Xanthine oxidase can generate nitric oxide from nitrate in ischaemia.
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Anaerobiosis↗
Biomedical subjects
D R Blake
Publications and source records attributed to D R Blake.
Elevated circulating plasma NADH oxidising activity of xanthine oxidoreductase in plasma.
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Electron Spin Resonance Spectroscopy↗
Expression of xanthine oxidase activity in human endothelial cells as a function of cell density.
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Cell Division↗
Putative analgesic activity of repeated oral doses of vitamin E in the treatment of rheumatoid arthritis. Results of a prospective placebo controlled double blind trial.
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.
Administration, Oral↗
Intra-articular pressure profile of the knee joint in a spectrum of inflammatory arthropathies.
OBJECTIVES: The intra-articular pressure (IAP) rises significantly after isometric quadriceps contraction in patients with rheumatoid synovitis, a process that may temporarily impede synovial blood flow and cause oxidative injury. In acute traumatic knee effusions (ATE) pressure rises are trivial. This study compared the IAP profiles of patients with ATE with three different populations--an acute synovitis on the background of a chronic inflammatory arthropathy, a chronic low grade inflammatory arthropathy, and an acute intermittent inflammatory arthropathy. The study objective was to discover if the pressure profiles observed in these groups reflect an influence of the inflammatory process or time or both. METHODS: Thirty three patients were studied. These were divided into four subgroups; group 1: five acute traumatic knee effusions (ATE); group 2: acute effusions on the background of a chronic inflammatory arthropathy: seven rheumatoid arthritis (RA), five psoriatic arthritis (PsA); group 3: seven osteoarthritis (OA) and group 4: acute effusions on the background of an intermittent inflammatory arthropathy: seven pyrophosphate arthropathy (PA), one amyloid (AA), one Behcet's (B). IAP was measured (mm Hg) at rest and during isometric quadriceps contraction using the hand held portable 295-1 intra-compartmental pressure monitor system (Stryker UK). The volume of synovial fluid aspirated was recorded. RESULTS: Expressed as medians (inter-quartile range). Resting IAP was; ATE 6 (2-12), RA 8 (5-47), PsA 18 (11-31), OA 17 (7-21), PA 25 (9-29), AA 14, and B 12. IAP increased in all subjects during isometric contraction; ATE 9 (7-16), RA 56 (33-150), PsA 52 (43-85), OA 56 (20-116), PA 53 (41-65), AA 47, B 57 and the IAP rise was significant (p < 0.05) in all except the ATE group (p > 0.05). The volume of synovial fluid aspirated in groups 2, 3, and 4 correlated significantly with the magnitude of the IAP change (r = 0.45, p < 0.05). CONCLUSION: The IAP rise during isometric quadriceps contraction is a feature of all patients with an inflammatory based effusion irrespective of the duration of the effusion. This is not the case in patients with an ATE. In inflammatory synovitis the rise in intra-articular pressure with isometric quadriceps contraction relates to effusion volume. It is concluded that the inflammatory process prevents reflex muscle inhibition, a locally protective mechanism that minimises the potential for intermittent ischaemia/oxidative injury.
Acute Disease↗
Multicomponent investigations of the hydrogen peroxide- and hydroxyl radical-scavenging antioxidant capacities of biofluids: the roles of endogenous pyruvate and lactate. Relevance to inflammatory joint diseases.
High field proton (1H) nuclear magnetic resonance (NMR) analysis of biofluids (health human blood sera and inflammatory knee-joint synovial fluids) has been employed to evaluate the hydrogen peroxide (H2O2)- and hydroxyl radical (0OH)- scavenging antioxidant capacities of a range of polar, low-molecular-mass endogenous metabolites therein. Data obtained indicate that consumption of H2O2 by pyruvate (generating acetate and CO2 via an oxidative decarboxylation reaction) and 0OH radical by lactate (generating pyruvate, and subsequently acetate and CO2) may serve to protect alternative biofluid components (e.g., macromolecules) against reactive oxygen species-mediated oxidative damage in vivo. The mechanistic, physiological and potential therapeutic implications of these results are discussed with special reference to inflammatory joint diseases.
Adult↗
High resolution 1H NMR investigations of the reactivities of alpha-keto acid anions with hydrogen peroxide.
The chemical reactivity of various alpha-keto acid anions (beta-hydroxypyruvate, beta-phenylpyruvate, 2-ketobutyrate and 2-ketoglutarate) with hydrogen peroxide (H2O2) was investigated at physiological pH (7.4) and a temperature of 25 degrees C. The initial concentration of the alpha-keto acid anions was kept constant at 1.00 mM whilst that of added H2O2 was varied from 0.25 to 1.00 mM, and the rate and extent of these reactions was evaluated using 1H NMR spectroscopy. At all H2O2 concentrations utilised, the order of reactivity of the alpha-keto acid anions was beta-hydroxypyruvate > beta-phenylpyruvate > 2-ketobutyrate > 2-ketoglutarate. The results obtained are in agreement with a proposed mechanism for these reactions, involving nucleophilic attack of the mono-deprotonated peroxide species (HO2-) at the C-2 carbonyl group carbon centre. The antioxidant capacity of such alpha-keto acids is discussed in terms of their potential use as therapeutic agents in clinical conditions where H2O2 has been shown to play a critical role in the disease process, i.e., those involving 'oxidative stress'.
Butyrates↗
Modified low density lipoprotein and cytokines mediate monocyte adhesion to smooth muscle cells.
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.
Antibodies, Monoclonal↗
Ascorbate promotes low density lipoprotein oxidation in the presence of ferritin.
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.
Arteriosclerosis↗
Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite.
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 (<250 microM) 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.
Amino Acid Sequence↗
Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue.
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.
Aged↗
Innervation and neurokinin receptors during angiogenesis in the rat sponge granuloma.
Angiogenesis is an essential component of wound healing and inflammation. In the rat subcutaneous sponge implantation model, angiogenesis can be enhanced by administration of the sensory neuropeptide, substance P. We have used quantitative in vitro receptor autoradiography and immunohistochemistry to investigate the development of endogenous neurovascular regulatory systems in the newly-formed granulation tissue of this model. The fraction of endothelial cells immunoreactive for proliferating cell nuclear antigen, endothelial fractional area, and 133Xe clearance were used as measures of endothelial proliferation, neovascularization, and blood flow, respectively. Endothelial proliferation occurred predominantly in tissues surrounding the sponge, and peaked before neovascularization of sponge stroma and the establishment of sponge blood flow. Substance P-containing sensory nerves and specific, high affinity substance P binding sites with characteristics of neurokinin receptors of the NK1 subclass, were localized to microvessels surrounding the sponge at all time points. Lower density substance P binding sites were localized to newly formed microvessels within the sponge stroma, progressively increasing in density from day 4 to day 14. Nerve fibres were observed in the stroma of only 2 of 6 sponges at day 14, and none at earlier time points. These data support the hypothesis that substance P-enhanced angiogenesis in this model results from a direct action on microvascular NK1 receptors. Neovascularization is a sequential process, with early endothelial proliferation followed by new vessel formation and increased blood flow, with maturation of endogenous neurovascular regulatory systems occurring late in this process in inflamed tissues.
Animals↗
The effect of intra-articular capsaicin on nerve fibres within the synovium of the rat knee joint.
The aim of this study was to establish the effects of intra-articular capsaicin (pelargonic acid vallinylamide) on synovial innervation of the rat knee. Rats were sacrificed 1, 2, 4 and 7 days after intra-articular injection of capsaicin and joint tissues stained with either conventional haematoxylin and eosin (H and E) or with specific antibodies to the calcitonin gene-related peptide (CGRP), substance P (both of which are markers for primary afferent fibres), the C-flanking peptide of neuropeptide Y (CPON) (localised in postganglionic sympathetic fibres), or protein gene product 9.5 (a pan-neuronal marker). At lower concentrations (0.1% and 0.25%), capsaicin produced no change in peptide staining pattern or histological appearance. At 0.5% capsaicin, there was complete loss of nerve fibres showing positive staining for CGRP and substance P at all time points. Staining for CPON and protein gene product 9.5 was still present, but decreased, 1 and 2 days after treatment and virtually absent at 4 and 7 days. These findings provide evidence for partially selective denervation induced by 0.5% capsaicin, in contrast to 1% capsaicin which abolished staining for all peptide markers, indicating a total ablation of nerve fibres. A consistent but unexpected finding was the presence of a severe inflammatory response in joints treated with 0.5% and 1% capsaicin. An influx of polymorphonuclear leucocytes was found to occur within 4 h of injection, with progressive appearance of mononuclear cells after this time. We conclude that it is difficult to specifically deplete sensory nerve fibres from the synovium by means of local capsaicin injection. Although selective loss of staining for sensory nerve fibres could be achieved by injection of 0.5% capsaicin, there was progressive non-specific loss of post-ganglionic autonomic fibres which may be related to the severe inflammatory response provoked by the higher doses of capsaicin.
Animals↗
Adriamycin stimulates proliferation of human lymphoblastic leukaemic cells via a mechanism of hydrogen peroxide (H2O2) production.
It is becoming clear that adriamycin cytotoxicity may be mediated by semiquinone-free radicals derived from the drug itself and reactive oxygen species (ROS). Recent evidence supports the concept that low concentrations of ROS are able to stimulate cell proliferation, and, based on the observation that subtoxic concentrations of adriamycin can also induce cell proliferation, we hypothesize that low concentrations of adriamycin stimulate cell proliferation by a ROS generation mechanism. We have employed spin-trapping and electron spin resonance (ESR) spectroscopy to investigate the nature of the adriamycin-generated ROS. The spin trap 3,5-dibromo-4-nitrosobenzenesulphonate (DBNBS), which is oxidized in the presence of H2O2 and peroxidase enzymes, was used to produce a characteristic three-line spectrum, and it was found that an identical spectrum was produced by human lymphoblastic leukaemic cells (CCRF-CEM cells) after exposure to adriamycin. We tested our hypothesis further by exposing CCRF-CEM cells to subtoxic concentrations of adriamycin (10(-8), 10(-9) and 10(-10) M) and low concentrations of H2O2 (10(-8), 10(-9) and 10(-10) M) and subsequently monitored cell proliferation. We found that low concentrations of both adriamycin and H2O2 significantly stimulate CCRF-CEM cell proliferation. We therefore conclude that subtoxic concentrations of adriamycin are likely to induce cell proliferation via an H2O2 mediated mechanism.
Cell Division↗
Measuring outcome in rheumatoid arthritis--which measures are suitable for routine clinical use?
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Arthritis, Rheumatoid↗
A synthetic 10-kD heat shock protein (hsp10) from Mycobacterium tuberculosis modulates adjuvant arthritis.
The heat shock protein, hsp10, is an abundant protein in Mycobacterium tuberculosis (Mtb), its nucleotide sequence encoding a protein of 99 amino acids with a molecular mass of 10.7 kD. This sequence is phylogenetically conserved, being represented by the GroES homologue of Escherichia coli. Hsp10 and GroES are members of the chaperonin 10 family of molecular chaperones, and GroEs is necessary for the optimal activity of GroEL, a member of the chaperonin 60 family and the E. coli homologue of mycobacterial hsp65. Since hsp65 has been implicated in both experimental and human rheumatoid arthritis, we aimed to assess the immunomodulatory effects of its co-chaperonin, hsp10, in experimental arthritis. Our results show that an aqueous solution of a mycobacterial hsp10 delayed the onset and severity of adjuvant-induced arthritis in rodents when administered after disease induction but before joint involvement occurred. This biological activity was specific for the hsp10 of Mtb, since neither GroES not the rat homologue was effective. Using synthetic hsp10 fragments, the activity was localized to the N-terminal region of the molecule. Assessment of circulating antibody levels to mycobacterial hsp10 and hsp65 indicated that all arthritic rats had increased titres to both hsp10 and hsp65: hsp10-treated rats showed further elevation of this humoral response not only to hsp10 but also to hsp65 when compared with the untreated arthritic control. This is the first report of the immunomodulatory activity of mycobacterial hsp10 in experimental arthritis, and exhibits a potential role for this co-chaperonin in pathophysiological situations.
Adjuvants, Immunologic↗
Thrombin receptor expression in rheumatoid and osteoarthritic synovial tissue.
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.
Arthritis, Rheumatoid↗