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D R Blake

Publications and source records attributed to D R Blake.

At least 37 records · Page 2Linked to original sources
PubMed2001

Localization of 3-nitrotyrosine to rheumatoid and normal synovium.

OBJECTIVE: To determine the localization of 3-nitrotyrosine (3-NT), a footprint marker of peroxynitrite (ONOO-) and other reactive nitrogen species, to the inflamed human synovium and to compare this with normal synovial and nonsynovial tissue of human and animal origin. METHODS: Monoclonal and polyclonal antibodies were used to investigate for 3-NT, inducible nitric oxide synthase (iNOS), macrophage marker CD68, and the vascular smooth muscle marker alpha-actin by avidin-biotin immunocytochemistry. RESULTS: In the inflamed synovium, 3-NT was found in the vascular smooth muscle and macrophages. In normal human synovium, 3-NT was present in the vascular smooth muscle and some lining cells and was not associated with immunoreactivity for iNOS. Similarly, 3-NT could be demonstrated in the vascular smooth muscle cells of normal rats and iNOS knockout mice. It was not present in the vascular smooth muscle of healthy, nonsynovial tissue. CONCLUSION: The synovial vasculature in histologically normal human and naive rodent synovium was alone among the normal tissues studied in exhibiting iNOS-independent immunoreactivity for 3-NT. These findings suggest a physiologic role for ONOO- in normal synovial vascular function.

Actins↗
PubMed2000

Antibacterial properties of xanthine oxidase in human milk.

Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.

Anti-Bacterial Agents↗
PubMed2000

Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase.

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and </=6.0 for xanthine. Involvement of the molybdenum site of XO in nitrite reduction was shown by the fact that alloxanthine inhibits xanthine oxidation competitively with nitrite. Strong preference for Mo=S over Mo=O was shown by the relatively very low NADH-nitrite reductase activity shown by desulfo-enzyme. The FAD site of XO was shown not to influence nitrite reduction in the presence of xanthine, although it was clearly involved when NADH was the reducing substrate. Apparent production of NO decreased with increasing oxygen tensions, consistent with reaction of NO with XO-generated superoxide. It is proposed that XO-derived NO fulfills a bactericidal role in the digestive tract.

Anaerobiosis↗
PubMed2000

Qualitative study of interpretation of reassurance among patients attending rheumatology clinics: "just a touch of arthritis, doctor?".

OBJECTIVES: To examine commonly used methods of reassurance by clinicians and explore their effect on patients. DESIGN: Qualitative study of tape recordings of in-depth, semistructured interviews with patients before and after consultation and of their consultations with doctors. SETTING: NHS specialist rheumatology clinics in two large British cities. PARTICIPANTS: 35 patients selected by consultant rheumatologists from general practitioner referral letters (28 women, 7 men; 24 with inflammatory arthropathies, 11 other rheumatological complaints). MAIN OUTCOME MEASURES: Patients' perceptions of reassurance. RESULTS: Reassurance was an important part of consultations, whether the diagnosis was clear or uncertain. Clinicians tried to reduce anxiety by emphasising the mildness, early stage, or non-seriousness of the disorder and the likelihood that patients would recover. Patients interpreted reassurance in the context of their own views and perceptions. Doctors' emphasis on the mildness or earliness of the condition raised the spectre of future pain and disability rather than providing reassurance. Patients who felt that their problems were properly acknowledged felt more reassured. CONCLUSIONS: Typical patterns of reassurance were not successful because of the differences in perspective of patients and doctors. A key to successful reassurance seemed to be the doctor's ability to acknowledge patients' perspectives of their difficulties.

Adult↗
PubMed2000

Effects of iron and iron chelation in vitro on mucosal oxidant activity in ulcerative colitis.

BACKGROUND: Reactive oxygen species may be pathogenic in ulcerative colitis. Oral iron supplements anecdotally exacerbate inflammatory bowel disease and iron levels are elevated in the inflamed mucosa. Mucosal iron may enhance hydroxyl ion production via Fenton chemistry. Conversely, the iron chelator, desferrioxamine, is reportedly beneficial in Crohn's disease. AIMS: To assess the in vitro effects of exogenous iron and of iron chelators on the production of reactive oxygen species by colonic biopsies from normal control subjects and patients with ulcerative colitis. METHODS: Luminol-amplified chemiluminescence was used to measure mucosal reactive oxygen species production both before and after addition in vitro of ferric citrate (100 microM), desferrioxamine (1 mM) and 1,10-phenanthroline (1 mM). RESULTS: Ferric citrate had no effect on the chemiluminescence produced by human colonic mucosa. However, desferrioxamine and phenanthroline reduced chemiluminescence by 47% (n=7, P=0.018) and by 26% (n=10, P=0.005), respectively, in inactive ulcerative colitis, and by 44% (n=9, P=0. 008) and 42% (n=11, P=0.006) in active disease. CONCLUSION: The lack of effect of ferric citrate suggests that sufficient free iron is already present in inflamed biopsies to drive the Fenton reaction maximally. The effects of desferrioxamine and 1,10-phenanthroline on the chemiluminescence of biopsies from patients with ulcerative colitis suggest that a clinical trial of topical iron chelation in active disease is indicated.

Adult↗
PubMed1999

Effect of faradic products on direct current-stimulated calvarial organ culture calcium levels.

Calcium release from mouse calvarial organ cultures was used to analyse the well-described biological effects of constant direct current (20 microA) in combination with Faradic products generated at a titanium wire cathode. Constant 20-microA direct current stimulation alone, delivered by agar salt bridges, consistently lowered the media calcium levels. Direct exposure of calvariae to a titanium cathode and its faradic products resulted in further lowering of media calcium levels and also a significant increase in the media pH. Hydrogen peroxide is a faradic product of the titanium cathode, micromolar amounts being generated by our system over 24 hr. H(2)O(2) is pro-resorptive whereas elevated pH stimulates osteoblast activity. We propose that where bone tissue is in direct contact with metal wire cathodes, the faradic products, hydrogen peroxide and hydroxyl ion, are significant factors which, in their own right, further contribute to accelerated remodelling and improved clinical outcome.

Animals↗
PubMed1999

Examination of the metabolic status of rat air pouch inflammatory exudate by high field proton NMR spectroscopy.

High field proton (1H) NMR spectroscopy was employed to investigate the metabolic status of rat air pouch inflammatory exudates obtained subsequent to the induction of inflammation with carrageenan, and the 1H NMR profiles of these fluids were compared and contrasted with those of inflammatory human synovial fluid, rat plasma and human serum. The characteristic biochemical features obtained from 1H NMR analysis of these exudates consisted of (1) substantially elevated levels of lactate (11.40+/-1.46x10-3 mol dm-3 for samples collected at a time point of 24 h post induction) with little or no NMR-detectable glucose, data consistent with a hypoxic environment and consequent anaerobic metabolism in the inflamed air pouch, and (2) high levels of the ketone body 3-d-hydroxybutyrate, providing evidence for an increased utilization of fats for energy by lymphocytes, the predominant leucocytes present in this environment. These phenomena represent a pathological extreme of the abnormal metabolic status of inflammatory human synovial fluids.

Air↗
PubMed1999

Nitrite determination in human plasma and synovial fluid using reactions of nitric oxide with 3, 5-dibromo-4-nitrosobenzenesulphonate (DBNBS).

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

Arthritis, Rheumatoid↗
PubMed1999

Use of spun urine to enhance detection of Trichomonas vaginalis in adolescent women.

BACKGROUND: Diagnosis of Trichomonas vaginalis infection is traditionally performed by microscopic examination of vaginal fluid. Although this technique is relatively insensitive compared with culture, it is widely used because of its lower cost and immediate results. OBJECTIVE: To assess the utility of microscopic examination of spun urine as a means of increasing the sensitivity of microscopic diagnosis of T. vaginalis. DESIGN AND SETTING: Retrospective observational study performed in a hospital-based adolescent clinic. SUBJECTS: Female patients enrolled between July 1995 and August 1996 into a larger study evaluating diagnosis of vaginal infections (N = 686). To be included, subjects had to have a positive culture for T. vaginalis (n = 97); those who did not have a spun urine examination were excluded (n = 22). MAIN OUTCOME MEASURE: Microscopic examination of vaginal fluid and spun urine for presence of motile trichomonads. Using a positive Trichomonas culture as the reference standard, the sensitivity of vaginal fluid alone was compared with vaginal fluid plus spun urine. The McNemar test for paired samples was used to test the statistical significance of the difference in sensitivities. RESULTS: Ninety-seven subjects had culture results positive for Trichomonas. Of these, 75 (77%) had a spun urine examination performed. Subjects were aged 13 to 22 years and all were African American. Seventy-three percent of the infections were detected by vaginal fluid specimen, 64% by spun urine, and 85% by either vaginal specimen or spun urine. The difference in sensitivity between vaginal specimen alone and vaginal specimen plus spun urine was 12% (95% confidence interval, 3%-21%; P<.005). Nine patients who would not have been diagnosed by examination of vaginal fluid alone were diagnosed with the addition of spun urine examination. CONCLUSION: Microscopic examination of a spun urine specimen performed in conjunction with microscopic examination of a vaginal fluid specimen improves the detection rate of T. vaginalis.

Adolescent↗
PubMed1999

Simultaneous analysis of nitrite, nitrate and the nicotinamide nucleotides by capillary electrophoresis: application to biochemical studies and human extracellular fluids.

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

Body Fluids↗
PubMed1999

Antioxidant effects of aminosalicylates and potential new drugs for inflammatory bowel disease: assessment in cell-free systems and inflamed human colorectal biopsies.

BACKGROUND: The therapeutic efficacy of 5-aminosalicylic acid in inflammatory bowel disease may be related to its antioxidant properties. AIM: To compare in vitro the antioxidant effects of conventional drugs (5-aminosalicylic acid, corticosteroids, metronidazole), with new aminosalicylates (4-aminosalicylic acid, balsalazide) and other potential therapies (ascorbate, N-acetylcysteine, glutathione, verapamil). METHODS: Compounds were assessed for efficacy in reducing the in vitro production of reactive oxygen species by cell-free systems (using xanthine/xanthine oxidase, with or without myeloperoxidase) and by colorectal biopsies from patients with ulcerative colitis using luminol-amplified chemiluminescence. RESULTS: 5-aminosalicylic acid and balsalazide were more potent antioxidants than 4-aminosalicylic acid or N-acetyl-5-aminosalicylic acid in cell-free systems. 5-aminosalicylic acid (20 mM) and balsalazide (20 mM) inhibited rectal biopsy chemiluminescence by 93% and 100%, respectively, compared with only 59% inhibition by 4-aminosalicylic acid (20 mM). Hydrocortisone, metronidazole and verapamil had no significant effect on chemiluminescence in any system. Ascorbate (20 mM) inhibited chemiluminescence by 100% in cell-free systems and by 60% in rectal biopsies. N-acetyl cysteine (10 mM), and both oxidized and reduced glutathione (10 mM), completely inhibited chemiluminescence in cell-free systems, but not with rectal biopsies. CONCLUSIONS: The antioxidant effects of compounds varies between cell-free systems and inflamed colorectal biopsies. The effect of drugs on the chemiluminescence produced by these two assay systems is useful for screening potentially new antioxidant treatments for inflammatory bowel disease. Ascorbate seems worth further study as a novel therapy.

Adrenal Cortex Hormones↗
PubMed1999

The role of N-acetylcysteine in protecting synovial fluid biomolecules against radiolytically-mediated oxidative damage: a high field proton NMR study.

High field proton (1H) NMR spectroscopy has been employed to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory synovial fluid samples against oxidative damage arising from gamma-radiolysis (5.00 kGy) in the presence of atmospheric O2. Although oxidation of urate to allantoin by radiolytically-generated *OH radical was readily circumventable by pre-treatment of synovial fluids with N-acetylcysteine (1.00 or 3.00 x 10(-3) mol x dm(-3)) or cysteine (1.00, 2.00 or 5.00 x 10(-3) mol x dm(-3)), both thiols offered only a limited protective capacity with respect to hyaluronate depolymerisation and the production of formate from carbohydrates in general. Radiolytic products generated from the added thiols (predominantly their corresponding disulphides) were simultaneously detectable in 1H Hahn spin-echo spectra of gamma-irradiated synovial fluids, permitting a quantitative evaluation of the radioprotective capacity of these agents. It is concluded that the multicomponent analytical ability of high field 1H NMR spectroscopy provides much useful molecular information regarding mechanisms associated with the radioprotectant actions of thiols in intact biofluids.

Acetylcysteine↗
PubMed1999

Tumour necrosis factor alpha stimulated rheumatoid synovial microvascular endothelial cells exhibit increased shear rate dependent leucocyte adhesion in vitro.

OBJECTIVE: To investigate endothelial cell adhesion molecule expression and leucocyte adhesion to endothelial cells isolated from the microvasculature of rheumatoid arthritic synovial tissue (SMEC) in comparison with similar cells isolated from healthy subcutaneous adipose tissue (ADMEC) or from umbilical veins (HUVEC). METHODS: Cultured endothelial cells were treated with tumour necrosis factor alpha (TNFalpha) for 2-24 hours before the assessment of cell surface E-selectin, vascular (VCAM-1) or intercellular cell adhesion molecule-I (ICAM-1) expression. Neutrophil and T lymphocyte adhesion to TNFalpha treated endothelial cells was assessed using static and shear dependent assay systems. RESULTS: VCAM-1 expression by SMEC was significantly less sensitive to TNFalpha stimulation than HUVEC or ADMEC. E-selectin expression by SMEC appeared to be more sensitive to TNFalpha stimulation and maximal expression was about 30% greater in comparison with HUVEC or ADMEC. Sensitivity to TNFalpha induction and maximal ICAM-1 expression was similar in all three endothelial cell types. Static neutrophil adhesion to TNFalpha stimulated SMEC was significantly increased in comparison with HUVEC, however this phenomenon was dependent on the presence of neutralising antibodies to ICAM-1. At shear rates in excess of 2.4 dynes/cm(2) significantly more neutrophils and, predominantly CD45RO+, T lymphocytes adhered to TNFalpha stimulated SMEC than HUVEC. CONCLUSION: Rheumatoid synovial endothelial cells differentially regulate E-selectin and VCAM-1. The increased ability of TNFalpha stimulated synovial endothelial cells to support leucocyte adhesion may help to explain the leucocyte, in particular CD45RO+ T-lymphocyte, recruitment observed in the rheumatoid synovium.

Arthritis, Rheumatoid↗
PubMed1998

Pulsed electromagnetic fields simultaneously induce osteogenesis and upregulate transcription of bone morphogenetic proteins 2 and 4 in rat osteoblasts in vitro.

Pulsed electromagnetic fields (PEMF) are successfully employed in the treatment of a variety of orthopaedic conditions, particularly delayed and nonunion fractures. In this study, we examined PEMF effects on in vitro osteogenesis by bone nodule formation and on mRNA expression of bone morphogenetic proteins 2 and 4 by reverse-transcriptase polymerase chain reaction (RT-PCR) in cultured rat calvarial osteoblasts. PEMF exposure induced a significant increase in both the number (39% over unexposed controls) and size (70% larger compared to unexposed controls) of bone-like nodules formed. PEMF also induced an increase in the levels of BMP-2 and BMP-4 mRNA in comparison to controls. This effect was directly related to the duration of PEMF exposure. This study shows that clinically applied PEMF have a reproducible osteogenic effect in vitro and simultaneously induce BMP-2 and -4 mRNA transcription. This supports the concept that the two effects are related.

Animals↗
PubMed1998

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity.

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

Anaerobiosis↗
PubMed1998

Xanthine oxidoreductase in human mammary epithelial cells: activation in response to inflammatory cytokines.

Xanthine oxidoreductase (XOR) in human mammary epithelial cells was shown to have low true specific activity, similar to that in breast milk. Enzymic activity was increased in response to inflammatory cytokines; increases of 2-2.5-fold being seen with TNF-alpha and IL-1beta and of approximately 8-fold with IFN-gamma. No significant increase was seen with IL-6. A combination of IFN-gamma and TNF-alpha, or of these two cytokines plus IL-1beta, led to responses representing the sum of those obtained by using the individual cytokines. The 8-fold increase in enzymic activity, stimulated by IFN-gamma, corresponded to only a 2-3-fold increase in specific mRNA, suggesting the possibility of post-translational activation; a possibility strongly supported by the corresponding 2-3-fold rise in XOR protein, as determined by ELISA. In no case was cytokine-induced activation accompanied by changes in the oxidase-dehydrogenase ratio of XOR. These data strongly support a role for XOR in the inflammatory response of the human mammary epithelial cell, and provide further evidence of post-translational activation of a low activity form of human XOR, similar to that previously observed in vivo for the breast milk enzyme.

Breast↗