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D R Blake

Publications and source records attributed to D R Blake.

At least 55 records · Page 3Linked to original sources
PubMed1998

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.

Animals↗
PubMed1998

Xanthine oxidoreductase is asymmetrically localised on the outer surface of human endothelial and epithelial cells in culture.

Subcellular localisation of xanthine oxidoreductase (XOR) was determined by indirect immunofluorescence using confocal microscopy in human endothelial and epithelial cell lines and in primary cultures of human umbilical vein endothelial cells. XOR was diffusely distributed throughout the cytoplasm but with higher intensity in the perinuclear region. In non-permeabilised cells, XOR was clearly seen to be asymmetrically located on the outer surfaces, showing, in many cases, a higher intensity on those faces apposed by closely neighbouring cells. Such specific distribution suggests a functional role for the enzyme in cell-cell interactions, possibly involving signalling via reactive oxygen species.

Cell Line↗
PubMed1998

In vivo absorption, metabolism, and urinary excretion of alpha,beta-unsaturated aldehydes in experimental animals. Relevance to the development of cardiovascular diseases by the dietary ingestion of thermally stressed polyunsaturate-rich culinary oils.

Thermal stressing of polyunsaturated fatty acid (PUFA)- rich culinary oils according to routine frying or cooking practices generates high levels of cytotoxic aldehydic products (predominantly trans-2-alkenals, trans,trans-alka-2,4-dienals, cis,trans-alka-2, 4-dienals, and n-alkanals), species arising from the fragmentation of conjugated hydroperoxydiene precursors. In this investigation we demonstrate that typical trans-2-alkenal compounds known to be produced from the thermally induced autoxidation of PUFAs are readily absorbed from the gut into the systemic circulation in vivo, metabolized (primarily via the addition of glutathione across their electrophilic carbon-carbon double bonds), and excreted in the urine as C-3 mercapturate conjugates in rats. Since such aldehydic products are damaging to human health, the results obtained from our investigations indicate that the dietary ingestion of thermally, autoxidatively stressed PUFA-rich culinary oils promotes the induction, development, and progression of cardiovascular diseases.

Aldehydes↗
PubMed1998

Why do we need new treatments for rheumatoid arthritis?

Rheumatoid arthritis is a chronic systemic autoimmune inflammatory disease characterized by progressive joint damage. The classical treatments of the disease such as myocrisin and sulphasalazine, are not always effective at controlling the disease. This has necessitated the development of novel agents for treating rheumatoid arthritis. Most of these drugs are biological in nature and are targeted at specific sites of the inflammatory cascade of reactions. A number of clinical trials have been conducted. The clinical effects that have been observed are transient, necessitating repeated treatments and the risk of vaccination effects. Many of these agents have to be administered parenterally, production costs are very high. Consequently, chemical entities which can be taken orally need to be developed. Since the immune system is very complex with pleiotropic cytokines and redundancy in some of the regulatory networks, it may therefore be necessary to use multiple agents targeted at different specific sites of the inflammatory cascade or that different agents could be given at different stages of the disease, to induce disease remission and maintain the response to therapy. Cytokines such as tumour necrosis factor (TNF) and interleukin 1 (IL-1) play important physiological roles in the host's defence systems against infections and malignancy. The chronic inhibition of these cytokines by targeted therapies may therefore lead to the development of side effects. Thus, carefully controlled long-term studies will be required to assess the safety of selective targeting of processes involved in inflammation. A more recent novel approach is to target hypoxic tissues with bioreductive agents. Thus, some of the established rheumatoid arthritis treatments could be linked to bioreductive agents and released in hypoxic tissues where inflammation is occurring. This review summarizes the important developments in the therapy of rheumatoid arthritis. There is no doubt that despite these developments we need to develop new and advanced treatment modalities for rheumatoid arthritis.

Antirheumatic Agents↗
PubMed1998

Inactivation of antithrombin III in synovial fluid from patients with rheumatoid arthritis.

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

Aged↗
PubMed1998

Evaluation of vaginal infections in adolescent women: can it be done without a speculum?

OBJECTIVE: Given that highly sensitive urine-based nucleic acid amplification tests may eliminate the need for speculum exam to diagnose gonorrhea and chlamydia cervicitis, we sought to determine if vaginal infections could be diagnosed without using a speculum. METHODS: Matched pairs of vaginal specimens were collected from participants before and during speculum exam for diagnosis of trichomoniasis, bacterial vaginosis, and vulvovaginal candidiasis. Females age 12 to 22 years presenting to the Johns Hopkins adolescent primary care clinics who required a pelvic examination were eligible to participate. A convenience sample of 686 patients was recruited between July 1995 and August 1996. Paired vaginal specimens were evaluated with blinded microscopic evaluation. Analysis consisted of: 1) comparison of collection method sensitivities; and 2) assessment of proportions of infections detected by one method that were also detected by the other method. RESULTS: Sensitivities of speculum and nonspeculum collection methods were 75% and 77% (difference = -2%; 95% confidence interval, -11%, 7%) for trichomoniasis, 64% and 68% (difference = -4% [-10%, 3%]) for bacterial vaginosis, and 85% and 80% (difference = 5% [-12%, 22%]) for vulvovaginal candidiasis. The speculum method identified 88% (trichomoniasis), 90% (bacterial vaginosis), and 81% (vulvovaginal candidiasis) of infections detected by the nonspeculum method. The nonspeculum method identified 91% (trichomoniasis), 95% (bacterial vaginosis), and 76% (vulvovaginal candidiasis) of infections detected by the speculum method. CONCLUSIONS: Vaginal infections can be adequately diagnosed without a speculum. Once urine-based diagnosis of gonorrhea and chlamydia becomes well established, it may be possible to perform evaluations for uncomplicated genitourinary complaints without using a speculum.

Adolescent↗
PubMed1998

A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor.

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

Allopurinol↗
PubMed1998

Focally regulated endothelial proliferation and cell death in human synovium.

Angiogenesis and vascular insufficiency each may support the chronic synovial inflammation of rheumatoid arthritis. We have shown by quantitative immunohistochemistry and terminal uridyl deoxynucleotide nick end labeling that endothelial proliferation and cell death indices were each increased in synovia from patients with rheumatoid arthritis compared with osteoarthritic and noninflamed controls, whereas endothelial fractional areas did not differ significantly among disease groups. Markers of proliferation were associated with foci immunoreactive for vascular endothelial growth factor and integrin alpha(v)beta3, whereas cell death was observed in foci in which immunoreactivities for these factors were weak or absent. No association was found with thrombospondin immunoreactivity. The balance between angiogenesis and vascular regression in rheumatoid synovitis may be determined by the focal expression of angiogenic and endothelial survival factors. Increased endothelial cell turnover may contribute to microvascular dysfunction and thereby facilitate persistent synovitis.

Adult↗
PubMed1997

Protection of rats from adjuvant arthritis by immunization with naked DNA encoding for mycobacterial heat shock protein 65.

OBJECTIVE: To assess the feasibility of vaccination with naked DNA encoding for mycobacterial heat shock protein 65 (hsp65) in the modulation of experimental arthritis. METHODS: Adjuvant arthritis (AA) was induced in Lewis rats preimmunized, intramuscularly, with a plasmid encoding for hsp65 (pCMV3.65). Clinical scores were recorded for 3-4 weeks, and histologic and radiologic parameters were evaluated. Cellular and antibody reactivity to hsp65 was assessed. The expression of the hsp65 gene was investigated in injected muscles. RESULTS: The pCMV3.65-treated rats were significantly protected from disease development in comparison with the control groups. This finding correlated with the results of histologic and radiologic examinations of the involved joints. The message for hsp65 was detected in injected muscles. T cell proliferation and antibodies to this protein were found to be elevated in pCMV3.65-treated rats when compared with both the arthritic control (AA-induced) and the naive (did not receive adjuvant) animals. CONCLUSION: We have demonstrated for the first time that naked DNA delivery is feasible in controlling experimental autoimmune disease. Although the actual mechanism of protection has not been fully elucidated, this simple and versatile approach could represent a useful tool in dissecting basic autoimmune mechanisms.

Animals↗
PubMed1997

A modified form of low-density lipoprotein with increased electronegative charge is present in rheumatoid arthritis synovial fluid.

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

Aged↗
PubMed1997

Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma.

1. The vasoconstrictor peptide antiotensin II (AII) can stimulate angiogenesis, an important process in wound healing, tumour growth and chronic inflammation. To elucidate mechanisms underlying AII-enhanced angiogenesis, we have studied a subcutaneous sponge granuloma model in the rat by use of 133Xe clearance, morphometry and quantitative in vitro autoradiography. 2. When injected directly into the sponge, AII (1 nmol day-1) increased 133Xe clearance from, and fibrovascular growth in sponge granulomas, indicating enhanced angiogenesis 6 to 12 days after implantation. This AII-enhanced angiogenesis was inhibited by daily doses (100 nmol/sponge) of the specific but subtype non-selective AII receptor antagonist (Sar1, Ile8)AII, and by the selective non-peptide AT1 receptor antagonists losartan and DuP 532. In contrast, AII-enhanced neovascularization was not inhibited by the AT2 receptor antagonist PD123319, nor was it mimicked by the AT2 receptor agonist CGP42112A (each at 100 nmol/sponge day-1). 3. AI (1 nmol/sponge day-1), the angiotensin converting enzyme (ACE) inhibitors captopril (up to 100 micrograms/sponge day-1) and lisinopril (40 micrograms/sponge day-1), or AII receptor antagonists did not affect angiogenesis in the absence of exogenous AII. 4. [125I]-(Sar1, Ile8)AII binding sites with characteristics of AT1 receptors were localized to microvessels and to non-vascular cells within the sponge stroma from 4 days after implantation, and were at higher density than in skin throughout the study. 5. [125I]-(Sar1, Ile8)AII binding sites with characteristics of AT2 receptors were localized to non-vascular stromal cells, were of lower density and appeared later than did AT1 sites. 6. The ACE inhibitor [125I]-351A bound to sites with characteristics of ACE, 14 days after sponge implantation. [125I]-351A bound less densely to sponge stroma than to skin. 7. We propose that AII can stimulate angiogenesis, acting via AT1 receptors within the sponge granuloma. AT1 and AT2 receptors and ACE develop sequentially during microvascular maturation, and the role of the endogenous angiotensin system in angiogenesis will depend on the balanced local expression of its various components. Pharmacological modulation of this balance may provide novel therapeutic approaches in angiogenesis-dependent diseases.

1-Sarcosine-8-Isoleucine Angiotensin II↗