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Many oral bacteria reduce inorganic nitrate, a natural part of a vegetable-rich diet, into nitrite that acts as a precursor to nitric oxide, a regulator of vascular tone and neurotransmission. Aging is hallmarked by reduced nitric oxide production with associated detriments to cardiovascular and cognitive function. This study applied a systems-level bacterial co-occurrence network analysis across 10-day dietary nitrate and placebo interventions to test the stability of relationships between physiological and cognitive traits and clusters of co-occurring oral bacteria in older people. Relative abundances of Proteobacteria increased, while Bacteroidetes, Firmicutes and Fusobacteria decreased after nitrate supplementation. Two distinct microbiome modules of co-occurring bacteria, that were sensitive to nitrate supplementation, showed stable relationships with cardiovascular (Rothia-Streptococcus) and cognitive (Neisseria-Haemophilus) indices of health across both dietary conditions. A microbiome module (Prevotella-Veillonella) that has been associated with pro-inflammatory metabolism was diminished after nitrate supplementation, including a decrease in relative abundance of pathogenic Clostridium difficile. These nitrate-sensitive oral microbiome modules are proposed as potential pre- and probiotic targets to ameliorate age-induced impairments in cardiovascular and cognitive health.

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, Graphical abstract fx1, Highlights • Dietary nitrate alters the salivary microbiome of healthy young and old adults. • Nitrate supplementation decreases relative abundances of Prevotella and Veillonella. • Nitrate supplementation increases relative abundances of Rothia and Neisseria. • The salivary microbiome modulates nitric oxide bioavailability and blood pressure.

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., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

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., Highlights • Oxidants can generate stable post-translational modifications (PTMs) on proteins. • Oxidative PTMs are recognised in evolutionarily-conserved innate immune responses. • These PTMs can represent neo-epitopes that break tolerance in autoimmune disease. • Antibodies targeting these PTMs in diseases e.g. RA and SLE, can be biomarkers., Graphical abstract

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 O2) and co-factors. Recently an additional, O2-independent, NO generating pathway has been identified, where nitrite (NO2 −) can undergo a simple one electron reduction to yield NO. NO2 − is produced endogenously from the oxidation of NO and also from the reduction of dietary nitrate (NO3 −) by facultative bacteria residing on the tongue. Recent data show that dietary NO3 − supplementation, which increases the circulating plasma [NO2 −], reduces the O2 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 th...

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) ~2 kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90 min (97.5% O(2) at 2.4 ATA; PO(2) ~237 kPa). 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.

Non-technical summary Reduced atmospheric O2 availability (hypoxia) impairs muscle oxidative energy production and exercise tolerance. We show that dietary supplementation with inorganicnitratereducesmarkersofmusclefatigueandimproveshigh-intensityexercisetolerance in healthy adults inhaling air containing14.5% O2.Inthebody,nitratecanbeconvertedtonitrite and nitric oxide. These molecules can improve muscle efficiency and also dilate blood vessels allowing more O2 to be delivered to active muscle. These results suggest that dietary nitrate could be beneficial during exercise at moderate to high altitude and in conditions where O2 delivery to muscle is reduced such as in pulmonary, cardiovascular and sleep disorders. Abstract Exercise in hypoxia is associated with reduced muscle oxidative function and impaired exercisetolerance.Wehypothesisedthatdietarynitratesupplementation(whichincreasesplasma (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; H-BR) or 0.75 L of nitrate-depleted beetroot juice as a placebo (0.006mmol nitrate; H-PL). Muscle metabolism was assessed using calibrated 31 P-MRS. Plasma (nitrite) was elevated (P

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.

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. Multiple Sclerosis 2007; 13: 955—961. http://msj.sagepub.com

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

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 favourably 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 ageing-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.

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.

This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License.-- et al., [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., The authors acknowledge financial support from the European Cooperation in Science and Technology (COST Action BM1203/EU-ROS) (P.G.W., N.Z., T.R., A.C., H.E.P., H.H.H.W.S., P.G.), the Peptide Research Network of Excellence, European Cross-border Cooperation Programme INTERREG IV A France (Channel)-England, ERDF (P.G.), the Toyota foundation, Denmark (H.E.P.), a Senior Principal Research Fellowship from the National Health and Medical Research Council and National Health and Medical Research Council Project Grants 1020776 and 1060804 (R.S.), and National Institutes of Health Grant HL116263 (S.S.D.). A.K. is supported by a grant from the Defence Science and Technology Laboratory, United Kingdom, to P.G.W. and E.L.T.

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.

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.

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.

Summary Background Nitric oxide (NO) is a critical negative regulator of platelets that is implicated in the pathology of thrombotic diseases. Platelets generate NO, but the presence and functional significance of NO synthase (NOS) in platelets is unclear. Inorganic nitrate/nitrite is increasingly being recognized as a source of bioactive NO, although its role in modulating platelets during health and vascular dysfunction is incompletely understood. Methods We investigated the functional significance and upstream sources of NO–cGMP signaling events in platelets by using established methods for assessing in vitro and in vivo platelet aggregation, and assessed the bioconversion of inorganic nitrate to nitrite during deficiency of endothelial NOS (eNOS). Results The phosphodiesterase 5 (PDE5) inhibitor sildenafil inhibited human platelet aggregation in vitro. This inhibitory effect was abolished by a guanylyl cyclase inhibitor and NO scavengers, but unaffected by NOS inhibition. Inorganic nitrite drove cGMP-mediated inhibition of human platelet aggregation in vitro and nitrate inhibited platelet function in eNOS−/− mice in vivo in a model of thromboembolic radiolabeled platelet aggregation associated with an enhanced plasma nitrite concentration as compared with wild-type mice. Conclusions Platelets generate transient, endogenous cGMP signals downstream of NO that are primarily independent of NOS and may be enhanced by inhibition of PDE5. Furthermore, nitrite can generate transient NO–cGMP signals in platelets. The absence of eNOS leads to enhanced plasma nitrite levels following nitrate administration in vivo, which negatively impacts on platelet function. Our data suggest that inorganic nitrate exerts an antiplatelet effect during eNOS deficiency, and, potentially, that dietary nitrate may reduce platelet hyperactivity during endothelial dysfunction.

In this chapter, bromelain, a crude multi-component extract isolated from the stems of the pineapple plant, takes us on a tour through rather unusual therapeutic approaches and pharmacological mechanisms. Bromelain acts on several targets that address edematous, inflammatory, and immunological processes. Therefore, many indications were treated with or are conceivable to be treated by use of this phytomedicine. Bromelain teaches us that we should keep an open mind toward at first sight exotic therapeutic approaches. A plea for the use of the potential of natural products that are well established in medical therapy for decades or even centuries.

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.

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 (

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.

Introduction Biological Systems Relevant for Redox Signaling and Control Cellular Generation of Oxidants: Relation to Oxidative Stress The Chemical Basis of Biological Redox Control Protein Glutathiolation Structure and Function of the Human Peroxiredoxin-Based Antioxidant System: the Interplay between Peroxiredoxins, Thioredoxins, Thioredoxin Reductases, Sulfiredoxins and Sestrins Hydrogen Peroxide and Cysteine Protein Signaling Pathways Protein Tyrosine Phosphatases as Mediators of Redox Signaling Hypoxia-Induced Gene Regulation through Hypoxia Inducible Factor-1a Eicosanoid-Based Signaling Redox-Controlled Transcription Factors and Gene Expression Nitric Oxide Regulation in Redox Signaling Is Hydrogen Sulfide a Regulator of Nitric Oxide Bioavailability in the Vasculature? Aspects of Nox/Duox Signaling Photodynamic Therapy with Aminolevulinic Acid and Iron Chelators: A Clinical Example of Redox Signaling Oxidative Stress and Apoptosis Redox Regulation of Apoptosis in Immune Cells Redox Control in Human Disease with a Special Emphasis on the Peroxiredoxin-Based Antioxidant System Free Radicals and Mammalian Aging

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.

Objectives Peroxiredoxin (PRDX) thiol-based antioxidant enzymes have been identified in several neurological diseases. Astrocytes, multifunctional supporting cells, become activated during pathological processes and develop reactive and/or scarring phenotypes featuring characteristic changes in protein expression. Astrocyte activation can be beneficial although scarring astrocytes inhibit repair. Little is known about astrocytes expression of the PRDX family. However altered expression or inactivation of PRDXs could contribute to pathology. This preliminary study investigated PRDXs and related redox enzymes in a human astrocyte culture system that mimics the phenotypes of quiescent, reactive/scarring astrocytes in vivo. Methods Human astrocytes, isolated from post mortem subventricular deep white matter were grown in conditions inducing quiescence or activation. RNA was extracted and cDNA obtained by reverse transcription PCR. Expression levels of PRDXs 2,3,5, sulfiredoxin (SRX), thioredoxin (TRX) and thioredoxin reductase (TR) in quiescent and activated astrocytes was assessed by qPCR. Results Quiescent astrocytes expressed low basal PRDX 2, 3 and 5 mRNA. Following activation, PRDX 3 mRNA levels were unchanged. However, a threefold increase of PRDXs 2 and 5 expression was seen. Conversely high basal levels of SRX, TRX and TR mRNA in quiescent astrocytes, were decreased in activated astrocytes. Changes in PRDX expression are linked to astrocyte phenotype.

A key feature of inflammatory diseases such as rheumatoid arthritis (RA) is the increased expression of certain genes which encode “inflammatory” proteins. These include a variety of adhesion molecules, as well as cytokines such as tumour necrosis factor a (TNFa) and interleukin-1 (IL-1). The proteinases involved in inflammatory tissue destruction include the metalloproteinases; collagenases, gelatinases and stromelysins. An important characteristic of gene expression is the control of gene transcription by specific proteins, transcription factors, which bind to short DNA sequence elements located adjacent to the promoter or in enhancer regions of genes. Once bound to DNA, transcription factors interact with each other and with the proteins of the transcriptional apparatus itself (e.g. RNA polymerase) to regulate gene expression. Recently, there has been considerable interest in the idea that transcription factors may be useful targets for novel therapeutic strategies in the treatment of human diseases, including inflammatory diseases [1].

Acute inflammation is the host response to infection and tissue damage, and serves to limit harm to the body. However, dysregulated or chronic inflammation underlies the pathology of many diseases, such as autoimmune disease. Elevated H 2 S levels are present in the joints of rheumatoid arthritis patients, and increased levels of H 2 S and the H 2 S synthesising enzyme CSE are observed in vitro and in vivo following exposure to an inflammatory stimulus. However, the role of H 2 S in inflammation is unclear, with pro-and anti-inflammatory effects observed. The method of H 2 S delivery may be, at least partially, responsible for these discrepancies. For instance, fast release donors that deliver an instant bolus of H 2 S (e.g. NaSH/Na 2 S) invariably have pro-inflammatory effects in vivo , whereas slow-release donors that deliver H 2 S in a manner comparable to endogenous production are predominantly anti-inflammatory. Recently, attention has focused on the slow-release H 2 S donor GYY4137, with down-regulation of pro-inflammatory mediators and pro-inflammatory enzyme activity, as well as reduced joint swelling observed, supporting an anti-inflammatory role of H 2 S in vitro and in vivo . Here the effects of GYY4137 and two novel slow-release H 2 S donors, AP105 and AP106, which release H 2 S at different rates (AP105 > AP106 > GYY4137), were compared by examining their effects on pro-inflammatory mediators, pro-inflammatory enzyme activity and NF- κ B signalling. Also, the effect on the proliferation of primary human synoviocytes (HFLS) and Jurkat T-cells, whose hyper-proliferation contributes to joint inflammation, were investigated. Following stimulation of HFLS with a cocktail of pro-inflammatory cytokines (TNF- α , IL-1 β , IFN- γ ) in the presence or absence of H 2 S donors, pro-inflammatory mediator levels were measured in the culture media. Significantly decreased IL-6 levels ( p In-vitro recombinant enzyme activity assays showed that TNF- α converting enzyme (TACE) and cycloxygenase-2 (COX-2) activity were significantly inhibited by all three slow-release H 2 S donors (200–1000 μM). However, whereas small, significant decreases in matrix metalloprotease-2 (MMP-2) activity were observed with AP105 and AP106 (250 μM or 500 μM), significantly increased MMP-2 activity was observed with GYY4137 (250 μM). Jurkat T-cell numbers were significantly decreased following 72 h culture in the presence of AP105 (62.5 μM or 250 μM) or AP106 (250 μM), whereas significantly increased Jurkat T-cell numbers were observed with 62.5 μM AP106. GYY4137 had no effect on Jurkat proliferation, and there was no significant effect on cell viability with any of the donors, except a small decrease with AP105 or AP106 at 250 μM (3.43% and 2.93% c.f. control, respectively). A significant decrease in HFLS numbers was observed with AP105 (250 μM or 500 μM) after 72 h, either in standard culture or following stimulation with TNF- α , with no significant effect of AP106 or GYY4137 under the same conditions. Following cytokine stimulation of HFLS there was no effect of GYY4137, AP105 or AP106 on I- κ B phosphorylation at early time-points. With exceptions, the down-regulation of pro-inflammatory mediators, pro-inflammatory enzyme activity and reduced T-cell and HFLS cell proliferation observed supports an anti-inflammatory role for H 2 S from slow-release H 2 S donors. This work was supported by the Wellcome Trust.

Hydrogen sulfide (H2S) is emerging as important mediator in disparate physiological and pathophysiological processes [1] . These studies have highlighted the therapeutic potential for the pharmacological manipulation of H2S. Indeed, H2S donor molecules such as GYY4137, ATB-346, SPRC and ATB-337 have been shown to regulate blood pressure in experimental induced hypertension and to inhibit tissue damage, oedema and inflammatory signalling in sepsis, colitis and arthritis and many of these studies will be reviewed elsewhere at this meeting. The development and characterisation of appropriate H2S donors such as these are crucial as commonly used sulfide salts such as NaSH or Na2S, generate H2S (and Na+) as an instantaneous bolus rather than model the slow and sustained enzymatic generation of H2S from CSE, CBS or 3-MST [1] , [2] . As with NO and CO, it is likely that the tissue and/or cellular responsiveness to H2S may well be dependent upon the manner in which cells/tissues are exposed to this gas [2] . However, pharmacological tools which generate H2S at different rates (analogous to the NONOates in the nitric oxide field) are currently unavailable. We have attempted to overcome this limitation and synthesised several novel compounds capable of generating H2S at different rates (e.g. AP67, AP72, AP105). These compounds will enable researchers to characterise the physiology and pharmacology of H2S in a variety of physiological and pathophysiological models. More recently we have synthesised novel dithiolethione (e.g. AP39) or thiohydroxybenzamide (e.g. AP123) derivatives containing a triphenylphosphonium moiety enabling specific delivery of sub-nanomolar concentrations of the compounds to mitochondria. The anti-hypertensive, anti-inflammatory, cytoprotective and anti-proliferative effects in vitro, ex vivo and in vivo of these compounds in comparison to other H2S donors will be discussed. These studies highlight the potential for H2S manipulation and mitochondrial delivery of H2S to regulate vascular and inflammatory signalling in health and disease.

Publisher Summary This chapter discusses evidence that many antioxidant compounds with anti-inflammatory properties are more likely to be acting through their effects on inflammatory cellular messenger systems than by direct prevention of freeradical-mediated damage to bio–molecules. Concentration is on the role of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) in mediating the activation of the transcription factor, nuclear factor- K B (NF- K B).Interest in the role of free radicals in diseases with an inflammatory component led to research into the role of free radicals; toward the putative role of ROI as damaging agents; on the oxidative modification of bio–molecules. The cyto–toxicity of free radicals and their reaction products were investigated, in relation to endothelial cell function. ROI can activate apoptosis, a programmed form of cell death.ROI appear to activate certain protein kinases involved in cellular signal transduction, and also activate, or suppress, various protein phosphatases.ROI have been implicated in the regulation of mammalian transcription factors and certain “heat shock,” or stress protein where ROI activity plays a critical role in regulating the expression of genes that encode either proteins with potential pro–inflammatory actions or proteins that may act in a protective fashion. The capacity of endogenous intracellular antioxidants or of repair mechanisms for oxidative DNA damage might determine the susceptibility of different cell populations to the induction of specific gene expression, killing, or even mutation. An increased susceptibility to the actions of ROI on target lymphocytes of patients with autoimmune disorders could thus be an important mechanism in the pathogenesis of diseases such as rheumatoid arthritis by inducing excessive inflammatory responses and increased clonal proliferation of auto–reactive lymphocytes via the activation of transcription factors. The chapter also discusses the action of anti-inflammatory drugs in the inhibition of NF- K B, and the cellular source of ROI Involved in NF- K B activation.

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.

Introduction Synthesis of H2S is markedly increased in knee joint synovial fluid (SF) joints from patients with inflammatory joint disease (IJD) compared to paired plasma and SF from age-matched disease controls. SF levels also negatively correlated with disease activity, SF total white cell and neutrophil count suggesting a possible anti-inflammatory role for induced H2S synthesis in the joint. These observations also highlight the pharmacological potential for H2S donor molecules in (IJD). Methods We have assessed the effect of GYY4137 (a slow-releasing H2S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We also examined the effects of GYY4137 on human recombinant iNOS, COX-1, COX-2 and tumour necrosis factor-α converting enzyme (TACE). We further examined the effect of GYY4137 in a complete Freund’s adjuvant (CFA) model of arthritis in the mouse. Results GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite ( NO 2 - ) , PGE2, TNF-αand IL-6 from HFLS and HAC, reduced the expression and catalytic activity of iNOS, COX-2, and reduced LPS-induced NF-kB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-1, COX-2, iNOS and TACE. In the CFA treated mouse, adjuvant induced a two fold increase in synovial H2S concentrations. GYY4137 (50 mg/kg, i.p.) injected 1 h prior to CFA increased knee joint swelling whilst an anti-inflammatory effect, as evidenced 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 h after CFA. GYY4137 was also anti-inflammatory when given 18 h after CFA. Conclusion The effects of H2S on the inflammatory process are highly complex. Not only is the anti-inflammatory effect determined by the donor used (’fast’ vs ’slow’ release donors) but also determined in vivo by the timing of the donor administration. Thus, whilst GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on arthritis in vivo depended on the timing of administration.

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.

Matrilysin is shown to rapidly inactivate α 1 PI, an inhibitor of elastase, by cleaving the Pro 357 -Met 358 peptide bond of its reactive centre. The rate of inactivation of α 1 PI by matrilysin is four times higher than stromelysin. Matrilysin cleaves oxidised α 1 PI at the Phe 352 -Leu 353 bond, whilst stromelysin cleaves oxidised α 1 PI at the Met 358 -Ser 359 bond. We conclude that matrilysin is a potent serpinase which could play a role in inflammatory tissue damage by proteolytically inactivating α 1 PI.

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.

to the editor: The letter of Lundberg et al. ([12][1]) does not relate directly to any of the articles we published in this journal ([1][2], [2][3], [6][4]). Rather, the correspondents make a general point on the potential risks to athletes who might choose to supplement their diet with nitrate or (

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.

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.

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.

Paradoxically, oxygen is both necessary for aerobic life and a toxic hazard at higher than atmospheric concentrations. In mammalian systems the controlled reduction of oxygen is essential for the production of energy. The quantum chemistry of oxygen (see below) provides for this molecule to undergo univalent reduction more readily than divalent reduction. Mammalian cells control this process by virtue of an array of enzymes — the mitochrondrial respiratory chain — which supplies electrons to O2 singly, This system is imperfect and up to 5% of the electrons ‘leak’ out, forming toxic O2 - radicals. Furthermore, other enzymes have the specific function of releasing toxic oxygen species under certain conditions. For example the bactericidal capacity of neutrophils is, in part, dependent on the generation of toxic oxygen species by a membrane NADPH oxidase. Thus, in some circumstances, the production of oxygen free radicals may be desirable. However, protective mechanisms normally deal effectively with toxic oxygen species when they escape their intended sphere of action. These ‘antioxidant defences’ consist of both enzymes and low-molecular-weight compounds which may either ‘scavenge’ oxygen radicals or ‘repair’ oxidative damage to biomolecules. Recent evidence suggests that many inflammatory disorders may be linked to an increase in the production of oxygen radicals and/or deficiencies of antioxidant defence systems.

to the editor: The points made by Derave and Taes ([2][1]) in relation to our recent article ([1][2]) are important. We are very much aware of the literature concerning nitrate toxicity, which is extensive and complex. Concern about dietary nitrate began in the 1970's ([5][3], [6][4]) with the

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.

The aim of this study was to construct a promoter containing DNA motifs for an endogenous transcription factor associated with inflammation along with motifs for pharmacological regulation factors. We demonstrate in transfected cells that expression of a gene of interest is induced by hypoxic conditions or through pharmacological induction, and also show pharmacological repression. In vivo studies utilised electroporation of plasmid to mouse paws, a delivery method shown to be effective by bioluminescence imaging. For gene therapy, the promoter was used to drive expression of IL-1Ra in a paw inflammation model with therapeutic effect observed which was further enhanced when the promoter was additionally induced with a pharmacological activator. One of the most important observations from this study was that promoter induction by hypoxia or inflammation could be prevented by the pharmacological repressor in the absence of doxycycline. These studies demonstrate that hybrid promoters enable pharmacological adjustment to the pathophysiological level of gene expression and, importantly, that they allow termination of gene expression even in the presence of pathophysiological stimuli. Fil: Subang, Maria C.. Queen Mary University of London; Reino Unido Fil: Fatah, Rewas. Queen Mary University of London; Reino Unido Fil: Bright, Carly. Queen Mary University of London; Reino Unido Fil: Blanco, Patricia. Fundación Instituto Leloir; Argentina Fil: Berenstein, Mariana Gisela. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Wu, Ying. University Of London; Reino Unido Fil: Podhajcer, Osvaldo Luis. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Winyard, Paul G.. University Of Exeter; Reino Unido Fil: Chernajovsky, Yuti. Queen Mary University of London; Reino Unido Fil: Gould, David. Queen Mary University of London; Reino Unido

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.

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are 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. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis 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. Exofacial thioredoxin-1 was also observed. 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 exofacially peroxiredoxin 2+ve (median, 39%). Prdx2 was also detected in human extracellular fluids. 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.

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.

Ceruloplasmin (CP) is a blue copper oxidase found in human serum. It has been suggested that CP functions as an antioxidant by virtue of its ability to scavenge the superoxide anion radical1 and/or its ability to catalyse the conversion of Fe(II) to Fe(III), thereby inhibiting the Fenton reaction2. However, the true physiological role of CP is still not clear. In attempting to define such a role it is important to recognise the relative lability of this protein3. Many of the changes in the physicochemical properties of CP3 which occur during isolation and storage can be ascribed to proteolysis3. However, this does not appear to account for all of the modifications which occur during the ageing of CP preparations.

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.