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Biomedical subjects

D R Blake

Publications and source records attributed to D R Blake.

At least 127 records · Page 7Linked to original sources
PubMed1993

Monoarthritis in the rat knee induces bilateral and time-dependent changes in substance P and calcitonin gene-related peptide immunoreactivity in the spinal cord.

Bilateral changes in the spinal cord and dorsal root ganglion content of the sensory peptides substance P and calcitonin gene-related peptide have been previously reported in animal models of arthritis which affect many joints within the body. The central nervous system has been implicated in the symmetry of joint involvement in human rheumatoid arthritis. We aimed to determine whether unilateral inflammation of the knee joint can also induce bilateral changes in the spinal cord. We have induced a monoarthritis in the knee joint of the rat and used quantitative immunocytochemistry to look at changes of these peptides in the dorsal horn of the spinal cord and the dorsal root ganglia. Furthermore we have examined the responses during the acute (three days) and the chronic (21 days) phases of the model. The data show that in the acute phase of the monoarthritis there is both an ipsilateral and contralateral response which increases the immunoreactive substance P and calcitonin gene-related peptide in the L4 level of the dorsal horn of the spinal cord. In the chronic phase of the monoarthritis, the contralateral side of the dorsal horn returned to control values whilst the ipsilateral side showed reduced amounts of immunoreactive substance P and calcitonin gene-related peptide compared to controls. We propose that the acute response, at three days, to unilateral inflammation is appropriate and has evolved to protect an organism against the original insult ipsilaterally, and the possibility of subsequent insult contralaterally.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

An optical hydroxyl radical sensor.

A hydroxyl radical (.OH) fibre-optic sensor has been developed. An .OH radical-sensitive reagent phase (nitrophenol) was immobilized onto XAD-7 methacrylate beads. Subsequently the beads were attached to the distal end of a polymethylmethacrylate fibre optic. Nitrocatechol, generated from the attack of .OH radical on nitrophenol, exhibits a strong absorption band in the visible region of the electromagnetic spectrum (lambda max = 510 nm). Here, reflectance spectroscopy was employed to monitor the concomitant intensity decrease in the reflectance spectrum upon .OH radical attack. The sensor exhibited excellent stability and linearity of response to .OH generated by a Fenton reaction system (EDTA, Fe(II) and H2O2) with H2O2 over the concentration range of 3.6 x 10(-6)-8.0 x 10(-2) M.

Fiber Optic Technology↗
PubMed1993

Free radicals in inflammation: second messengers and mediators of tissue destruction.

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.

Antioxidants↗
PubMed1993

Cellular biology of bone resorption.

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged. 1. The resorption of bone is the result of successive steps that can be regulated individually. 2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone. 3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation. 4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells. 5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO2 by carbonic anhydrase and secreted by an ATP-driven vacuolar H(+)-K(+)-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone. 6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca2+) generated as a result of osteoclastic bone resorption. 7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.

Animals↗
PubMed1993

Localization and characterization of neuropeptide Y binding sites in porcine and human colon.

1. We have used quantitative receptor autoradiography to investigate the localization and characteristics of binding sites for 125Iodine-Bolton Hunter-labelled human neuropeptide Y ([125I]-BH-NPY) in porcine and human colon, and compared the binding characteristics with those found in porcine spleen. 2. Saturable, specific, high affinity [125I]-BH-NPY binding was localized to myenteric ganglia in porcine and human colons, and to submucosal ganglia in porcine colon. 3. Specific [125I]-BH-NPY binding to porcine myenteric ganglia was reversible in the presence of guanosine 5'-O-(3-thiotriphosphate) and was inhibited by related peptides with the rank order of potency; porcine NPY = human NPY = peptide tyrosine tyrosine (PYY) >> pancreatic polypeptide. 4. The Y2 selective analogue, NPY (13-36), competed for [125I]-BH-NPY binding to porcine myenteric ganglia with greater potency than the Y1 selective analogue, [Leu31, Pro34] NPY, the difference being small, but significant. 5. The characteristics of [125I]-BH-NPY binding to porcine myenteric ganglia were similar to those observed concurrently to porcine splenic red pulp. 6. The small difference in inhibitory potencies between NPY(13-36) and [Leu31, Pro34]NPY observed in this study in comparison with previous studies was not explained by differential ligand depletion during incubations, but may be due to differences in methodology between binding studies performed on tissue sections and on membranes. 7. We conclude that specific [25I]-BH-NPY binding sites are present in the myenteric and submucosal ganglia of the colon and that these sites may act as functional receptors by which NPY and PYY modulate colonic motility and electrolyte transport.

Animals↗
PubMed1993

Osteoclast function and its control.

Bone resorption appears to be dependent on a range of processes. It requires an adequate number of osteoclasts to access bone mineral. These osteoclasts must be activated by a mechanism which is dependent upon prior osteoblastic stimulation. A range of factors then contribute to the formation of a functionally effective resorptive hemivacuole. These entail osteoclast adhesion to the bone surface leading to the formation of a sealing zone. Only then can subsequent processes such as H+ ion transport, enzyme secretion and matrix digestion become effective. Thus, any one process is potentially limiting to resorption and is a potential target for regulation. Long-range regulation takes place through the action of hormones, of which the mode of action of calcitonin has been the subject of recent investigations in isolated osteoclasts. Such studies have shown a possible involvement of distinguishable receptor subtypes, the occupancy of which may activate at least two types of triggering mechanism. It is likely that an eventual influence on motility properties through G protein mediation accounts for the actions of this hormone and of related peptides such as amylin and CGRP at the cellular level. Similar pathways may contribute to shorter range modulation of osteoclast activity by increases in ambient Ca2+. Finally, there is recent evidence for a contribution of endothelial cell-derived product to osteoclast regulation.

Animals↗
PubMed1993

Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases.

OBJECTIVES: To estimate the extent of genomic DNA damage and killing of lymphocytes by reactive oxygen intermediates in autoimmune diseases. METHODS: 8-Oxo-7-hydrodeoxyguanosine (8-oxodG), a promutagenic DNA lesion induced by reactive oxygen intermediates, was measured by high performance liquid chromatography, coupled with electrochemical detection, in hydrolysates of DNA which had been extracted from lymphocyte and polymorphonuclear leucocyte fractions of human blood. In addition, human primary blood lymphocytes stimulated by concanavalin A were assayed for cytotoxicity induced by hydrogen peroxide on day 0, by assessing cell proliferation during seven days of culture. RESULTS: Constitutive 8-oxodG was detectable (mean (2 SEM) moles 8-oxodG/10(6) moles deoxyguanosine) in DNA isolated from normal human blood lymphocytes (68 (8), n = 26) and polymorphonuclear leucocytes (118 (24), n = 24). Lymphocyte DNA from donors with the following inflammatory autoimmune diseases contained significantly higher levels of 8-oxodG than that from healthy donors: rheumatoid arthritis (98 (16)), systemic lupus erythematosus (137 (28)), vasculitis (100 (32)), and Behçet's disease (92 (19)). Lymphocyte 8-oxodG levels in non-autoimmune controls and patients with scleroderma were not significantly different from those of healthy controls. The levels of 8-oxodG were significantly higher in the DNA from normal polymorphonuclear leucocytes than in paired DNA samples from normal lymphocytes, but there were no differences between levels of 8-oxodG in polymorphonuclear leucocytes from normal subjects and the patients studied. Levels of 8-oxodG did not correlate with disease duration, disease severity, or age. Lymphocytes from patients with systemic lupus erythematosus and rheumatoid arthritis, but not those with scleroderma, also showed cellular hypersensitivity to the toxic effects of hydrogen peroxide. CONCLUSION: There was increased genomic DNA damage, and increased susceptibility to cytotoxic killing by hydrogen peroxide, in lymphocytes from patients with certain autoimmune diseases. These results might be explained by defective repair of DNA damage or by increased production of reactive oxygen intermediates in inflammation. Although more direct studies are needed, the evidence available favours the former explanation.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis.

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.

Arthritis, Rheumatoid↗
PubMed1993

High field proton NMR investigations of the metabolic profiles of multidrug-sensitive and -resistant leukaemic cell lines: evidence for diminished taurine levels in multidrug-resistant cells.

High field proton (1H) nuclear magnetic resonance (NMR) spectroscopy has for the first time been employed to investigate and compare the metabolic profiles of vinblastine-sensitive and -resistant T-lymphoid leukaemic cell lines (CCRF-CEM and CEM/VLB100 respectively) and evidence is presented for a significantly lower taurine content in the CEM/VLB100 resistant subline when expressed relative to that of its drug-sensitive parental counterpart. These data suggest differences in the nature and relative involvements of taurine biosynthetic pathways between the two cell lines, a phenomenon that may be related to their differing sensitivities towards chemotherapeutic agents such as adriamycin which promote the generation of cytotoxic reactive oxygen species (ROS) in vivo. However, the 1H NMR data obtained provided no evidence for an increased metabolic consumption of hypotaurine (a metabolic precursor of taurine with powerful .OH radical scavenging properties) in CCRF-CEM cells since differences observed in the hypotaurine: taurine concentration ratio between the drug-sensitive and -resistant cell lines were not statistically significant. Furthermore, hypotaurine is unlikely to compete with alternative endogenous .OH radical scavengers present such as lactate since its level in either of the two cell lines investigated (ca. 6.0 x 10(-8) mol./10(8) cells) is insufficient for it to act as an antioxidant in this context. The biochemical and therapeutic significance of these results are discussed.

Antineoplastic Agents↗
PubMed1993

Assessment of a human recombinant manganese superoxide dismutase in models of inflammation.

We evaluated a novel human recombinant preparation of manganese superoxide dismutase (MnSOD) for anti-inflammatory and anti-oxidant activity compared with a copper zinc (CuZn) SOD preparation. The results showed that administration of MnSOD (50, 100 and 200 micrograms kg-1) in the Freund's Complete Adjuvant (FCA) mediated paw oedema model suppressed the inflammation at 4 hours by 43, 25 and 43% (P < 0.001, P < 0.01 and P < 0.001 at respective doses). However, 24 hours post-challenge, MnSOD (50 and 100 micrograms kg-1), suppressed the inflammation by 19% (P < 0.001). In contrast, Mn SOD at higher doses (400-800 micrograms kg-1; 2 mgkg-1) exacerbated the inflammatory response at 4 hours. This pro-inflammatory response declined progressively by 24 hours. Furthermore, CuZn SOD produced no significant effects on the inflammatory response. In the carrageenan-induced synovitis model, Mn SOD (25 and 50 micrograms; intra-articular administration) exacerbated the inflammation at 48 hours. In contrast, Mn SOD at 5 micrograms produced a significant suppression (44%, P < 0.05) in knee joint swelling at 24 hours. The CuZn SOD preparation produced marked pro-inflammatory effects in the joints whilst it lacked activity in the FCA-mediated paw oedema model. These findings support a therapeutic potential of MnSOD in inflammatory disorders, however the compound has a complex pharmaco-dynamic profile.

Animals↗
PubMed1993

Microvascular substance P binding to normal and inflamed rat and human synovium.

The regulatory peptide substance P has been implicated in the development and persistence of inflammatory synovitis. The authors used quantitative in vitro receptor autoradiography to compare synovial binding of 125Iodine-Bolton Hunter-labeled substance P ([125I]BH-SP) in rats and humans and between uniflamed and persistently inflamed synovium. [125I]BH-SP binding to microvascular endothelium paralleled the distribution of substance P-immunoreactive nerves and had characteristics of the neurokinin (NK) 1 class of tachykinin receptor. Specific binding was inhibited by the selective NK1 receptor antagonist, FK888, and the dual NK1/NK2 receptor antagonist FK224, with Hill coefficients near unity. FK888 was > 1000 times and FK224 > 10 times more potent at inhibiting binding in human compared with rat synovium. Synovium from patients and rats with chronic arthritis contained heterogeneously distributed inflammatory cell infiltrates. For the 10 microvessels with the densest [125I]BH-SP binding in each section, no significant differences in binding density, affinity, or Ki values for substance P, FK888 or FK224 were found between synovium from naive and monoarthritic rats, nor between that from patients with rheumatoid arthritis or osteoarthritis. However, in both rat and human specimens, microscopic examination suggested that microvascular [125I]BH-SP binding in intensely infiltrated regions of synovium was less dense than in adjacent, less infiltrated areas. It was concluded that NK1 receptors are similarly distributed in rat and human synovium but show major differences in selectivity for antagonists such as FK888. NK1 receptors in synovium may mediate proinflammatory actions of locally released substance P; defective neurovascular regulation may contribute to the persistence of chronic arthritis.

Adult↗
PubMed1993

Neuropeptide degrading enzymes in normal and inflamed human synovium.

Regulatory peptides, including neuropeptides, are metabolized by membrane-bound peptidases. We have localized the membrane peptidases angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPPIV), and aminopeptidase M (APM) in normal and inflamed human synovium by immunohistochemistry and enzyme histochemistry. ACE was localized to endothelial cells of all vessels, whereas endothelial DPPIV and APM were restricted to veins and capillaries of some cases. Perivascular spindle-shaped cells stained positively for APM, but rarely for ACE and DPPIV. Synovial lining cells were universally positive for APM but rarely for ACE or DPPIV, whereas a subintimal layer of spindle-shaped cells was frequently positive for all three enzymes, particularly APM. Staining for each enzyme was also observed on some stromal cells. Inflamed synovium displayed increased cellularity with corresponding increases in membrane peptidase staining. APM-positive, perivascular spindle-shaped cells and DPPIV-positive lymphocytes frequently bore HLA class II antigens. This distribution of membrane peptidases supports the hypothesis of a functional compartmentalization of vascular peptidergic systems, with the activities of vasoactive peptides localized to their sites of release. Furthermore, metabolic pathways for regulatory peptides may vary between different structures within human synovium, and these enzymes may be of different relative importance in normal and inflamed tissues. The implied regional control of regulatory peptide activity by membrane peptidases suggests novel potential approaches to the pharmacological manipulation of inflammation by specific enzyme inhibitors.

Adolescent↗
PubMed1993

Species and tissue specificity of vasoactive regulatory peptides.

Peptide regulatory systems are increasingly being implicated in inflammatory processes through their actions on vascular function. Peptides released by sensory nerves (substance P and calcitonin gene-related peptide) and autonomic nerves (neuropeptide Y), or generated by endothelium (angiotensin II) or injured tissues (bradykinin), regulate vascular tone, permeability and proliferation. Peptide systems comprise multiple components responsible for generation, release and metabolism of peptides, as well as receptor expression, combinations of which act together to produce a particular biological effect. Each of these components may vary between species, between tissues and during disease, and the particular combination of these components contributes to the specificity of peptide regulation of tissue function. Understanding the species and tissue specificity of peptide-induced responses is important in developing animal models of human disease and in developing drugs for subsequent use in man. In this paper we review some of the evidence for and implications of species and tissue specificity of vasoactive peptide systems.

Animals↗
PubMed1993

Antioxidants as antirheumatics.

Reactive oxygen species (ROS) are generated by activated leukocytes and during ischaemia-reperfusion damage, such as that which occurs in the joint during exercise. Activities of endogenous scavengers in the synovial fluid are insufficient to deal with this excessive oxidative stress. Therapy with naturally occurring antioxidants, however, has been disappointing. This is due predominantly to poor pharmacokinetics. Studies on traditional anti-rheumatic drugs and the actions of novel compounds suggest that targeting to the joint coupled with defined mechanisms of action may lead to the development of effective antioxidant antirheumatics.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1993

An imaginative approach to synovitis--the role of hypoxic reperfusion damage in arthritis.

The rheumatoid joint is hypoxic. The loss of the physiologic defense mechanism, reflex muscle inhibition, allows the generation of high intraarticular pressures, particularly during exercise. Hypoxia alters the biochemistry of the synovium and encourages the production of reactive oxygen species (ROS) on reperfusion of blood. In excess, ROS damage tissues, and the products of oxidative damage are detectable in rheumatoid synovial fluid. In addition to damaging proteins, carbohydrates and lipids, cellular and structural damage also occurs.

Arthritis↗