David R. Blake

Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions

Xanthine oxidoreductase (XOR) catalyses the reduction of the therapeutic organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric oxide (NO) under hypoxic conditions in the presence of NADH. Generation of nitric oxide is not detectable under normoxic conditions and is inhibited by the molybdenum site‐specific inhibitors, oxypurinol and (−)BOF 4272. These enzymic reactions provide a mechanism for generation of NO under hypoxic conditions where nitric oxide synthase does not function, suggesting a vasodilatory role in ischaemia.

Body perception disturbance: A contribution to pain in complex regional pain syndrome (CRPS)

Abstract In spite of pain in the CRPS limb, clinical observations show patients pay little attention to, and fail to care for, their affected limb as if it were not part of their body. Literature describes this phenomenon in terms of neurological neglect‐like symptoms. This qualitative study sought to explore the nature of the phenomenon with a view to providing insights into central mechanisms and the relationship with pain. Twenty‐seven participants who met the IASP CRPS classification were interviewed using qualitative methods to explore feelings and perceptions about their affected body parts. These semi‐structured interviews were analysed utilising principles of grounded theory. Participants revealed bizarre perceptions about a part of their body and expressed a desperate desire to amputate this part despite the prospect of further pain and functional loss. A mismatch was experienced between the sensation of the limb and how it looked. Anatomical parts of the CRPS limb were erased in mental representations of the affected area. Pain generated a raised consciousness of the limb yet there was a lack of awareness as to its position. These feelings were about the CRPS limb only as the remaining unaffected body was felt to be normal. Findings suggest that there is a complex interaction between pain, disturbances in body perception and central remapping. Clinically, findings support the use of treatments that target cortical areas, which may reduce body perception disturbance and pain. We propose that body perception disturbance is a more appropriate term than ‘neglect‐like’ symptoms to describe this phenomenon.

Arterial stiffness and central blood pressure, as determined by pulse wave analysis, in rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is associated with increased cardiovascular mortality for reasons which are insufficiently understood. Chronic inflammation may impair vascular function and lead to an increase of arterial stiffness, an important determinant of cardiovascular risk. Objective: To investigate the augmentation index (AIx) as a measure of arterial stiffness in patients with RA, free of cardiovascular disease or risk factors, by means of a matched cohort pilot study. Method: Patients with a diagnosis of RA, aged 50 years or younger, were screened for the absence of clinical cardiovascular disease and risk factors, such as smoking, hypercholesterolaemia, hypertension, and excessive systemic steroid use. Suitable subjects were assessed by non-invasive radial pulse wave analysis to determine their AIx. These data were compared with those from healthy controls, matched closely for sex, age, mean peripheral blood pressure, heart rate, and height. Results: 14 suitable patients (11 female; mean (SD) age 42 (6) years, mean RA duration 11 (6) years; mean C reactive protein 19 (15) mg/l, no clinical systemic rheumatoid vasculitis) and matched controls were identified. The RA group had a higher mean (SD) AIx and mean (SD) central blood pressure (BP) than the control group: AIx 26.2 (6.7) v 18.9 (10.8)%, p=0.028; mean central BP 91.3 (7.8) v 88.2 (8.9) mm Hg, p<0.0001, by two tailed, paired t test. Conclusions: This preliminary study suggests that RA is associated with increased arterial stiffness and central BP, independently of clinically manifest cardiovascular disease or risk factors. This may contribute to the increased cardiovascular mortality in RA.

Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite

Peroxynitrate (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 μM ONOO− reducing the inhibitory activity of TIMP‐1 towards gelatinase‐A by 50%. High concentrations of ONOO− (500 μM‐5 mM) caused protein fragmentation whilst lower concentrations (<250 μM) inactivated TIMP‐1 without altering the molecular weight. Inactivation could be blocked by ONOO− scavengers but not by hydroxyl radical scavengers. Our results show that ONOO− is capable of inactivating TIMP‐1, a process which could potentiate metalloproteinase‐mediated tissue breakdown.

Self-perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation.

When human IgG is exposed to free radical generating systems such as ultraviolet irradiation, peroxidizing lipids, or activated human neutrophils, characteristic auto-fluorescent monomeric and polymeric IgG is formed (excitation [Ex], 360 nm, emission [Em], 454 nm). 1 h ultraviolet irradiation of IgG results in the following reductions in constituent amino acids; cysteine (37.0%), tryptophan (17.0%), tyrosine (10.5%), and lysine (3.6%). The fluorescent IgG complexes, when produced in vitro, can stimulate the release of superoxide from normal human neutrophils. In the presence of excess unaltered IgG, further fluorescent damage to IgG occurs. Measurement and isolation of fluorescent monomeric and polymeric IgG by high performance liquid chromatography, from in vitro systems and from fresh rheumatoid sera and synovial fluid, indicates that identical complexes are present in vivo; all these fluorescent complexes share the property of enhancing free radical production from neutrophils. The results described in this study support the hypothesis that fluorescent monomeric and aggregated IgG may be formed in vivo by oxygen-centered free radicals derived from neutrophils, and that in rheumatoid inflammation this reaction may be self-perpetuating within the inflamed joint.

Intra-articular injections of 750 kD hyaluronan in the treatment of osteoarthritis: a randomised single centre double-blind placebo-controlled trial of 91 patients demonstrating lack of efficacy.

OBJECTIVE--To determine the safety and efficacy of intra-articular injections of hyaluronan in the treatment of osteoarthritis of the knee. METHODS--A randomised double-blind placebo-controlled trial was carried out on 91 patients with radiologically confirmed osteoarthritis of the knee who were recruited from the outpatient clinics. RESULTS--It was found that weekly intraarticular injections of 20 mg of hyaluronan of M(r) = 750,000 (Hyalgan) in 2 ml of buffered saline performed no better than the inert vehicle alone over a five week period. The principal side effects of a transient increase in pain and swelling in the affected knee was observed in 47% of the treatment group compared with 22% of the placebo group. A few patients with radiologically mild disease treated with Hyalgan appeared to experience medium to long-term symptomatic improvement over matched placebo controls as judged by a delayed return to previous NSAID therapy or analgesia other than paracetamol. Patient numbers in the survival groups, however, were too small to be meaningful. CONCLUSION--It is concluded that intraarticular administration of this preparation of 750 kD hyaluronan offers no significant benefit over placebo during a five week treatment period, but incurs a significantly higher morbidity, and therefore has no place in the routine treatment of osteoarthritis.

Reactive oxygen species and iron—a dangerous partnership in inflammation

Cells of nearly all forms of life require well-defined amounts of iron for survival, replication and expression of differentiated processes. It has a central role in erythropoiesis but is also involved in many other intracellular processes in the tissues of the body. It is the fourth most abundant element in the Earths crust and the most abundant transition metal in living organisms for which its characteristic chemistry endows it with a series of properties enabling it to fulfil certain biological reactions especially those involving redox mechanisms. It is involved in the transport of oxygen, in electron transfer, in the synthesis of DNA, in oxidations by oxygen (O2) and hydrogen peroxide (H2O2) and in many other processes maintaining normal structure and function of virtually all mammalian cells. Because an iron atom can exist in two valency states, ferrous and ferric, iron became the primordial partner of oxygen in evolution. However, as de Sousa et al. (1989) state, such long standing partnerships have to use protective devices to ensure that the toxicity of neither partner is expressed in the presence of the other. Here, we discuss this dangerous partnership and its relevance to inflammation. The main themes of this review are the known roles of iron in the generation of reactive oxygen intermediates and new developments, including iron and transcription and the reaction of iron with nitric oxide. We also consider the widening recognition of the importance of oxygen metabolites in hypoxia-reperfusion injury and disease of the skin and joint.

The Determination of Dehydroascorbic Acid and Ascorbic acid in the Serum and Synovial Fluid of Patients with Rheumatoid Arthritis (RA)

Using a novel high performance liquid chromatography (HPLC) determination of ascorbic acid and dehydroascorbic acid, we have measured the relative amounts of ascorbate and dehydroascorbate in 20 normal controls and in paired sera and synovial fluid from 13 patients with rheumatoid arthritis (RA). In complete contrast to previous published findings we were able to detect dehydroascorbate in normal human sera (12.0 +/- 3.7 mumol/l), while the mean and range of ascorbate measured in normals was 69.6 +/- 20.6 mumol/l. These findings were completely reversed in rheumatoid sera (21.8 +/- 8.6 mumol/l and 5.1 +/- 5.0 mumol/l for dehydroascorbate and ascorbic acid respectively). In several rheumatoid sera no ascorbate could be detected. In paired synovial fluid and serum samples, there was always more dehydroascorbate detected in synovial fluids than in the corresponding sera (p less than 0.01). The data suggests that the reduced level of ascorbate and increased level of dehydroascorbate may be a reflection of the increased antioxidant and free-radical scavenging activity of the vitamin in RA, especially within the inflamed joint.

Quantitative assessment of the rheumatoid synovial microvascular bed by gadolinium-DTPA enhanced magnetic resonance imaging.

OBJECTIVE To examine the relation between rate of synovial membrane enhancement, intra-articular pressure (IAP), and histologically determined synovial vascularity in rheumatoid arthritis, using gadolinium-DTPA enhanced magnetic resonance imaging (MRI). METHODS Dynamic gadolinium-DTPA enhanced MRI was performed in 31 patients with knee synovitis (10 patients IAP study, 21 patients vascular morphometry study). Rate of synovial membrane enhancement was quantified by line profile analysis using the image processing package ANALYZE. IAP was measured using an intra-compartmental pressure monitor system. Multiple synovial biopsy specimens were obtained by a blind biopsy technique. Blood vessels were identified immunohistochemically using the endothelial cell marker QBend30 and quantified (blood vessel numerical density and fractional area). RESULTS Median blood vessel numerical density and fractional area were 77.5/mm2 (IQR; 69.3–110.7) and 5.6% (IQR; 3.4–8.5) respectively. The rate of synovial membrane enhancement (median 2.74 signal intensity units/s, IQR 2.0–3.8) correlated with both blood vessel numerical density (r = 0.46, p < 0.05) and blood vessel fractional area (r = 0.55, p < 0.02). IAP did not influence the rate of enhancement. CONCLUSIONS Gadolinium-DTPA enhanced MRI may prove to be a valuable technique for evaluating drugs that influence angiogenesis.

ANTIOXIDANTS, REDOX-REGULATED TRANSCRIPTION FACTORS, AND INFLAMMATION

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.