr] (an index of metabolic perturbation, measured by (31)P-MRS) in hypoxia would be accelerated after dietary nitrate (NO3 (-)) supplementation. In a double-blind crossover design, eight moderately trained subjects underwent 5 days of NO3 (-) (beetroot juice, BR; 8.2 mmol/day NO3 (-)) and placebo (PL; 0.003 mmol/day NO3 (-)) supplementation in four conditions: normoxic PL (N-PL), hypoxic PL (H-PL; 13% O2), normoxic NO3 (-) (N-BR), and hypoxic NO3 (-) (H-BR). The single-leg knee-extension protocol consisted of 10 min of steady-state exercise and 24 s of high-intensity exercise. The [PCr] recovery τ was greater in H-PL (30 ± 4 s) than H-BR (22 ± 4 s), N-PL (24 ± 4 s) and N-BR (22 ± 4 s) (P < 0.05) and the maximal rate of mitochondrial ATP resynthesis (Qmax) was lower in the H-PL (1.12 ± 0.16 mM/s) compared with H-BR (1.35 ± 0.26 mM/s), N-PL (1.47 ± 0.28 mM/s), and N-BR (1.40 ± 0.21 mM/s) (P < 0.05). The τ of postexercise T2. signal intensity was greater in H-PL (47 ± 14 s) than H-BR (32 ± 10 s), N-PL (38 ± 9 s), and N-BR (27 ± 6 s) (P < 0.05). The postexercise [PCr] and T2. recovery τ were correlated in hypoxia (r = 0.60; P < 0.05), but not in normoxia (r = 0.28; P > 0.05). These findings suggest that the NO3 (-)-NO2 (-)-NO pathway is a significant modulator of muscle energetics and O2 delivery during hypoxic exercise and subsequent recovery.

BACKGROUND: in this substudy of the effect of dietary nitrate on blood pressure, endothelial function, and insulin sensitivity in type 2 diabetes, we report the development of a novel nitrate depleted beetroot juice for use clinical trials and determine if dietary nitrate supplementation improved cognitive function in patients with type 2 diabetes mellitus. METHODS: Beetroot juice was treated with the anion exchange resin Purolite A520e. UV-vis-spectrophotometry, and a blind taste test were performed along with determination of sugar content, measurement of ascorbate and dehydroascorbate, the ionic composition of juice and Proton NMR. Subsequently, 27 patients, age 67.2±4.9 years, (18 male) were recruited for a double blind, randomised, placebo-controlled crossover trial. Participants were randomised to begin in either order beetroot juice (nitrate content 7.5 mmol per 250 ml) or placebo (nitrate depleted beetroot juice nitrate content 0.002 mmol per 250 ml). At the end of each 2 week supplementation period cognitive function was assessed using E-prime, E-Studio software with 5 separate tests being performed. The tests utilised in the present study have been adapted from the Cambridge Neuropsychological Test Automated Battery (CANTAB). RESULTS: the differences in the UV-vis spectra were comparable to the natural variation found in differing cultivars. There were no discernable differences in taste, sugar content, or Proton NMR. Ascorbate and dehydroascorbate were undetectable in either juice. After 2 weeks of beetroot juice simple reaction time was significantly quicker in the active arm at 327±40 ms versus 341.8±52.7 ms in the placebo arm, mean difference 13.9±25.6 ms (95% CI 3.8-24.0 ms), p=0.009. No other measures of cognitive function differed between treatment arms. CONCLUSION: We have developed an effective placebo beetroot juice for use in trials of supplementation of dietary nitrate. Two weeks supplementation of the diet with 7.5 mmol of nitrate per day caused a significant improvement in simple reaction time in individuals with T2DM.

We investigated the effects of dietary nitrate (NO3 (-)) supplementation on the concentration of plasma nitrite ([NO2 (-)]), oxygen uptake (V̇o2) kinetics, and exercise tolerance in normoxia (N) and hypoxia (H). In a double-blind, crossover study, 12 healthy subjects completed cycle exercise tests, twice in N (20.9% O2) and twice in H (13.1% O2). Subjects ingested either 140 ml/day of NO3 (-)-rich beetroot juice (8.4 mmol NO3; BR) or NO3 (-)-depleted beetroot juice (PL) for 3 days prior to moderate-intensity and severe-intensity exercise tests in H and N. Preexercise plasma [NO2 (-)] was significantly elevated in H-BR and N-BR compared with H-PL (P < 0.01) and N-PL (P < 0.01). The rate of decline in plasma [NO2 (-)] was greater during severe-intensity exercise in H-BR [-30 ± 22 nM/min, 95% confidence interval (CI); -44, -16] compared with H-PL (-7 ± 10 nM/min, 95% CI; -13, -1; P < 0.01) and in N-BR (-26 ± 19 nM/min, 95% CI; -38, -14) compared with N-PL (-1 ± 6 nM/min, 95% CI; -5, 2; P < 0.01). During moderate-intensity exercise, steady-state pulmonary V̇o2 was lower in H-BR (1.91 ± 0.28 l/min, 95% CI; 1.77, 2.13) compared with H-PL (2.05 ± 0.25 l/min, 95% CI; 1.93, 2.26; P = 0.02), and V̇o2 kinetics was faster in H-BR (τ: 24 ± 13 s, 95% CI; 15, 32) compared with H-PL (31 ± 11 s, 95% CI; 23, 38; P = 0.04). NO3 (-) supplementation had no significant effect on V̇o2 kinetics during severe-intensity exercise in hypoxia, or during moderate-intensity or severe-intensity exercise in normoxia. Tolerance to severe-intensity exercise was improved by NO3 (-) in hypoxia (H-PL: 197 ± 28; 95% CI; 173, 220 vs. H-BR: 214 ± 43 s, 95% CI; 177, 249; P = 0.04) but not normoxia. The metabolism of NO2 (-) during exercise is altered by NO3 (-) supplementation, exercise, and to a lesser extent, hypoxia. In hypoxia, NO3 (-) supplementation enhances V̇o2 kinetics during moderate-intensity exercise and improves severe-intensity exercise tolerance. These findings may have important implications for individuals exercising at altitude.

Together with carbon monoxide (CO), nitric oxide (NO) and hydrogen sulfide (H2S) form a group of physiologically important gaseous transmitters, sometimes referred to as the "gaseous triumvirate". The three molecules share a wide range of physical and physiological properties: they are small gaseous molecules, able to freely penetrate cellular membranes; they are all produced endogenously in the body and they seem to exert similar biological functions. In the cardiovascular system, for example, they are all vasodilators, promote angiogenesis and protect tissues against damage (e.g. ischemia-reperfusion injury). In addition, they have complex roles in inflammation, with both pro- and anti-inflammatory effects reported. Researchers have focused their efforts in understanding and describing the roles of each of these molecules in different physiological systems, and in the past years attention has also been given to the gases interaction or "cross-talk". This review will focus on the role of NO and H2S in inflammation and will give an overview of the evidence collected so far suggesting the importance of their cross-talk in inflammatory processes.

Tissue inflammation results in the production of numerous reactive oxygen, nitrogen and chlorine species, in addition to the products of lipid and sugar oxidation. Some of these products are capable of chemically modifying amino acids. This in turn results in changes to the structure and function of proteins. Increasing evidence demonstrates that such oxidative post-translational modifications result in the generation of neo-epitopes capable of eliciting both innate and adaptive immune responses. In this paper, we focus on how free radicals and related chemical species generated in inflammatory environments modulate the antigenicity of self-proteins, resulting in immune responses which involve the generation of autoantibodies against key autoantigens in autoimmune diseases. As examples, we will focus on Ro-60 and C1q in systemic lupus erythematosus, along with type-II collagen in rheumatoid arthritis. This review also covers some of the emerging literature which demonstrates that neo-epitopes generated by oxidation are conserved, as exemplified by the evolutionarily conserved pathogen-associated molecular patterns (PAMPs). We discuss how these observations relate to the pathogenesis of both human autoimmune diseases and inflammatory disease, such as atherosclerosis. The potential for these neo-epitopes and the immune responses against them to act as biomarkers or therapeutic targets is also discussed.

Synthesis and bioavailability of the endogenous gasomediator hydrogen sulfide (H2S) is perturbed in many disease states, including those involving mitochondrial dysfunction. There is intense interest in developing pharmacological agents to generate H2S. We have synthesised a novel H2S donor molecule coupled to a mitochondria-targeting moiety (triphenylphosphonium; TPP+) and compared the effectiveness of the compound against a standard non-TPP+ containing H2S donor (GYY4137) in the inhibition of oxidative stress-induced endothelial cell death. Our study suggests mitochondria-targeted H2S donors are useful pharmacological tools to study the mitochondrial physiology of H2S in health and disease. © 2014 the Partner Organisations.

Dietary supplementation with beetroot juice (BR), containing approximately 5-8 mmol inorganic nitrate (NO3(-)), increases plasma nitrite concentration ([NO2(-)]), reduces blood pressure, and may positively influence the physiological responses to exercise. However, the dose-response relationship between the volume of BR ingested and the physiological effects invoked has not been investigated. In a balanced crossover design, 10 healthy men ingested 70, 140, or 280 ml concentrated BR (containing 4.2, 8.4, and 16.8 mmol NO3(-), respectively) or no supplement to establish the effects of BR on resting plasma [NO3(-)] and [NO2(-)] over 24 h. Subsequently, on six separate occasions, 10 subjects completed moderate-intensity and severe-intensity cycle exercise tests, 2.5 h postingestion of 70, 140, and 280 ml BR or NO3(-)-depleted BR as placebo (PL). Following acute BR ingestion, plasma [NO2(-)] increased in a dose-dependent manner, with the peak changes occurring at approximately 2-3 h. Compared with PL, 70 ml BR did not alter the physiological responses to exercise. However, 140 and 280 ml BR reduced the steady-state oxygen (O2) uptake during moderate-intensity exercise by 1.7% (P = 0.06) and 3.0% (P < 0.05), whereas time-to-task failure was extended by 14% and 12% (both P < 0.05), respectively, compared with PL. The results indicate that whereas plasma [NO2(-)] and the O2 cost of moderate-intensity exercise are altered dose dependently with NO3(-)-rich BR, there is no additional improvement in exercise tolerance after ingesting BR containing 16.8 compared with 8.4 mmol NO3(-). These findings have important implications for the use of BR to enhance cardiovascular health and exercise performance in young adults.

The breakdown of human immune tolerance to self-proteins occurs by a number of mechanisms, including posttranslational modifications of host molecules by reactive oxygen, nitrogen, or chlorine species. This has led to great interest in detecting serum autoantibodies raised against small quantities of oxidatively modified host proteins in patients with autoimmune inflammatory diseases, such as rheumatoid arthritis. Here, we provide protocols for the preparation and chemical characterization of oxidatively modified protein antigens and procedures for their use in immunoblotting and ELISAs that detect autoantibodies against these antigens in clinical samples. These gel electrophoresis- and plate reader-based immunochemical methods sometimes suffer from low analytical specificity and/or sensitivity when used for serum autoantibody detection. This is often because a single solid-phase protein (antigen) is exposed to a complex mixture of serum proteins that undergo nonspecific binding. Therefore more sensitive/specific techniques are required to detect autoantibodies specifically directed against oxidatively modified proteins. To address this, we describe novel affinity chromatography protocols by which purified autoantibodies are isolated from small volumes (

Hyperbaric oxygen has proven to be a useful treatment for chronic wounds. However, therapeutic conditions vary between treatment centers, and we wished to investigate the effects of different treatment pressures on cells under inflammatory conditions. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O2 at 1 atmosphere absolute (atm abs); PO2 ~ 2 kPa) in the presence of 0.5 μg/ml lipopolysaccharide and 1 ng/ml TNF-alpha for 24 hours, then treated with normobaric oxygen (NBO2; 95%O2/5%CO2 at 1.0 atm abs; PO2 ~ 96.3 kPa), hyperbaric oxygen (HBO2) at 1.5 atm abs (1.5HBO2; 96.7%O2/3.3%CO2 at 1.5 atm abs; PO2 ~ 147 kPa) and HBO2 at 2.4 atm abs (2.4 HBO2; 97.9% O2/2.1% CO2 at 2.4 atms; PO2 ~238 kPa). The mRNA expression of 92 genes was then analyzed, and we identified changes in genes involved in adhesion molecule expression, angiogenesis and tissue remodeling, intracellular signaling, and cellular oxygen responses and redox signaling. We noted differences in expression between different treatment pressures, highlighting the need for further research into the use of different therapeutic protocols in the treatment of inflammatory conditions such as chronic wounds.

Diets rich in green, leafy vegetables have been shown to lower blood pressure (BP) and reduce the risk of cardiovascular disease. Green, leafy vegetables and beetroot are particularly rich in inorganic nitrate. Dietary nitrate supplementation, via sequential reduction to nitrite and NO, has previously been shown to lower BP and improve endothelial function in healthy humans. We sought to determine if supplementing dietary nitrate with beetroot juice, a rich source of nitrate, will lower BP and improve endothelial function and insulin sensitivity in individuals with type 2 diabetes (T2DM). Twenty-seven patients, age 67.2±4.9 years (18 male), were recruited for a double-blind, randomized, placebo-controlled crossover trial. Participants were randomized to begin, in either order, a 2-week period of supplementation with 250ml beetroot juice daily (active) or 250ml nitrate-depleted beetroot juice (placebo). At the conclusion of each intervention period 24-h ambulatory blood pressure monitoring, tests of macro- and microvascular endothelial function, and a hyperinsulinemic isoglycemic clamp were performed. After 2 weeks administration of beetroot juice mean ambulatory systolic BP was unchanged: 134.6±8.4mmHg versus 135.1±7.8mmHg (mean±SD), placebo vs active-mean difference of -0.5mmHg (placebo-active), p=0.737 (95% CI -3.9 to 2.8). There were no changes in macrovascular or microvascular endothelial function or insulin sensitivity. Supplementation of the diet with 7.5mmol of nitrate per day for 2 weeks caused an increase in plasma nitrite and nitrate concentration, but did not lower BP, improve endothelial function, or improve insulin sensitivity in individuals with T2DM.

Dietary nitrate (NO(3)(-)) supplementation has been shown to reduce resting blood pressure and alter the physiological response to exercise in young adults. We investigated whether these effects might also be evident in older adults. In a double-blind, randomized, crossover study, 12 healthy, older (60-70 yr) adults supplemented their diet for 3 days with either nitrate-rich concentrated beetroot juice (BR; 2 × 70 ml/day, ∼9.6 mmol/day NO(3)(-)) or a nitrate-depleted beetroot juice placebo (PL; 2 × 70 ml/day, ∼0.01 mmol/day NO(3)(-)). Before and after the intervention periods, resting blood pressure and plasma [nitrite] were measured, and subjects completed a battery of physiological and cognitive tests. Nitrate supplementation significantly increased plasma [nitrite] and reduced resting systolic (BR: 115 ± 9 vs. PL: 120 ± 6 mmHg; P < 0.05) and diastolic (BR: 70 ± 5 vs. PL: 73 ± 5 mmHg; P < 0.05) blood pressure. Nitrate supplementation resulted in a speeding of the Vo(2) mean response time (BR: 25 ± 7 vs. PL: 28 ± 7 s; P < 0.05) in the transition from standing rest to treadmill walking, although in contrast to our hypothesis, the O(2) cost of exercise remained unchanged. Functional capacity (6-min walk test), the muscle metabolic response to low-intensity exercise, brain metabolite concentrations, and cognitive function were also not altered. Dietary nitrate supplementation reduced resting blood pressure and improved Vo(2) kinetics during treadmill walking in healthy older adults but did not improve walking or cognitive performance. These results may have implications for the enhancement of cardiovascular health in older age.

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells (HUVEC) and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and TNF-α, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced ˙NO-related protein modifications. The transnitrosating agent SNCEE (600µM) also reduced neutrophil adhesion to HUVEC monolayers, and the iNOS inhibitor 1400W (10µM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400W (10 μM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation. © 2013 by the Wound Healing Society.

Dietary nitrate supplementation, which enhances nitric oxide (NO) bioavailability, has previously been shown to contribute to improved exercise performance by reducing both oxygen cost and energy expenditure. In contrast, previous studies have indicated that NO can lower force production in vitro. To examine the role of dietary nitrates in regulating force generation under normal physiological conditions, we undertook an extended nitrate supplementation regime and determined force output and energy cost with a repeated isometric maximum voluntary contraction (MVC) protocol. In a double-blind, randomized, crossover design, eight participants received 0.5 l/day of nitrate-rich (BR) or nitrate-depleted (PL) beetroot juice for 15 days and completed an exercise protocol consisting of 50 MVCs at 2.5 h, 5 days and 15 days after the beginning of the supplementation period. No significant reduction in force output was determined for BR relative to PL for the peak contraction, the mean or the end force, and no significant time effect was found over the course of the supplementation period. There was a reduction in the mean PCr cost of exercise averaged over the BR supplementation trials, but this did not reach statistical significance for end exercise (BR 15.10 ± 4.14 mM, PL 17.10 ± 5.34 mM, P = 0.06) or the mean throughout the protocol (BR 15.96 ± 4.14 mM, PL 17.79 ± 4.51 mM, P = 0.06). However, a significant reduction in PCr cost per unit force output was found for BR at end exercise (P = 0.04). These results indicate that, under normal physiological conditions, increased NO bioavailability is not associated with a reduction of force-generating capability in human skeletal muscle and confirm that nitrate supplementation reduces the PCr cost of force production. © 2013 Springer-Verlag Berlin Heidelberg.

Dietary nitrate supplementation, which enhances nitric oxide (NO) bioavailability, has previously been shown to contribute to improved exercise performance by reducing both oxygen cost and energy expenditure. In contrast, previous studies have indicated that NO can lower force production in vitro. To examine the role of dietary nitrates in regulating force generation under normal physiological conditions, we undertook an extended nitrate supplementation regime and determined force output and energy cost with a repeated isometric maximum voluntary contraction (MVC) protocol. In a double-blind, randomized, crossover design, eight participants received 0.5 l/day of nitrate-rich (BR) or nitrate-depleted (PL) beetroot juice for 15 days and completed an exercise protocol consisting of 50 MVCs at 2.5 h, 5 days and 15 days after the beginning of the supplementation period. No significant reduction in force output was determined for BR relative to PL for the peak contraction, the mean or the end force, and no significant time effect was found over the course of the supplementation period. There was a reduction in the mean PCr cost of exercise averaged over the BR supplementation trials, but this did not reach statistical significance for end exercise (BR 15.10 ± 4.14 mM, PL 17.10 ± 5.34 mM, P = 0.06) or the mean throughout the protocol (BR 15.96 ± 4.14 mM, PL 17.79 ± 4.51 mM, P = 0.06). However, a significant reduction in PCr cost per unit force output was found for BR at end exercise (P = 0.04). These results indicate that, under normal physiological conditions, increased NO bioavailability is not associated with a reduction of force-generating capability in human skeletal muscle and confirm that nitrate supplementation reduces the PCr cost of force production.

The role of hydrogen sulfide (H2 S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H2 S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund's adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1-0.5 mM) decreased LPS-induced production of nitrite (NO2 (-) ), PGE2 , TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.

Introduction: the aim of this study was to test the naturally occurring organosulfur compound dipropyltetrasulfide (DPTTS) found in plants, which has antibiotic and anti-cancer properties, as a treatment of HOCl-induced systemic sclerosis in the mouse.
Methods: the pro-oxidative, anti-proliferative and cytotoxic effects of DPTTS were evaluated ex vivo on fibroblasts from normal and HOCl-mice. In vivo, the anti-fibrotic and immunomodulating properties of DPTTS were evaluated in the skin and lungs of HOCl-mice.
Results: H2O2 production was higher in fibroblasts derived from HOCl-mice than in normal fibroblasts (P

Background: Recent findings suggest a role of oxidative stress in the pathogenesis of Behcet's disease (BD), but the utility of oxidative stress-associated assays in offering diagnostic information or in the monitoring of disease activity is largely unassessed. Objective and methods. We aimed to measure oxidative and inflammatory markers, along with the markers of reactive nitrogen species, S-nitrosothiols and 3-nitrotyrosine, in BD patients (n = 100) and healthy volunteers (n = 50). These markers were evaluated in regard to their role in the pathogenesis of BD as well as their relation to clinical presentation, disease activity and duration. Results: Median values for erythrocyte sedimentation rate (ESR), C-reactive protein, leukocyte count, and IL-18 levels, as well as myeloperoxidase (MPO) activity, were statistically higher in the patient group compared to controls. Some inflammation markers (ESR, neutrophil and leukocyte counts) were statistically higher (p < 0.05) in the active period. In contrast, oxidative stress-associated measures (erythrocyte lipid peroxidation, antioxidant enzymes and measures of serum antioxidant capacity), revealed no statistically significant differences between the median values in BD patients versus healthy control subjects (p > 0.05 in all statistical comparisons), nor was there any difference in median levels of these oxidative stress markers in active disease versus disease remission. S-nitrosothiols and 3-nitrotyrosine were undetectable in BD plasma. Conclusions: the application of oxidative stress-associated measures to BD blood samples offered no supplemental diagnostic or disease activity information to that provided by standard laboratory measures of inflammation. S-nitrosothiols and 3-nitrotyrosine appeared not to be markers for active BD; thus the search for biochemical markers that will indicate the active period should be continued with larger studies. © 2012 Akcay et al; licensee BioMed Central Ltd.

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1β, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1β, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood
lymphocytes and extracellular fluid in healthy subjects and rheumatoid arthritis patients is poorly
described. In the present study, the median intracellular peroxiredoxin 2 protein content of
lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with
healthy subjects’ lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca.
8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. In the total
lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed
surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17+ve
lymphocytes were peroxiredoxin 2+ve (median, 39%). We suggest that crucial inflammatory cell
subsets, i.e. interleukin-17+ve T cells, exhibit increased exofacial redox-regulating enzymes and
that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic
inflammation.

OBJECTIVE: to determine whether MPO contributes to oxidative stress and disease activity in RA and whether it produces hypochlorous acid in SF. METHODS: Plasma and where possible SF were collected from 77 RA patients while 120 healthy controls supplied plasma only. MPO and protein carbonyls were measured by ELISAs. 3-Chlorotyrosine in proteins and allantoin in plasma were measured by mass spectrometry. RESULTS: Plasma MPO concentrations were significantly higher in patients with RA compared with healthy controls [10.8 ng/ml, inter-quartile range (IQR): 7.2-14.2; P3.2) and those with low disease activity (LDA; DAS-28 ≤ 3.2) (HDA 27.9 ng/ml, 20.2-34.1 vs LDA 22.1 ng/ml, 16.9-34.9; P>0.05). There was a significant relationship between plasma MPO and DAS-28 (r=0.35; P=0.005). Plasma protein carbonyls and allantoin were significantly higher in patients with RA compared with the healthy controls. MPO protein was significantly higher in SF compared with plasma (median 624.0 ng/ml, IQR 258.4-2433.0 vs 30.2 ng/ml, IQR 25.1-50.9; P

Nitric oxide (NO) is a potent signalling molecule that influences an array of physiological responses. It was traditionally assumed that NO was derived exclusively via the nitric oxide synthase (NOS) family of enzymes. This complex reaction requires a five electron oxidation of L-arginine and is contingent on the presence of numerous essential substrates (including O 2) and co-factors. Recently an additional, O 2-independent, NO generating pathway has been identified, where nitrite (NO 2-) can undergo a simple one electron reduction to yield NO. NO 2- is produced endogenously from the oxidation of NO and also from the reduction of dietary nitrate (NO 3-) by facultative bacteria residing on the tongue. Recent data show that dietary NO 3- supplementation, which increases the circulating plasma [NO 2-], reduces the O 2 cost of submaximal exercise in healthy humans. This finding is striking given that efficiency during moderate-intensity exercise has been considered to be immutable. There is evidence that the muscle ATP turnover at a fixed work rate is reduced and the mitochondrial P/O ratio is increased following NO 3- supplementation, which offers important insights into the physiological bases for the reduced V̇O 2 during exercise. NO 3- supplementation has also been shown to improve exercise performance in both healthy and patient populations. Therefore, dietary NO 3- supplementation may represent a practical and cost-effective method to improve exercise efficiency and exercise tolerance in humans. Given that a NO 3--rich diet may have numerous cardiovascular and other health benefits, dietary NO 3- intake may have important implications for human lifelong health and performance. © 2012 Copyright European College of Sport Science.

PURPOSE: Dietary nitrate supplementation has been shown to reduce the O2 cost of submaximal exercise and to improve high-intensity exercise tolerance. However, it is presently unknown whether it may enhance performance during simulated competition. The present study investigated the effects of acute dietary nitrate supplementation on power output (PO), VO2, and performance during 4- and 16.1-km cycling time trials (TT). METHODS: After familiarization, nine club-level competitive male cyclists were assigned in a randomized, crossover design to consume 0.5 L of beetroot juice (BR; containing ∼ 6.2 mmol of nitrate) or 0.5 L of nitrate-depleted BR (placebo, PL; containing ∼ 0.0047 mmol of nitrate), ∼ 2.5 h before the completion of a 4- and a 16.1-km TT. RESULTS: BR supplementation elevated plasma [nitrite] (PL = 241 ± 125 vs BR = 575 ± 199 nM, P < 0.05). The VO2 values during the TT were not significantly different between the BR and PL conditions at any elapsed distance (P > 0.05), but BR significantly increased mean PO during the 4-km (PL = 279 ± 51 vs BR = 292 ± 44 W, P < 0.05) and 16.1-km TT (PL = 233 ± 43 vs BR = 247 ± 44 W, P < 0.01). Consequently, BR improved 4-km performance by 2.8% (PL = 6.45 ± 0.42 vs BR = 6.27 ± 0.35 min, P < 0.05) and 16.1-km performance by 2.7% (PL = 27.7 ± 2.1 vs BR = 26.9 ± 1.8 min, P < 0.01). CONCLUSIONS: These results suggest that acute dietary nitrate supplementation with 0.5 L of BR improves cycling economy, as demonstrated by a higher PO for the same VO2 and enhances both 4- and 16.1-km cycling TT performance.

The field of free radical biology and medicine continues to move at a tremendous pace, with a constant flow of ground-breaking discoveries. The following collection of papers in this issue of Biochemical Society Transactions highlights several key areas of topical interest, including the crucial role of validated measurements of radicals and reactive oxygen species in underpinning nearly all research in the field, the important advances being made as a result of the overlap of free radical research with the reinvigorated field of lipidomics (driven in part by innovations in MS-based analysis), the acceleration of new insights into the role of oxidative protein modifications (particularly to cysteine residues) in modulating cell signalling, and the effects of free radicals on the functions of mitochondria, extracellular matrix and the immune system. In the present article, we provide a brief overview of these research areas, but, throughout this discussion, it must be remembered that it is the availability of reliable analytical methodologies that will be a key factor in facilitating continuing developments in this exciting research area.

Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90min (97.5% O(2) at 2.4 ATA; PO(2) ~237kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.

Exercise in hypoxia is associated with reduced muscle oxidative function and impaired
exercise tolerance. We hypothesised that dietary nitrate supplementation (which increases
plasma [nitrite] and thus NO bioavailability) would ameliorate the adverse effects of hypoxia
on muscle metabolism and oxidative function. In a double-blind, randomised crossover study,
nine healthy subjects completed knee-extension exercise to the limit of tolerance (Tlim), once
in normoxia (20.9% O2; CON) and twice in hypoxia (14.5% O2). During 24 h prior to the
hypoxia trials, subjects consumed 0.75 L of nitrate-rich beetroot juice (9.3 mmol nitrate; HBR)
or 0.75 L of nitrate-depleted beetroot juice as a placebo (0.006 mmol nitrate; H-PL).
Muscle metabolism was assessed using calibrated 31P-MRS. Plasma [nitrite] was elevated
(P

Dietary supplementation with beetroot juice (BR) has been shown to reduce resting blood pressure and the O(2) cost of submaximal exercise and to increase tolerance to high-intensity cycling. We tested the hypothesis that the physiological effects of BR were consequent to its high NO(3)(-) content per se, and not the presence of other potentially bioactive compounds. We investigated changes in blood pressure, mitochondrial oxidative capacity (Q(max)), and physiological responses to walking and moderate- and severe-intensity running following dietary supplementation with BR and NO(3)(-)-depleted BR [placebo (PL)]. After control (nonsupplemented) tests, nine healthy, physically active male subjects were assigned in a randomized, double-blind, crossover design to receive BR (0.5 l/day, containing ∼6.2 mmol of NO(3)(-)) and PL (0.5 l/day, containing ∼0.003 mmol of NO(3)(-)) for 6 days. Subjects completed treadmill exercise tests on days 4 and 5 and knee-extension exercise tests for estimation of Q(max) (using (31)P-magnetic resonance spectroscopy) on day 6 of the supplementation periods. Relative to PL, BR elevated plasma NO(2)(-) concentration (183 ± 119 vs. 373 ± 211 nM, P < 0.05) and reduced systolic blood pressure (129 ± 9 vs. 124 ± 10 mmHg, P < 0.01). Q(max) was not different between PL and BR (0.93 ± 0.05 and 1.05 ± 0.22 mM/s, respectively). The O(2) cost of walking (0.87 ± 0.12 and 0.70 ± 0.10 l/min in PL and BR, respectively, P < 0.01), moderate-intensity running (2.26 ± 0.27 and 2.10 ± 0.28 l/min in PL and BR, respectively, P < 0.01), and severe-intensity running (end-exercise O(2) uptake = 3.77 ± 0.57 and 3.50 ± 0.62 l/min in PL and BL, respectively, P < 0.01) was reduced by BR, and time to exhaustion during severe-intensity running was increased by 15% (7.6 ± 1.5 and 8.7 ± 1.8 min in PL and BR, respectively, P < 0.01). In contrast, relative to control, PL supplementation did not alter plasma NO(2)(-) concentration, blood pressure, or the physiological responses to exercise. These results indicate that the positive effects of 6 days of BR supplementation on the physiological responses to exercise can be ascribed to the high NO(3)(-) content per se.

INTRODUCTION: Rheumatoid arthritis (RA) is considered a T cell driven autoimmune disease, therefore, the ability of B cell depleting biologics, e.g. anti-CD20 antibodies, to alleviate RA is unclear. This study examined the proportions of IL-17-secreting lymphocytes in the blood of healthy subjects and RA patients and determined if Th17 cells belong to a CD20+ subset of T cells. METHODS: Fluorescence-activated cell sorting and confocal microscopy verified CD3, CD4/CD8 and CD20-staining of T cells. IL-17 secretion was determined using a commercial assay. RESULTS: in healthy subjects and RA patients blood, the median percentage of total CD20+ lymphocytes was similar (7.5%; n = 6 and 10.3%; n = 9, respectively) and comprised predominantly of B cells (~ 86%). However, 2-4% of CD3+ T cells from both healthy subjects (n = 7) and RA (n = 8) individuals co-expressed CD20. The peripheral blood of healthy subjects contained few IL-17-secreting CD20+ T cells (< 0.1%; n = 6). In contrast, in RA blood a median and interquartile range % of, 24.2%; IQR 28.5 of IL-17-secreting T cells were CD20+ (n = 9; p = 0.02). CONCLUSIONS: in the blood of RA patients, a greater proportion of Th17 cells are of a CD20+ phenotype compared to healthy individuals. These cells may represent an additional target for anti-CD20 therapies.

Hydrogen sulfide is rapidly gaining ground as a physiological mediator of inflammation, but
there is no clear consensus as to its precise role in inflammatory signaling. This article discusses
the disparate anti-inflammatory (‘the good’) and proinflammatory (‘the bad’) effects of
endogenous and pharmacological H2S in disparate animal model and cell culture systems. We
also discuss ‘the ugly’, such as problems of using wholly specific inhibitors of enzymatic H2S
synthesis, and the use of pharmacological donor compounds, which release H2S too quickly to
be physiologically representative of endogenous H2S synthesis. Furthermore, recently developed
slow-release H2S donors, which offer a more physiological approach to understanding the
complex role of H2S in acute and chronic inflammation (‘the promising’) are discussed.

We investigated the influence of the antioxidant N-acetylcysteine (NAC) on plasma nitrite concentration ([NO₂⁻]), pulmonary oxygen uptake (V(O₂)) kinetics and exercise tolerance. Eight males completed 'step' moderate- and severe-intensity cycle exercise tests following infusion of either NAC (125 mg kg⁻¹ h⁻¹ for 15 min followed by 25 mg kg⁻¹ h⁻¹ until the termination of exercise) or Placebo (PLA; saline). Following the initial loading phase, NAC infusion elevated plasma free sulfhydryl groups compared to placebo (PLA: 4 ± 2 vs. NAC: 13 ± 3 μ M g⁻¹; P < 0.05) and this elevation was preserved throughout the protocol. The administration of NAC did not significantly influence plasma [NO₂⁻] or V(O₂) kinetics during either moderate- or severe-intensity exercise. Although NAC did not significantly alter severe-intensity exercise tolerance at the group mean level (PLA: 776 ± 181 vs. NAC: 878 ± 284 s; P > 0.05), there was appreciable inter-subject variability in the response: four subjects had small reductions in exercise tolerance with NAC compared to PLA (-4%, -8%, -11%, and -14%) while the other four showed substantial improvements (+24%, +24%, +40%, and +69%). The results suggest that exercise-induced redox perturbations may contribute to fatigue development in recreationally-active adults.

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO•, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.

Many sulfur compounds are known to exhibit widespread antimicrobial activity. The latter is often the result of an intricate redox biochemistry whereby reactive sulfur species, such as organic polysulfanes, interact with pivotal cellular signaling pathways. The S8 unit in elemental sulfur resembles certain aspects of the chemistry of polysulfanes. As a consequence, water-soluble S8-sulfur nanoparticles are active against some smaller organisms, including nematodes, yet are non-toxic against human cells. In contrast, selenium and tellurium nanoparticles are less active. Together, the ease of production of the sulfur nanoparticles, their chemical stability in aqueous dispersion, amenable physical properties and selective toxicity, turn sulfur nanoparticles into promising antimicrobial prototypes for medical as well as agricultural applications.

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca(2+) homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca(2)(+)-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.

It has recently been reported that dietary nitrate (NO(3)(-)) supplementation, which increases plasma nitrite (NO(2)(-)) concentration, a biomarker of nitric oxide (NO) availability, improves exercise efficiency and exercise tolerance in healthy humans. We hypothesized that dietary supplementation with L-arginine, the substrate for NO synthase (NOS), would elicit similar responses. In a double-blind, crossover study, nine healthy men (aged 19-38 yr) consumed 500 ml of a beverage containing 6 g of l-arginine (Arg) or a placebo beverage (PL) and completed a series of "step" moderate- and severe-intensity exercise bouts 1 h after ingestion of the beverage. Plasma NO(2)(-) concentration was significantly greater in the Arg than the PL group (331 ± 198 vs. 159 ± 102 nM, P < 0.05) and systolic blood pressure was significantly reduced (123 ± 3 vs. 131 ± 5 mmHg, P < 0.01). The steady-state O(2) uptake (VO(2)) during moderate-intensity exercise was reduced by 7% in the Arg group (1.48 ± 0.12 vs. 1.59 ± 0.14 l/min, P < 0.05). During severe-intensity exercise, the Vo(2) slow component amplitude was reduced (0.58 ± 0.23 and 0.76 ± 0.29 l/min in Arg and PL, respectively, P < 0.05) and the time to exhaustion was extended (707 ± 232 and 562 ± 145 s in Arg and PL, respectively, P < 0.05) following consumption of Arg. In conclusion, similar to the effects of increased dietary NO(3)(-) intake, elevating NO bioavailability through dietary L-Arg supplementation reduced the O(2) cost of moderate-intensity exercise and blunted the VO(2) slow component and extended the time to exhaustion during severe-intensity exercise.

Dietary nitrate (NO(3)(-)) supplementation with beetroot juice (BR) over 4-6 days has been shown to reduce the O(2) cost of submaximal exercise and to improve exercise tolerance. However, it is not known whether shorter (or longer) periods of supplementation have similar (or greater) effects. We therefore investigated the effects of acute and chronic NO(3)(-) supplementation on resting blood pressure (BP) and the physiological responses to moderate-intensity exercise and ramp incremental cycle exercise in eight healthy subjects. Following baseline tests, the subjects were assigned in a balanced crossover design to receive BR (0.5 l/day; 5.2 mmol of NO(3)(-)/day) and placebo (PL; 0.5 l/day low-calorie juice cordial) treatments. The exercise protocol (two moderate-intensity step tests followed by a ramp test) was repeated 2.5 h following first ingestion (0.5 liter) and after 5 and 15 days of BR and PL. Plasma nitrite concentration (baseline: 454 ± 81 nM) was significantly elevated (+39% at 2.5 h postingestion; +25% at 5 days; +46% at 15 days; P < 0.05) and systolic and diastolic BP (baseline: 127 ± 6 and 72 ± 5 mmHg, respectively) were reduced by ∼4% throughout the BR supplementation period (P < 0.05). Compared with PL, the steady-state Vo(2) during moderate exercise was reduced by ∼4% after 2.5 h and remained similarly reduced after 5 and 15 days of BR (P < 0.05). The ramp test peak power and the work rate at the gas exchange threshold (baseline: 322 ± 67 W and 89 ± 15 W, respectively) were elevated after 15 days of BR (331 ± 68 W and 105 ± 28 W; P < 0.05) but not PL (323 ± 68 W and 84 ± 18 W). These results indicate that dietary NO(3)(-) supplementation acutely reduces BP and the O(2) cost of submaximal exercise and that these effects are maintained for at least 15 days if supplementation is continued.

Introduction Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles. Methods Materials and Methods Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real time polymerase chain reaction (QRT-PCR) analysis.
Results and Discussion the mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p

Blood concentrations of hydrogen sulfide (H(2)S) are markedly elevated in several animal models of inflammation. Pharmacological inhibition of H(2)S synthesis reduces inflammation and swelling, suggesting that H(2)S is a potential inflammatory mediator. However, it is currently unknown whether H(2)S synthesis is perturbed in human inflammatory conditions or whether H(2)S is present in synovial fluid. We analyzed paired plasma and synovial fluid (SF) aspirates from rheumatoid arthritis (RA; n= 20) and osteoarthritis (OA; n= 4) patients and plasma from age matched healthy volunteers (n= 20). Median plasma H(2)S concentrations from healthy volunteers and RA and OA patients were 37.6, 36.6, and 37.6 microM, respectively. In RA patients, median synovial fluid H(2)S levels (62.4 microM) were significantly higher than paired plasma (P= 0.002) and significantly higher than in synovial fluid from OA patients (25.1 microM; P= 0.009). SF H(2)S levels correlated with clinical indices of disease activity (tender joint count, r= 0.651; P < 0.05) and markers of chronic inflammation; Europhile count (r=-0.566; P < 0.01) and total white cell count (r=-0.703; P < 0.01). Our study shows for the first time that H(2)S is present in synovial fluid and levels correlated with inflammatory and clinical indices in RA patients.

The purpose of this study was to elucidate the mechanistic bases for the reported reduction in the O(2) cost of exercise following short-term dietary nitrate (NO(3)(-)) supplementation. In a randomized, double-blind, crossover study, seven men (aged 19-38 yr) consumed 500 ml/day of either nitrate-rich beet root juice (BR, 5.1 mmol of NO(3)(-)/day) or placebo (PL, with negligible nitrate content) for 6 consecutive days, and completed a series of low-intensity and high-intensity "step" exercise tests on the last 3 days for the determination of the muscle metabolic (using (31)P-MRS) and pulmonary oxygen uptake (Vo(2)) responses to exercise. On days 4-6, BR resulted in a significant increase in plasma [nitrite] (mean +/- SE, PL 231 +/- 76 vs. BR 547 +/- 55 nM; P < 0.05). During low-intensity exercise, BR attenuated the reduction in muscle phosphocreatine concentration ([PCr]; PL 8.1 +/- 1.2 vs. BR 5.2 +/- 0.8 mM; P < 0.05) and the increase in Vo(2) (PL 484 +/- 41 vs. BR 362 +/- 30 ml/min; P < 0.05). During high-intensity exercise, BR reduced the amplitudes of the [PCr] (PL 3.9 +/- 1.1 vs. BR 1.6 +/- 0.7 mM; P < 0.05) and Vo(2) (PL 209 +/- 30 vs. BR 100 +/- 26 ml/min; P < 0.05) slow components and improved time to exhaustion (PL 586 +/- 80 vs. BR 734 +/- 109 s; P < 0.01). The total ATP turnover rate was estimated to be less for both low-intensity (PL 296 +/- 58 vs. BR 192 +/- 38 microM/s; P < 0.05) and high-intensity (PL 607 +/- 65 vs. BR 436 +/- 43 microM/s; P < 0.05) exercise. Thus the reduced O(2) cost of exercise following dietary NO(3)(-) supplementation appears to be due to a reduced ATP cost of muscle force production. The reduced muscle metabolic perturbation with NO(3)(-) supplementation allowed high-intensity exercise to be tolerated for a greater period of time.

There has now been a great deal written about inorganic nitrate in both the popular press and in scientific journals. Papers in the 1970s warned us that inorganic nitrate could theoretically be metabolised in the human body to N-nitroso compounds, many of which are undoubtedly carcinogenic. More recently there is evidence that nitrate can undergo metabolic conversion to nitrite and nitric oxide and perform a useful protective function to prevent infection, protect our stomach, improve exercise performance and prevent vascular disease.

OBJECTIVE: the binding of FasL (CD95L) to its receptor, Fas (CD95), induces apoptosis. Studies have shown that in patients with rheumatoid arthritis (RA), T lymphocytes are resistant to FasL-induced apoptosis in vivo but are susceptible to FasL-induced apoptosis in vitro. Dysfunction in this mechanism may be an important contributor to the pathophysiology of RA. Thus, the present study was undertaken to determine which factors might inhibit FasL-Fas binding in vivo and those that would inhibit apoptosis of T lymphocytes in an in vitro model system. METHODS: Human Jurkat T cells rendered apoptotic by FasL exposure were analyzed by flow cytometry. Necrosis was determined according to measurement of lactate dehydrogenase release. Quantification of calreticulin in plasma and synovial fluid and of calreticulin-FasL binding was performed by enzyme-linked immunosorbent assay. Measurement of nitrite/nitrate in the plasma and synovial fluid was carried out by chemiluminescence assay. RESULTS: Extracellular calreticulin was present at a significantly higher concentration in the plasma (median 10.3 ng/ml, interquartile range [IQR] 14.8 ng/ml) and synovial fluid (median 10.3 ng/ml, IQR 12.0 ng/ml) of RA patients (each P < 0.05) compared with the plasma (median 3.1 ng/ml, IQR 1.3 ng/ml) and synovial fluid (median 2.9 ng/ml, IQR 0.9 ng/ml) of patients with psoriatic arthritis and the plasma of healthy control subjects (median 2.9 ng/ml, IQR 0.9 ng/ml). Calreticulin concentrations in the synovial fluid correlated with the tender and swollen joint counts and the activity scores on the 28-joint Disease Activity Score assessment. Calreticulin also bound directly to FasL. In vitro, calreticulin (2-16 ng/ml) inhibited FasL-induced apoptosis of Jurkat T cells. CONCLUSION: Calreticulin was present at higher concentrations in the plasma and synovial fluid of RA patients. Calreticulin had the capacity to bind directly to FasL and to inhibit FasL-mediated apoptosis of Jurkat T cells, and thus might play a role in inhibiting apoptosis of inflammatory T cells in RA.

Reactive oxygen intermediates (ROIs) play a key role in a number of human diseases either by inducing cell death, cellular proliferation, or by acting as mediators in cellular signaling. Therefore, their measurement in vivo and in cell culture is desirable but technically difficult and often troublesome. To address some of the key methodological issues in examining the formation of ROI in cells and mitochondria, this chapter discusses the following: (a) the cellular sources of ROI and their enzymatic removal, (b) common methods used to determine cellular and mitochondrial ROI such as chemiluminescence, electron paramagnetic resonance spectroscopy, fluorescence, and enzymatic techniques, and (c) some common problems associated with these assays and the interpretation of data. We also provide some simple protocols for the estimation of ROI production in cells and mitochondria, and when measuring ROI in cells and mitochondria, we emphasize the need for thorough understanding of results obtained and their interpretation.

Pharmacological sodium nitrate supplementation has been reported to reduce the O2 cost of submaximal exercise in humans. In this study, we hypothesized that dietary supplementation with inorganic nitrate in the form of beetroot juice (BR) would reduce the O2 cost of submaximal exercise and enhance the tolerance to high-intensity exercise. In a double-blind, placebo (PL)-controlled, crossover study, eight men (aged 19-38 yr) consumed 500 ml/day of either BR (containing 11.2 +/- 0.6 mM of nitrate) or blackcurrant cordial (as a PL, with negligible nitrate content) for 6 consecutive days and completed a series of "step" moderate-intensity and severe-intensity exercise tests on the last 3 days. On days 4-6, plasma nitrite concentration was significantly greater following dietary nitrate supplementation compared with PL (BR: 273 +/- 44 vs. PL: 140 +/- 50 nM; P < 0.05), and systolic blood pressure was significantly reduced (BR: 124 +/- 2 vs. PL: 132 +/- 5 mmHg; P < 0.01). During moderate exercise, nitrate supplementation reduced muscle fractional O2 extraction (as estimated using near-infrared spectroscopy). The gain of the increase in pulmonary O2 uptake following the onset of moderate exercise was reduced by 19% in the BR condition (BR: 8.6 +/- 0.7 vs. PL: 10.8 +/- 1.6 ml.min(-1).W(-1); P < 0.05). During severe exercise, the O2 uptake slow component was reduced (BR: 0.57 +/- 0.20 vs. PL: 0.74 +/- 0.24 l/min; P < 0.05), and the time-to-exhaustion was extended (BR: 675 +/- 203 vs. PL: 583 +/- 145 s; P < 0.05). The reduced O2 cost of exercise following increased dietary nitrate intake has important implications for our understanding of the factors that regulate mitochondrial respiration and muscle contractile energetics in humans.

OBJECTIVES: to investigate the effect of hyperbaric oxygen (HBO) on platelet physiology. DESIGN AND METHODS: Human platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation. RESULTS: HBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa). CONCLUSIONS: Exposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.

Post-translational modifications play a central role in determining the function of proteins. Such protein modifications come in a great variety of guises, and include phosphorylation, proteolysis, glycosylation, citrullination and oxidative modifications. In relation to inflammatory autoimmune diseases, some post-translational modifications appear to result in the generation of new antigens, and hence autoantibodies. Examples include: the induction of peptide immunogenicity by the spontaneous conversion of aspartic acid residues to isoaspartic acid; granzyme B-mediated cleavage of SLE autoantigens; the oxidative modification--on the surface of apoptotic cells--of lipids and proteins, rendering them immunogenic; and the presence of antibodies to oxidatively modified type II collagen and C1q in RA and SLE patients, respectively. The measurement of autoantibodies to citrullinated proteins has been verified as a very useful diagnostic tool in RA. Proteomics techniques, in principle, allow the detection of all types of in vivo protein modifications, and the increasing application of such technologies to the study of rheumatological diseases will further our understanding of autoantigenicity.

S-Nitroso moieties, such as the S-nitroso group within S-nitrosated albumin, constitute a potential endogenous reservoir of nitric oxide (NO.) in human tissues and other biological systems. Moreover, S-nitroso compounds are under investigation as therapeutic agents in humans. Therefore, it is important to be able to detect S-nitrosothiols (RSNOs) in human extracellular fluids, such as plasma and synovial fluid, as well as other biological samples. This chapter describes a method for the determination of S-nitrosothiols in biofluids. The method is based on electron paramagnetic resonance (EPR) spectrometry, in combination with spin trapping using a ferrous ion complex of the iron chelator N-methyl-d-glucamine dithiocarbamate under alkaline conditions. This iron complex mediates the decomposition of RSNO to NO. as well as spin trapping the generated NO. The resulting spin adduct has a unique EPR signal that can be quantified.

Rheumatoid arthritis is an autoimmune-mediated inflammatory disease of unknown etiology, and is characterized by joint pain and soft-tissue swelling. The role of dietary antioxidants in the prevention and amelioration of symptoms in inflammatory joint disease has been of interest for many years. Epidemiological studies provide evidence of a link between dietary antioxidant intake and the likelihood of developing inflammatory arthritis. Interventional studies of antioxidant supplementation in established disease have been inconclusive overall; however, the quality of such studies has often been poor. The pathways by which antioxidant compounds might act are now better understood. In this Review, we explore not only some of the accepted mechanisms of antioxidant function but also outline some concepts that could aid further investigation of the potential therapeutic role of dietary antioxidants in inflammatory arthritis.

Plasma S-nitrosothiols (RSNOs) may act as a circulating form of nitric oxide that affects vascular function and platelet aggregation. Their role in liver ischemia/reperfusion (I/R) injury is largely unknown. The aim of the present study was to investigate the changes in plasma RSNOs following liver I/R injury. Two groups of New Zealand white rabbits were used (n=6, each): the I/R group underwent 60 min lobar liver ischemia and 7 h reperfusion, while the sham group underwent laparotomy but no liver ischemia. Serial RSNO levels were measured in plasma by electron paramagnetic resonance (EPR) spectrometry, nitrite/nitrates by capillary electrophoresis, hepatic microcirculation by laser Doppler flowmetry, redox state of hepatic cytochrome oxidase by near-infrared spectroscopy, liver iNOS mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) and the oxidation of dihydrorhodamine to rhodamine by fluorescence. The effect of the antioxidant N-acetylcysteine (NAC) on RSNOs formation and DHR oxidation was tested in a third group of animals (n=6) undergoing lobar liver I/R. Hepatic I/R was associated with a significant increase in plasma RSNOs, plasma nitrites, hepatic iNOS mRNA expression, impairment in hepatic microcirculation, decrease in the redox state of cytochrome oxidase, and significant production of rhodamine. The changes were more obvious during the late phase of reperfusion (>4 h). NAC administration decreased plasma RSNOs and oxidation of DHR to RH (P

Oxidative stress is implicated in the pathogenesis of multiple sclerosis (MS). Defence against oxidative damage is mediated by antioxidants. Peroxiredoxin V (PRDX V) is an intracellular anti-oxidant enzyme with peroxynitrite reductase activity. It is increased during inflammation, when free radical production intensifies, and is protective in an animal model of brain injury. However, little is known about PRDX V expression in the human brain. We investigated PRDX V expression in white matter from normal human brain (n = 5) and MS patients (n = 18), using immunohistochemistry and immunoblotting. A global increase in PRDX V was evident in MS normal-appearing white matter (NAWM) but the most striking increase was in astrocytes in MS lesions. PRDX V- positive hypertrophic reactive astrocytes were seen in acute lesions where inflammation was present. Yet surprisingly, in chronic lesions (CL), where inflammation has abated and a glial scar formed, there was strong PRDX V staining of post-reactive, scar astrocytes. Furthermore, immunoblotting analysis of tissue from two MS cases confirmed a substantial increase in PRDX V expression in CL compared with NAWM from the same individual. This might indicate ongoing oxidative stress despite the absence of histologically defined inflammation. Further investigations of this phenomenon will be of interest for therapeutic targeting.

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

The last decade has witnessed an increased interest in cysteine modifications such as sulfenic and sulfinic acids, thiyl radicals, sulfenyl-amides and thiosulfinates, which come together to enable redox sensing, activation, catalysis, switching and cellular signalling. While glutathionylation, sulfenyl-amide formation and disulfide activation are examples of relatively simple redox responses, the sulfinic acid switch in peroxiredoxin enzymes is part of a complex signalling system that involves sulfenic and sulfinic acids and interacts with kinases and sulfiredoxin. Although the in vivo evaluation of sulfur species is still complicated by a lack of appropriate analytical techniques, research into biological sulfur species has gained considerable momentum and promises further excitement in the future.

Nitric oxide (NO) is a small, highly reactive, diffusible free radical which has been implicated in many physiological and pathophysiological processes. It has either pro-apoptotic or anti-apoptotic effects on cells, depending upon a host of factors. This review outlines some of the regulatory molecules and organelles involved in the apoptotic pathways that can be influenced by the presence of NO, including p53, Bcl-2, caspases, mitochondria, and heat shock proteins. The effects of NO on the apoptosis of tumour cells are also examined.

OBJECTIVE: the inflammatory mediator substance P (SP) acts principally through the neurokinin (NK1) receptor. We assessed the influence of SP on production of NO and its possible role in the pathogenesis of rheumatoid arthritis (RA). METHODS: the effect of SP (0.1-100 nM) on concentrations of the NO metabolite, nitrite, produced by synovial fibroblasts from RA patients was studied. For comparison, the effects of TNF-alpha (0.57 pM-5.7 nM) and IL-1beta (0.57 pM-5.7 nM) were also studied. In parallel studies, footpad inflammation was induced in NK1 receptor knock-out (KO) and wild-type (WT) mice, and swelling and NO metabolite levels were measured. RESULTS: in cultured synoviocytes, SP, TNF-alpha and IL-1beta induced significantly increased nitrite concentrations. Consistent with a role for NO in SP-mediated inflammatory reactions, the plasma NO metabolite level in WT mice was significantly increased at 3 days following an injection of 10 mg/ml Mycobacterium tuberculosis, but there was no significant change in NK1 KO mice. These results were paralleled by the changes in footpad swelling in WT mice compared to NK1 KO mice. CONCLUSION: SP, like TNF-alpha and IL-1beta, induces NO in both rheumatoid synoviocytes and experimental models of inflammation. Treatments directed against SP may have important and hitherto unrecognised anti-inflammatory effects.

OBJECTIVE: Collagen-induced arthritis is a commonly accepted model of rheumatoid arthritis (RA). However, it has been difficult to substantiate the involvement of autoimmunity to type II collagen (CII) in the pathogenesis of RA. The aim of this investigation was to determine if CII, modified by reactive oxidant species present within the inflamed joint, could generate neoantigenic epitopes. METHODS: Oxidants that play a role in acute and chronic inflammation and are present in the rheumatoid joint (hydroxyl radical, hypochlorous acid, and peroxynitrite) were used for modification of native CII. In addition, CII was glycated with ribose, since nonenzymatic oxidative reactions by glycation are evident in RA. Modifications were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 3-dimensional fluorescence followed by enzyme-linked immunosorbent assay (ELISA) and Western blotting, using, as probes, sera from patients with RA and from patients with other inflammatory and noninflammatory joint diseases. RESULTS: Only 1 RA serum sample showed strong binding to native CII. In contrast, binding to modified CII was increased in 14 of 31 RA sera, of which 7 were strong binders and 7 were moderate binders. Among the non-RA serum samples, only 1 yielded a strong reaction to modified CII and 5 of 41 were moderate binders. Samples that showed the strongest binding to modified CII in ELISA also showed strong binding to various fragmented or aggregated forms of CII in Western blots, as well as strong binding to fragmented CII present in RA synovial fluid. CONCLUSION: When modified by conditions found within the inflamed joint, CII acts as an autoantigen in RA.

In human tissues, S-nitrosothiols (RSNOs) are generated by the nitric oxide (NO.)-dependent S-nitrosation of thiol-containing species. Here, a novel electron paramagnetic resonance spectrometry assay for RSNOs is described, together with its application to studies of human health and disease. The assay involves degrading RSNOs using N-methyl-d-glucamine dithiocarbamate (MGD) at high pH and spin trapping the NO. released using (MGD)2-Fe2+. Because dietary nitrate might contribute to tissue RSNOs, the assay was used to monitor the effect of Na15NO3 ingestion on plasma and gastric juice RSNOs in healthy human volunteers. Na15NO3 ingestion (2 mmol) increased gastric RS15NO concentrations (p

Living cells maintain a delicate balance between oxidizing and reducing species, and many disorders such as rheumatoid arthritis and Alzheimer's disease have been associated with a disturbed intracellular 'redox equilibrium'. The past few years have witnessed accelerated research into how natural redox responses and antioxidant defence systems are activated and how they restore a healthy redox balance. To function properly, many of these processes rely on a powerful sulfur redox chemistry, which is best exemplified by the complex, newly emerging cysteine-based redox regulation of the glutathione and thioredoxin pathways. Other redox systems based on oxidatively activated amino acid side chains in proteins are also becoming increasingly important, but are still barely understood or explored.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of rheumatoid arthritis (RA), while antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD) and catalase, block radical-induced events. The present study tested if the ex vivo transfer of EC-SOD and catalase genes alone or in combination in the knee joint of rats with monoarticular antigen-induced arthritis (AIA) was anti-inflammatory, and examined the potential mechanisms involved. Synoviocytes isolated from female Wistar rats were immortalized with a retroviral vector SUV19.5. These cells were permanently transfected with an EC-SOD expression plasmid (pEC-SODZeo) or a catalase expression plasmid (pCatalaseZeo) to create cells overexpressing EC-SOD or catalase, as measured by RT-PCR and Western blots. The cells were engrafted in knee joints of animals at the time of the induction of AIA. Three gene transfer groups, an EC-SOD group, a catalase group and a combined therapy group (EC-SOD and catalase) were included in these experiments. Animals in the control group were engrafted with synoviocytes transfected with the plasmid pZeoSV2 without an insert. Clinical and histological assessments were performed, as well as tissue measurements of SOD, catalase and gelatinase activities. Ex vivo gene transfer of EC-SOD and catalase into rat knee joints produced about a six- to seven-fold increase in EC-SOD activity and a two- to three-fold increase in catalase activity compared with the control animals. Rats treated with cells overexpressing EC-SOD, catalase or a combination of EC-SOD and catalase showed significant suppression of knee joint swelling, decreased infiltration of inflammatory cells within the synovial membrane and reduced gelatinase activity in knee joints, compared with animals receiving cells transfected with the plasmid alone. No statistically significant difference was found between the groups treated with cells overexpressing EC-SOD, catalase or a combination of both. Gene therapy involving the local intra-articular overexpression of two antioxidant enzymes, EC-SOD and catalase, was anti-inflammatory in AIA. One mechanism appears to be the suppression of gelatinase activities by both EC-SOD and catalase.

BACKGROUND: Inactivation of the elastase inhibitor, alpha1 proteinase inhibitor (alpha1PI), may be of pathogenic significance in inflammatory diseases like periodontal disease. Two key mechanisms of inactivation appear to be (a) the formation of an alpha1PI-elastase complex and (b) proteolytic cleavage by elastase or other enzymes such as metalloproteinases of host origin or enzymes of bacterial origin. Based on the different heat stabilities of the intact, complexed and proteolytically cleaved forms of alpha1PI, an enzyme-linked immunosorbent assay (ELISA) that allowed the simultaneous measurement of native and inactive forms of alpha1PI was developed. METHODS: the ELISA method described employs a commercially available antibody and represents a rapid, reproducible and sensitive method for studying alpha1PI inactivation in human inflammatory diseases. The assay was applied to normal human plasma and to human extracellular fluids obtained from patients with inflammatory diseases such as adult periodontitis and rheumatoid arthritis. Samples from patients with osteoarthritis, a "non-inflammatory" joint disease, were also studied. RESULTS: the findings expressed as the mean percentage (+/-SD) of the total alpha1PI that was inactivated were as follows: gingival crevicular fluid from adult periodontitis patients: 73.5+/-16.6% (n=12); normal human plasma: 8.4+/-4.9% (n=13); knee-joint synovial fluid (SF) from rheumatoid arthritis patients: 12.5+/-4.5% (n=15); plasma from rheumatoid arthritis patients: 8.0+/-1.8% (n=15); knee-joint SF from osteoarthritis patients: 8.6+/-8.2% (n=14); plasma from osteoarthritis patients: 5.7+/-4.8% (n=14). The results obtained by ELISA were in good agreement with those obtained by the semi-quantitative method of SDS-PAGE and Western blotting. CONCLUSIONS: We have shown that the differential heat stability of alpha1PI may be utilised as the basis for a rapid, sensitive and reproducible ELISA assay of alpha1PI inactivation. In gingival crevicular fluid from periodontal disease patients, alpha1PI is mainly inactivated and the extent of this inactivation is much higher than in inflammatory fluids from other chronic diseases such as rheumatoid arthritis. This assay could be useful in monitoring the progression of periodontal disease.

During the preparation of sodium 3,5-dibromo-4-nitrosobenzenesulphonate (DBNBS) of high purity for electron paramagnetic resonance (EPR) spin-trapping purposes, it was found that the material synthesised as part of the present study differed significantly from some commercially available samples of DBNBS. A thorough chemical characterisation of the contents of the various samples led to the conclusion that the preparations synthesised in the present study, as well as one of four commercially available samples, contained essentially pure DBNBS and had efficient spin-trapping activity. In contrast, the remaining three commercially available samples contained almost exclusively sodium 3,5-dibromo-4-nitrobenzenesulphonate, i.e. a one-oxygen oxidation product of DBNBS, and had little spin-trapping activity. The two compounds were readily separated by reverse-phase high performance liquid chromatography (HPLC). It was further found that the quality of DBNBS preparations may be determined by NMR spectrometry, IR spectrometry, fast atom bombardment-mass spectrometry (FAB-MS) and EPR spectrometry. In particular, UV-Visible spectroscopy may be used to determine A308/A280, which should be greater than 1.8 for a high purity DBNBS preparation.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are polygenic diseases. Polymorphisms in candidate genes have been studied for possible association with susceptibility to disease development. Aside from HLA polymorphisms, of particular interest are those in genes encoding cytokines, signaling molecules, and enzymes involved in the production and catabolism of oxygen and nitrogen radicals. Cytokines are involved in the modulation of the pathological process and have been the target for novel therapeutic interventions. Evidence for their involvement in RA and OA has been provided from genetic analyses in patient populations as well as from animal models of disease. Intracellular signaling cascades control cellular responses and thus regulate many aspects of the pathology manifested in rheumatic diseases. Deciphering the organization and activity of such signaling pathways in disease is underway. Polymorphisms have been identified in gene promoter regions regulating efficient binding of transcription factors, and in coding regions of genes whose products are involved in signal cascades relevant to RA. Among these are the NF-kappaB pathway, steroid receptors and the p53 tumor suppressor gene. Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) have also been implicated in rheumatic diseases. It is thought that excess, damaging, ROS/RNS may arise from an imbalance between the production and removal of these chemical species. Polymorphisms in genes that encode enzymes involved in either generating or degrading ROS/RNS may contribute to such an imbalance. In the last few years, polymorphisms in such genes have indeed been identified as risk factors for rheumatic diseases.

Although myoglobin protein radicals are thought important intermediates in peroxide-induced toxicity, the site of spin trapping of this radical in equine myoglobin using the trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) is unclear. We have combined EPR, electrophoretic adduct purification, and mass spectrometry approaches to unambiguously determine the site of trapping to be Tyr-103 and suggest that reports of trapping at Trp-7 or Trp-14 may be due to nonradical addition to proteolytically derived Trp-containing peptides with DBNBS. The technique developed here of combining electrophoretic separation of DBNBS adducts with MS of resultant peptides will also allow proteomic-like approaches to determining identities and sites of radical formation and translocation on complex mixtures of proteins.

Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), an oxidant was previously detected in the plasma of patients with renal failure and the synovial tissue of rheumatoid arthritis patients. This oxidant has been shown to react with DBNBS to give a 3-line electron paramagnetic resonance (EPR) spectrum, previously assigned to the DBNBS radical cation (DBNBS*(+). However, confusion has arisen as to whether this paramagnetic species is indeed DBNBS*(+) or, rather, the DBNBS sulfite radical adduct (DBNBS-SO(3)*(-)). In the present study, DBNBS*(+) (a(N)=1.32 mT) was distinguished from DBNBS-SO(3)*(-) (a(N)=1.32 mT with an additional splitting of a(H)=0.06 mT) by (a) using different EPR parameters, (b) determining the effect of addition of sulfite on the EPR spectrum resulting from the incubation of DBNBS with either human biofluids or the horseradish peroxidase (HRP)-hydrogen peroxide (H(2)O(2)) system, and (c) replacing DBNBS with its analogues (DBNBS-d(2,) DBNBS-15N and DBNBS-d(2)-15N) in the two systems.

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO(*)). The reaction between DBNBS and NO(*) yields a radical product which gives rise to an EPR signal consisting of three lines with an A(N) = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO(*) reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment-mass spectrometry (FAB-MS). Collectively, the FAB-MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO(*). A reaction scheme consistent with these results is presented.

Reactive oxygen intermediates (ROIs), such as hydrogen peroxide (H2O2), have been implicated as second messengers in the activation of NF-kappaB by a variety of stimuli, including tumour necrosis factor-alpha (TNF-alpha). The aim of the present study was to examine the effects of ROIs on NF-kappaB activation in primary human CD3+ T lymphocytes and human peripheral blood mononuclear cells (PBMCs). For comparison purposes, Jurkat T cells (subclones JR and JE6.1) were also investigated. Cells were incubated in the presence of either H2O2 or TNF-alpha and nuclear proteins were extracted. NF-kappaB binding was assessed by electrophoretic mobility shift assays (EMSAs). The concentration of H2O2 required to activate NF-kappaB in human primary CD3+ T lymphocytes was as low as 1 microM. In contrast, much higher concentrations of H2O2 were required to activate NF-kappaB in PBMCs and in the JR subclone of Jurkat T cells. H2O2-induced NF-kappaB activation was not observed in the JE6.1 subclone of Jurkat T cells. NF-kappaB was activated by TNF-alpha in all four cell types tested. In PBMCs and Jurkat T cells (subclones JR and JE6.1), this activation could be inhibited by pre-treatment with the antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC). Our results support a role for ROIs in NF-kappaB-DNA binding in human primary T lymphocytes.

OBJECTIVE: to investigate the activation of NF-kappaB in the carrageenan rat air pouch model of inflammation in a time course experiment, and the effect of dexamethasone on NF-kappaB activation. METHODS: Air pouch tissue treated with carrageenan (inflamed tissue) was obtained from rats killed at days 1, 2, 3, 6, 14, 21, 28 and 35 after carrageenan challenge. Tissue was also taken from non-carrageenan treated pouches (non-inflamed tissue) at day 3, and from inflamed tissues treated with dexamethasone. Tissue sections were wax embedded and stained with an "activity specific" monoclonal antibody raised against the nuclear localisation signal (NLS) of the p65 sub-unit of NF-kappaB. RESULTS: Cells containing activated NF-kappaB were detected in the intimal and sub-intimal regions of the air pouches as early as day 1. There was a significant increase in cells staining for activated NF-kappaB as the inflammation progressed. Initially cells staining were more prominent in the intimal versus sub-intimal region (p

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.

Several bone resorptive stimuli affect osteoclasts indirectly by modulating the production and release of osteoblastic factors. Using electrophoretic mobility shift assays, we found that not only tumour necrosis factor-alpha (TNF-alpha) but also interleukin-1beta and parathyroid hormone (PTH) caused dose and time-related increases in nuclear factor kappaB (NF-kappaB)-DNA binding in Saos-2 human osteoblastic (hOB) cells. Activation of NF-kappaB by TNF-alpha was reproduced in primary hOBs. In contrast, consistent with their previously reported lack of response to steroid hormones, Saos-2 cells did not respond to 1,25-dihydroxyvitamin D(3). We suggest that NF-kappaB activation in osteoblastic cells constitutes an important pathway in osteoblast-mediated resorptive signalling.

DBNBS (3,5-dibromo-4-nitrosobenzenesulphonate) reacts with nitric oxide (NO) produced from nitrite ions in acid solution to give a radical with a characteristic electron spin resonance spectrum, attributable to a 'DBNBS-NO' product, and comprising a triplet with alphaN=0.96 mT. This is identical with the spectrum obtained when NO, introduced from the gas phase, reacts with DBNBS. Under certain conditions, an additional signal is observed, attributable to oxidation of DBNBS to the radical cation, DBNBS*+ (a triplet with alphaN=1.32 mT). Conditions are described for the determination of nitrite, which avoid this DBNBS oxidation. The height of the low-field signal from the DBNBS-NO product is directly proportional to the nitrite concentration up to about 0.08 mM nitrite. The method has been applied to the measurement of nitrite concentrations in whole blood, plasma and synovial fluid taken from rheumatoid arthritis patients. In order to avoid the oxidation of DBNBS when analysing biological samples of this type, it is necessary to treat the specimen by ultrafiltration as soon as possible after collection and before addition of DBNBS.

A simple but rapid capillary electrophoresis method was developed for the measurement of nitrite and nitrate in human extracellular fluids and other aqueous solutions. The capabilities of the method were demonstrated by the measurement of endogenous nitrite and nitrate in plasma and serum samples from healthy volunteers, and serum and synovial fluid samples from rheumatoid arthritis patients. Furthermore, this method was used to simultaneously measure nicotinamide adenine dinucleotide, reduced (NADH), nicotinamide adenine dinucleotide (NAD+), nitrite, and nitrate, when studying the nitrite reductase activity of xanthine oxidase. The stability of nitrite was also investigated and it was found that when whole blood was spiked with nitrite and then processed, the nitrite was more stable in the plasma than in the serum. Our findings may help to explain the variations in basal nitrite concentrations reported in the literature.

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.

Nitric oxide (NO) synthesis is well-known to result from the oxidation of L-arginine by a family of NO synthases (NOS). However, under hypoxic conditions this mechanism of NO synthesis may be impaired and NO is formed by a NOS independent mechanism. This study was designed to examine the reduction of nitrite to NO by xanthine oxidase (XO) under hypoxia, because the bacterial nitrate/nitrite reductases have structural similarity to XO. We found that both purified and tissue containing XO catalyze the reduction of nitrite to NO, as demonstrated using a chemiluminescent NO meter. This redox reaction requires NADH as an electron donor, and is oxygen independent. The inhibitory profiles suggest that reduction of nitrite takes place at the molybdenum center of XO whilst NADH is oxidized at the FAD center. Heparin binding of XO caused an increase in the catalysis of nitrite reduction. The XO-catalyzed generation of NO may be important in redistribution of blood flow to ischaemic tissue as a supplement to NOS, since both nitrite and NADH have been shown to be elevated in hypoxic tissue.

OBJECTIVE: to investigate the thrombin inhibitory capacity of antithrombin III in the inflamed human joint. METHODS: Thrombin inhibitory capacity was measured, using a kinetic spectophotometric method, in matched plasma and synovial fluid samples of patients with rheumatoid arthritis (n = 22) and osteoarthritis (n = 16), together with normal control plasma samples (n = 13). In the same samples, the concentration of antithrombin III was also determined by the method of radial immunodiffusion. The combination of these measurements allowed the calculation of the specific thrombin inhibitory capacity of these samples. RESULTS: an increased concentration of antithrombin III in rheumatoid compared with osteoarthritic synovial fluid was noted (p < 0.05). However, there was a significant depression in the specific activity of antithrombin III in rheumatoid synovial fluid when compared with matched plasma samples (p < 0.001) or with osteoarthritic synovial fluid (p < 0.05). CONCLUSION: in rheumatoid synovial fluid the thrombin inhibitory capacity of antithrombin III is disproportionately depressed relative to the concentration of antithrombin III, indicating the inactivation of antithrombin III in the rheumatoid joint.

Reactive oxygen species (ROS) are pro-inflammatory factors in the pathogenesis of rheumatoid arthritis. During inflammation, the amount of low-density lipoprotein (LDL) in the inflamed joint is increased. LDL is known to be susceptible to oxidation by ROS. Oxidized LDL may serve as a mediator for joint damage, further exacerbating the inflammatory process. LDL isolated from synovial fluid and plasma from individual patients (paired samples) with rheumatoid arthritis or osteoarthritis was characterized by crossed immunoelectrophoresis. On analysis by this technique, synovial fluid LDL from most patients with rheumatoid arthritis contained two peaks: one corresponding to normal plasma native LDL, and the other having an increased electrophoretic mobility associated with oxidized LDL. Paired plasma LDL samples contained native LDL alone, as did paired synovial fluid and plasma LDL from patients with osteoarthritis. Thus, in addition to native LDL, a second form of LDL was shown to be present in rheumatoid synovial fluid, which had properties consistent with those of oxidized LDL.

OBJECTIVE: Vitamin E, the most potent naturally occurring lipid soluble antioxidant has been suggested to possess both anti-inflammatory and analgesic activity in humans. This double blind and randomised study used a broad spectrum of clinical and laboratory parameters to investigate whether there was any additional anti-inflammatory or analgesic effects, or both, of orally administered alpha-tocopherol in rheumatoid arthritis patients who were already receiving anti-rheumatic drugs. METHODS: Forty two patients were enrolled and treated with alpha-tocopherol (n = 20) at a dose of 600 mg twice a day (2 x 2 capsules) or with placebo (n = 22) for 12 weeks. The following parameters were measured: (1) Three clinical indices of inflammation--the Ritchie articular index, the duration of morning stiffness, and the number of swollen joints; (2) three measures of pain--pain in the morning, pain in the evening, and pain after chosen activity; (3) haematological and biochemical measures of inflammatory activity; (4) assays for the oxidative modification of proteins and lipids. RESULTS: all laboratory measures of inflammatory activity and oxidative modification were unchanged. Furthermore, the clinical indices of inflammation were not influenced by the treatment. However, the pain parameters were significantly decreased after vitamin E treatment when compared with placebo. CONCLUSION: the results provide preliminary evidence that vitamin E may exert a small but significant analgesic activity independent of a peripheral anti-inflammatory effect, but which complements standard anti-inflammatory treatment.

OBJECTIVE: the transcription factor nuclear factor kappaB (NF-kappaB) has been implicated in the inflammatory response and is known to be activated by a process involving reactive oxygen intermediates. The purpose of the present study was to demonstrate the presence and distribution of activated NF-kappaB in synovium samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and from autopsy subjects with no known history of arthritis. METHODS: Immunohistochemical staining was performed using both polyclonal and monoclonal "activity-specific" antibodies to the Rel-A (p65) subunit of NF-kappaB (anti-Rel-A nuclear location sequences). Histologic features of inflammation were also scored. RESULTS: Both antibodies demonstrated positive staining of synovial tissue, with a cellular distribution that was nuclear. The staining was associated with specific cell types within the tissue, in particular, type a synoviocytes and vascular endothelium. Notably, lymphoid aggregates were unstained. Using the monoclonal antibody, a further study was carried out to investigate the distribution of staining in tissues from patients with different disease activities and clinical diagnoses, as well as in normal control tissue obtained at autopsy. Patients with acute RA more commonly showed vessel staining (P = 0.05) and, conversely, showed less frequent staining of the synovial lining (P < 0.005) compared with OA patients. Synovial tissue from controls exhibited either no staining or only weak staining in the synovial lining. CONCLUSION: the activation of NF-kappaB in vascular endothelium and type a synovial lining cells is a feature of synovial tissue from both RA and OA patients. The distribution of this staining appears to be related to the clinical diagnosis.

Whilst catalytic iron has been implicated in the development of atherosclerosis by initiating low density lipoprotein (LDL) oxidation, the source of such iron remains uncertain. Here, we show that LDL oxidation in the presence of ferritin was stimulated by ascorbate (15-60 microM), whilst this effect was inhibited by the iron chelator desferrioxamine. Ascorbate also showed an antioxidant activity at high concentrations (125-250 microM). Our results suggest that the combination of ascorbate with ferritin may supply free iron for LDL oxidation in vivo.

OBJECTIVE: to assess whether the extent of LDL oxidation influences its cytotoxic effects, thus contributing to its atherogenic potential. DESIGN AND SETTING: the effects of native and modified LDL on cultured human coronary artery smooth muscle cells (SMC) and endothelial cells (ECs) were investigated. MAIN OUTCOME MEASURES: Four indices of cytotoxicity were studied: (i) chromium-51 release; (ii) 5-bromo-2'-deoxyuridine (BrDUrd) uptake; (iii) morphological appearance; and (iv) EC migration. RESULTS: (i) Minimally modified (mm) LDL (400 micrograms/ml) causes significant 51Cr release; the cytotoxic effect was significantly greater for copper oxidised (ox) LDL (400 micrograms/ml). Native LDL had no effect. (ii) BrDUrd uptake studies showed significant inhibition of cell proliferation by 100 micrograms/ml of oxLDL and to a lesser extent by mmLDL; native LDL had no effect. (iii) Morphological appearance was not altered by native LDL. Changes in cell morphology were induced by mmLDL (400 micrograms/ml), and were more pronounced with oxLDL in concentrations of > or = 200 micrograms/ml. (iv) EC migration was significantly inhibited by oxLDL (100 micrograms/ml), but not by native or mmLDL. CONCLUSION: the extent of oxidation of LDL determined its cytotoxicity to coronary artery cells. Native LDL had no cytotoxic effect. In contrast, oxLDL and to a lesser extent mmLDL caused cytotoxicity at concentrations to which cells in vivo might be exposed. This may contribute to the atherogenicity of modified LDL by enhancing cellular injury and inflammation, and by inhibiting re-endothelialisation of areas of coronary artery damaged during the atherogenic process.

Peroxynitrite (ONOO-) has recently been implicated in connective tissue destruction in vivo. We have studied the effect of ONOO- on the activity of tissue inhibitor of metalloproteinase-1 (TIMP-1) in vitro. The inactivation of TIMP-1 by ONOO- was dose dependent with 50 microM ONOO- reducing the inhibitory activity of TIMP-1 towards gelatinase-A by 50%. High concentrations of ONOO- (500 microM-5 mM) caused protein fragmentation whilst lower concentrations (

Monocyte adhesion to the arterial wall is a key event in the atherosclerotic process. We studied the interactions between human coronary arterial intimal smooth muscle cells (SMCs) and monocytes by examining (i) whether SMCs mediate monocyte adhesion when stimulated by oxidatively modified low density lipoprotein (LDL) or by the cytokines TNF alpha and IL-1, and (ii) the role of the adhesion molecules VCAM-1 and ICAM-1 (vascular cell and intercellular adhesion molecule, respectively) in this process. Preincubation of SMCs with both TNF alpha and IL-1 caused a significant 2-fold increase in VCAM-1 and ICAM-1 expression and a more than 9-fold increase in monocyte adhesion. The latter was significantly inhibited (by 1/3) by neutralising antibodies to VCAM-1 and ICAM-1. Modified LDL also induced a significant 3-fold increase in monocyte adhesion to SMCs, but did not induce VCAM-1 or ICAM-1 expression, nor was this adhesion inhibited by neutralising antibodies to VCAM-1 or ICAM-1. Oxidatively modified LDL, like the proinflammatory cytokines TNF alpha and IL-1, has the ability to enhance monocyte adhesion to human SMCs in vitro. LDL-induced monocyte adhesion to SMCs is distinct from that induced by TNF alpha and IL-1 in its lack of dependence on the classical adhesion pathways involving smooth muscle VCAM-1 and ICAM-1. SMCs are identified as a new cell population which may play an active role in recruiting monocytes to the arterial intima and atherosclerotic plaque.

OBJECTIVE: to investigate the possibility that synovial cells might respond to thrombin in the inflamed human joint, using immunohistochemical detection of thrombin receptors. METHODS: Frozen sections of synovial membrane from 20 patients with rheumatoid arthritis, 16 with osteoarthritis, and four normal controls were stained using a monoclonal antibody to the human thrombin receptor. Sections were also double stained for both receptors and non-specific esterase. RESULTS: Receptor positive cells were present in rheumatoid synovia, with some cells also staining positively for non-specific esterase. In contrast, both osteoarthritic and normal synovia contained very few cells expressing receptors. CONCLUSIONS: Thrombin may mediate important pathological changes during chronic inflammatory joint disease.

Oxidatively modified LDL (oLDL) is thought to play a key role in the pathogenesis of atherosclerosis. We have studied Cu(2+)-induced peroxidation reactions of LDL and have elucidated the sequence of events which subsequently occur within LDL particles by 1H-NMR spectroscopy. Studies of chloroform/methanol extracts show that LDL arachidonate is oxidised by Cu2+ at a higher rate and to a greater extent than linoleate, giving isomeric hydroperoxides with predominantly trans,trans double-bonds, whilst only cis,trans isomers were detected as intrinsic hydroperoxides in control LDL samples. These intrinsic hydroperoxides were not degraded during peroxidation, suggesting that they are not involved in the initiation of Cu(2+)-induced peroxidation. Aldehydes arising from the decomposition of hydroperoxides were also detected, as well as saturated fatty acids which were released into the external aqueous medium. Decomposition pathways of the two major isomeric hydroperoxides are discussed. Cu(2+)-induced oxidation of LDL cholesterol appears to occur only after hydroperoxide breakdown, with esterified cholesterol being oxidised to a greater extent than free cholesterol. Phospholipid hydrolysis appeared to parallel the peroxidation of arachidonic acid, and the released lysophosphatidylcholine may become associated with apoB. These results suggest that hydroperoxide breakdown (probably in phospholipids) may be a key event in the peroxidation process, leading to the oxidation of cholesterol and propagation into the core of LDL.

Depletion of antioxidants and the presence of products of free radical damage in plasma suggest that oxidative stress is increased in uremia. We have developed an application of electron spin resonance spectroscopy, and used this method to show that a stable oxidizing component or components of plasma accumulate in uremia. No oxidizing activity was detectable in plasma from subjects with normal renal function. The oxidant was detected by its capacity to oxidize the spin trap 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS). The oxidant was dialyzable from plasma, had an upper molecular weight limit of about 3,000 Daltons and was stable over many months. Physiological plasma concentrations of vitamin C, a water soluble congener of vitamin E and reduced glutathione were unable to inhibit the oxidizing capacity of uremic plasma. Thus, uremia is associated with accumulation of an endogenous oxidizing activity at much higher concentrations than in subjects with normal renal function.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is present in DNA isolated from human and murine cells. Other studies have reported artificially elevated levels in DNA extracted using phenol-based methods. This report shows that DNA, isolated by different methods, with or without the use of a phenol reagent, contains similar levels of 8-oxodG. This, taken with other studies of human tissues which show varying levels of 8-oxodG, indicates that, with due care in the extraction procedure, biologically significant amounts of this altered base are present in mammalian DNA.

Many drugs already in use for the treatment of skin disease, including steroids, retinoids, vitamin D derivatives and cyclosporin (CyA), exert effects on gene transcription. In this review we briefly outline how these drugs work, before proceeding to a consideration of how new drugs acting on the transcription process might be used to influence skin disease.

Matrilysin is shown to rapidly inactivate alpha 1PI, an inhibitor of elastase, by cleaving the Pro357-Met358 peptide bond of its reactive centre. The rate of inactivation of alpha 1PI by matrilysin is four times higher than stromelysin. Matrilysin cleaves oxidised alpha 1PI at the Phe352-Leu353 bond, whilst stromelysin cleaves oxidised alpha 1PI at the Met358-Ser359 bond. We conclude that matrilysin is a potent serpinase which could play a role in inflammatory tissue damage by proteolytically inactivating alpha 1PI.

OBJECTIVE: to examine the relationship between alpha 1-antitrypsin (alpha 1AT) specific activity and tumor necrosis factor alpha (TNF alpha) concentration in synovial fluid from 48 patients with rheumatoid arthritis. METHODS: the specific activity of alpha 1AT was calculated from the measurement of alpha 1AT concentration (by rocket immunoelectrophoresis) and elastase inhibitory capacity. TNF alpha was detected by enzyme-linked immunosorbent assay. RESULTS: TNF alpha concentrations correlated with the extent of alpha 1AT inactivation. CONCLUSION: Our findings are consistent with a role of elastase in TNF alpha release within the inflamed joint.

The rheumatoid joint is hypoxic. The loss of the physiologic defense mechanism, reflex muscle inhibition, allows the generation of high intraarticular pressures, particularly during exercise. Hypoxia alters the biochemistry of the synovium and encourages the production of reactive oxygen species (ROS) on reperfusion of blood. In excess, ROS damage tissues, and the products of oxidative damage are detectable in rheumatoid synovial fluid. In addition to damaging proteins, carbohydrates and lipids, cellular and structural damage also occurs.

In recent years it has become increasingly apparent that, in man, free radicals play a role in a variety of normal regulatory systems, the deregulation of which may play an important role in inflammation. As examples, we discuss the second messenger roles of: NO in the regulation of vascular tone, O2.- in fibroblast proliferation and H2O2 in the activation of transcription factors such as NF kappa B. Other control mechanisms, the physiological function of which may be perturbed in inflammation, include: the oxidative modification of low density lipoprotein, the oxidative inactivation of alpha-1-protease inhibitor, DNA damage/repair and heat shock protein synthesis. At sites of inflammation, increased free radical activity is associated with the activation of the neutrophil NADPH oxidase and/or the uncoupling of a variety of redox systems, including endothelial cell xanthine dehydrogenase. Although free radicals, thus produced, have the capacity to mediate tissue destruction, either alone or in concert with proteases, we argue that disturbances in the second messenger and regulatory activities of free radicals may also contribute significantly to the inflammatory process.

alpha 1-Antitrypsin (alpha 1AT) is known to be oxidised by reactive oxygen species both in vitro and in vivo, leading to its inactivation. We report here that synovial fluid (SF) alpha 1AT is inactivated during exercise of the knee-joints of rheumatoid arthritis (RA) patients. Sequential SF sampling from exercised RA patients showed a marked decrease in the mean activity of alpha 1AT after exercise with no change in the molecular forms of alpha 1AT. No such inactivation was found in the control (continuously resting) RA patients. We suggest that oxidation may contribute to alpha 1AT inactivation as a consequence of 'hypoxic-reperfusion' injury after exercise of the inflamed joint.

OBJECTIVES: to estimate the extent of genomic DNA damage and killing of lymphocytes by reactive oxygen intermediates in autoimmune diseases. METHODS: 8-Oxo-7-hydrodeoxyguanosine (8-oxodG), a promutagenic DNA lesion induced by reactive oxygen intermediates, was measured by high performance liquid chromatography, coupled with electrochemical detection, in hydrolysates of DNA which had been extracted from lymphocyte and polymorphonuclear leucocyte fractions of human blood. In addition, human primary blood lymphocytes stimulated by concanavalin a were assayed for cytotoxicity induced by hydrogen peroxide on day 0, by assessing cell proliferation during seven days of culture. RESULTS: Constitutive 8-oxodG was detectable (mean (2 SEM) moles 8-oxodG/10(6) moles deoxyguanosine) in DNA isolated from normal human blood lymphocytes (68 (8), n = 26) and polymorphonuclear leucocytes (118 (24), n = 24). Lymphocyte DNA from donors with the following inflammatory autoimmune diseases contained significantly higher levels of 8-oxodG than that from healthy donors: rheumatoid arthritis (98 (16)), systemic lupus erythematosus (137 (28)), vasculitis (100 (32)), and Behçet's disease (92 (19)). Lymphocyte 8-oxodG levels in non-autoimmune controls and patients with scleroderma were not significantly different from those of healthy controls. The levels of 8-oxodG were significantly higher in the DNA from normal polymorphonuclear leucocytes than in paired DNA samples from normal lymphocytes, but there were no differences between levels of 8-oxodG in polymorphonuclear leucocytes from normal subjects and the patients studied. Levels of 8-oxodG did not correlate with disease duration, disease severity, or age. Lymphocytes from patients with systemic lupus erythematosus and rheumatoid arthritis, but not those with scleroderma, also showed cellular hypersensitivity to the toxic effects of hydrogen peroxide. CONCLUSION: There was increased genomic DNA damage, and increased susceptibility to cytotoxic killing by hydrogen peroxide, in lymphocytes from patients with certain autoimmune diseases. These results might be explained by defective repair of DNA damage or by increased production of reactive oxygen intermediates in inflammation. Although more direct studies are needed, the evidence available favours the former explanation.

OBJECTIVE: Increased concentrations of lipid peroxidation products have been described in the serum and synovial fluid from patients with rheumatoid arthritis. A large proportion of the unsaturated lipids in human extracellular fluids is a component of low density lipoprotein (LDL). The oxidative modification of LDL, and its subsequent uptake by macrophages, has been implicated in the pathogenesis of atherosclerosis, but not of rheumatoid arthritis. This study aimed to assess whether oxidatively modified LDL was present in the rheumatoid synovium. METHODS: a polyclonal antiserum raised in rabbits against oxidised LDL (o-LDL) was used to perform an immunohistochemical study of a series of synovial biopsy specimens from patients with rheumatoid arthritis. RESULTS: Collections of positively stained macrophages, arranged in a linear fashion and with the morphological characteristics of foam cells--that is, 'fatty streaks', were identified around blood vessels within the intimal connective tissue. In addition, scattered, positively stained foam cells were present in association with deposits of fibrin. These staining patterns were absent from control synovial membranes (traumatic knee injuries). CONCLUSIONS: the findings in all rheumatoid patients studied suggest that atherosclerosis and rheumatoid arthritis have analogous pathogenetic features.

1. We have determined the antioxidant status of synovial fluid and serum of patients with inflammatory joint disease in terms of the biologically active lipid-soluble antioxidant, alpha-tocopherol. Synovial fluid concentrations of alpha-tocopherol were significantly lower relative to those of paired serum samples (P < 0.001). Serum levels of alpha-tocopherol in these patients did not differ significantly from those in control serum. 2. Lower concentrations of cholesterol, triacylglycerol and low-density lipoprotein were also observed in patients' synovial fluid compared with matched serum samples. However, multiple regression analysis of the data indicated that there remained a significant depletion of alpha-tocopherol, which was largely independent of these co-variables, in inflammatory synovial fluid. These findings are consistent with the consumption of alpha-tocopherol within the inflamed joint via its role in terminating the process of lipid peroxidation. 3. Nuclear magnetic resonance spectroscopic analysis of matched inflammatory synovial fluid and serum confirmed lower concentrations of triacylglycerol in synovial fluid together with evidence of a shortened mean triacylglycerol chain length. The latter metabolic difference suggests an increased utilization of triacylglycerols for energy within the inflamed joint.

The genetic toxicity of the antitumour antibiotic bleomycin (BLM) is thought to involve the formation of a reactive oxygen intermediate. 8-Oxo-7,8-dihydrodeoxyguanosine (oxo8dG), an oxidation product of deoxyguanosine, is one of the major products formed when isolated DNA is exposed to oxygen radical generating systems. Gamma-irradiation (10-500 Gy 60Co; 10 Gy/min) or BLM and Fe2+ (37.5-150 U/L and 0.5 mM, respectively) treatment of isolated DNA (0.25 mg/mL) increased oxo8dG above background. In the latter case, the effect was greater than that with Fe2+ (0.5 mM) alone and was dependent on the dose of BLM. When DNA was irradiated with 500 Gy60Co, deoxyguanosine oxidation was inhibited by antioxidants (ethanol: 37.5 and 98% inhibition at 2 and 20 mM, respectively; mannitol: 20.5, 60 and 92% inhibition at 0.1, 1.0 and 10 mM, respectively). Similarly the BLM-induced production of oxo8dG was inhibited (64%) by mannitol (10 mM). BLM also caused production of base propenals on interaction with isolated DNA. In contrast, oxo8dG was not induced above background concentration (27 mol oxo8dG/10(6) mol dG) in permeabilized (37 degrees) and non-permeabilized (4 degrees and 37 degrees) rat hepatocytes treated with BLM (260 U/L). Despite this, there was extensive BLM-induced unscheduled DNA synthesis (10 and 100 U/L) in non-permeabilized rat and human hepatocytes in the absence of hydroxyurea. These findings, in accord with other observations, draw into question the role of. OH in BLM-induced DNA damage and the mimicry of ionizing radiation in cellular systems.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin a plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.

1. We have determined the antioxidant status of synovial fluid and serum of patients with inflammatory joint disease in terms of the biologically active lipid-soluble antioxidant, α-tocopherol. Synovial fluid concentrations of α-tocopherol were significantly lower relative to those of paired serum samples (P < 0.001). Serum levels of α-tocopherol in these patients did not differ significantly from those in control serum. 2. Lower concentrations of cholesterol, triacylglycerol and low-density lipoprotein were also observed in patients' synovial fluid compared with matched serum samples. However, multiple regression analysis of the data indicated that there remained a significant depletion of α-tocopherol, which was largely independent of these co-variables, in inflammatory synovial fluid. These findings are consistent with the consumption of α-tocopherol within the inflamed joint via its role in terminating the process of lipid peroxidation. 3. Nuclear magnetic resonance spectroscopic analysis of matched inflammatory synovial fluid and serum confirmed lower concentrations of triacylglycerol in synovial fluid together with evidence of a shortened mean triacylglycerol chain length. The latter metabolic difference suggests an increased utilization of triacylglycerols for energy within the inflamed joint.

The proteinase inhibitory ability of alpha 1 antitrypsin was measured in 23 samples of rheumatoid arthritis synovial fluid, eight osteoarthritic synovial fluids and nine normal control serum samples. For each sample a detailed kinetic analysis was performed with porcine pancreatic elastase as the target proteinase. Samples were stored for less than 24 hours at 4 degrees C before analysis, which does not significantly alter the proportion of inactive alpha 1 antitrypsin. In rheumatoid synovial fluid the elastase inhibitory ability was disproportionately depressed relative to the immunochemically determined concentrations of alpha 1 antitrypsin.

alpha 1 Antitrypsin (alpha 1AT) is the main physiological inhibitor of neutrophil elastase, a serine protease which has been implicated in tissue degradation at inflammatory sites. We report here that the connective tissue metalloproteinase, stromelysin, cleaved alpha 1AT (54 kDa), producing fragments of approximately 50 kDa and 4 kDa, as shown by gel electrophoresis. The cleavage of alpha 1AT was accompanied by inactivation of its elastase inhibitory capacity. Isolation of the 4 kDa fragment by reversed-phase HPLC, followed by N-terminal amino acid sequencing, demonstrated that the cleavage of alpha 1AT occurred at the Pro357-Met358 (P2-P1) peptide bond, one peptide bond to the N-terminal side of the inhibitory site. We suggest that stromelysin may potentiate the activity of neutrophil elastase by proteolytically inactivating alpha 1AT.

We report the effects of oxidative stress generated by low-intensity u.v. irradiation (366 and 254 nm), dialysis against ascorbate and isolated stimulated neutrophils on some physicochemical properties of caeruloplasmin. Low-intensity u.v. irradiation resulted in a loss of ferroxidase activity and 610 nm absorption, changes previously reported to occur during storage and manipulation of caeruloplasmin. These alterations were found to correspond to aggregation of the protein, induction of visible fluorescence (excitation, 360 nm; emission, 454 nm), changes in c.d. spectra which were indicative of alterations in protein conformation, loss of half-cystine, tryptophan and tyrosine content and loss immunoreactivity. The changes in the far-u.v. c.d. spectrum of caeruloplasmin were more pronounced than those observed for u.v.-irradiated IgG. Similar c.d. changes and induction of fluorescence were observed following dialysis of caeruloplasmin against ascorbate or exposure to stimulated neutrophils. It is concluded that the lability of caeruloplasmin may arise from oxidative modification, in addition to the previously described susceptibility of this protein to proteolysis.

It was observed that during the storage of human extracellular fluids at -20 degrees C the azide-inhibitable ferroxidase activity of caeruloplasmin declined, whilst a new azide-resistant ferroxidase activity (ARFA) developed. The literature suggested that storage-induced ARFA might be due to either a poorly defined enzymatic activity of a low density lipoprotein (LDL) or to lipid peroxides formed within the different lipoprotein fractions. To study this further, the major lipoprotein classes were separated from human serum by density gradient centrifugation. After storage of the lipoprotein fractions, it was found that the LDL fraction had the highest specific activity of ARFA and the highest content of lipid peroxidation products, as assessed by diene conjugates. The ARFA of LDL correlated with its content of diene conjugates and TBA reactive material, which initially suggested that the Fe(II) oxidising activity of peroxidase LDL arose from the reduction of peroxides by Fe(II) in the classical reaction between the metal ion and free radical reduction of lipid peroxides. However, steady state kinetic analysis indicated an enzymic role of LDL in Fe(II) oxidation, with lipid peroxides acting as a substrate for the enzyme. These results indicate that LDL may contain a peroxidase activity, catalysing the oxidation of Fe(II) by lipid peroxides, as well as a ferrous oxidase activity where O2 is the oxidising substrate.

Three patients with rheumatoid disease were given the 'iron chelating' drug desferrioxamine (DFX), which also has an appreciable affinity for copper. The drug was injected cautiously, in lower doses than in patients with thalassaemia, and intramuscularly to evaluate its anti-inflammatory effects. Two of the three patients developed ocular abnormalities. One patient, who also received methyldopa, developed severe but reversible visual failure associated with an abnormal electro-oculogram (EOG); another showed reversible depression of the EOG. Analysis of the cerebrospinal fluid (CSF) of this patient showed an increase in phenanthroline detectable (non-caeruloplasmin-bound) copper. Analysis of the CSF of the third patient, who did not develop any clinical or electrophysiological ocular abnormalities, was normal. Haematological assessments indicated that all three patients probably had reduced iron stores. With in-vitro systems DFX was shown to mobilise copper from albumin and to facilitate copper movement across a cell membrane model, a property that was enhanced by methyldopa. Our observations are consistent with the concept that in rheumatoid patients low iron stores may result in binding of copper by DFX and that this may be of central importance in causing the ocular toxicity of DFX.

The oxidase activity of caeruloplasmin towards Fe II (ferroxidase) appears to be of physiological significance both in iron metabolism and as a serum anti-oxidant. We have developed an automated assay for ferroxidase using a centrifugal analyser. The method is quick and precise, and is not subject to interference by haemolysis, jaundice or lipaemia. Ferroxidase activities correlate significantly with caeruloplasmin and copper concentrations. Normal reference ranges have been measured.

The mechanism by which a synovial flare occurred in a patient with rheumatoid arthritis after intravenous infusion of iron-dextran was investigated. After the infusion, serum and synovial-fluid iron-binding capacity became saturated, giving rise to low-molecular-mass iron chelates with the capacity to cause oxidative damage ("bleomycin-iron"). At the same time lipid peroxidation and the concentration of oxidised ascorbic acid (dehydroascorbate) increased in both serum and synovial fluid, and red-cell glutathione fell. These changes corresponded closely to an exacerbation of rheumatoid synovitis. Hepatic function was transiently disturbed 7 days after the infusion, reflecting hepatic oxidant stress within the iron-loaded liver. Such changes provide clear evidence that iron-catalysed oxidative reactions influence the inflammatory process in human beings.

Inflammation induces the hepatic synthesis of the iron storage protein (apo)ferritin, which is released into the circulation, and behaves as an acute phase protein. The biological significance of the extracellular rise in serum (apo)ferritin is unknown. We have observed that (apo)ferritin will stimulate superoxide production from neutrophils in the presence of cytochalasin B across a physiologically appropriate concentration range. We therefore propose that extracellular ferritin has an important role in host defence against bacteraemia by stimulating oxidative metabolism.

Seven patients with rheumatoid disease were given the iron-chelating drug desferrioxamine (DFX) to evaluate its possible anti-inflammatory effects. Two of these patients, who also received the anti-emetic prochlorperazine, lost consciousness for 48-72 h and then fully recovered. Electroencephalography showed abnormalities of the type associated with metabolic disturbance. One of these patients showed pyramidal features and subsequently developed an optic neuropathy and pigmentary retinopathy. Analysis of his cerebrospinal fluid showed a decrease in loosely-bound (catalytic) iron and increase in loosely-bound (catalytic) copper, total iron and products of lipid peroxidation, with values approaching normal as the symptoms resolved. Subsequent in vivo/vitro studies clearly demonstrated that the neurological effects were due to a synergistic action of desferrioxamine and prochlorperazine, probably resulting in exceptional fluxes of intra/extra cellular iron/copper disturbing noradrenergic and serotonergic systems. Two other patients who did not receive prochlorperazine, developed retinal problems which later improved, one after only 15 g of desferrioxamine. Our observations suggest a new model for metabolic encephalopathy studies and provide insight into the mechanisms of pigmentary retinopathy.

One of the proposed mechanisms of vascular damage in connective tissue disease is the direct action of a cytotoxic serum factor inducing endothelial cell damage. The nature of this serum factor is unclear, but has been suggested to be a lipoprotein. Sera from patients with (1) systemic necrotising arteritis (polyarteritis nodosa, Wegener's granulomatosis, and necrotising arteritis associated with rheumatoid synovitis), (2) systemic or joint restricted rheumatoid disease, and (3) large vessel/giant cell arteritis have been examined for cytotoxicity to human cultured endothelial cells and azide-resistant ferroxidase-like activity (indicative of the oxidised lipoprotein content). Stored sera from patients with necrotising arteritis showed a significantly enhanced tendency to develop oxidised lipoprotein, which correlated closely with human endothelial cell cytotoxicity. Fresh sera also contained this factor, but to a lesser extent. It is argued that the cytotoxic factor detected in previous clinical studies is in part an in-vitro artefact, although its accelerated development in certain patient groups might suggest an excess of pro-oxidants that have developed in vivo.

No Cu(II) ion is measurable in human serum or synovial fluid by the phenanthroline assay. On storage of human serum or synovial fluid at 4 degrees C, phenanthroline-detectable copper appears, lipid peroxidation occurs, ferroxidase I activity declines and ferroxidase II activity rises, yet there is no fall in immunologically detectable caeruloplasmin. Storage of body fluids at -20 degrees C or -70 degrees C slows, but does not prevent, these deteriorative changes. It is suggested that the presence of low-molecular-mass Cu(II) ion complexes, ferroxidase II activity, "cytotoxic factors' and "immunosuppressive factors' in body fluids may be, in part or in whole, an artifact of the storage and handling of the fluids. A report [Blake, Blann, Bacon, Farr, Gutteridge & Halliwell (1983) Clin. Sci. 64, 551-553] that the caeruloplasmin present in rheumatoid synovial fluid is deficient in ferroxidase activity is shown to be such an artifact. It is strongly recommended that all such experiments be performed upon freshly taken fluid samples.

Exposure of human caeruloplasmin, an acute phase protein with antioxidant properties, to a mixture of xanthine/hypoxanthine and xanthine oxidase as a source of reactive oxygen intermediates decreased its ferroxidase and ascorbate oxidase activities and its ability to inhibit lipid peroxidation. Immunological reactivity was also altered. Exposure to hydrogen peroxide mimicked these effects. Exposure to low-intensity u.v. irradiation depressed caeruloplasmin's ability to inhibit iron-catalysed hyaluronic acid degradation. The results may explain the mechanism of the observed inactivation of caeruloplasmin within human rheumatoid synovial fluid.