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Biomedical subjects

J Lunec

Publications and source records attributed to J Lunec.

At least 163 records · Page 9Linked to original sources

An automated method for the kinetic measurement of ferroxidase activity.

The oxidase activity of caeruloplasmin towards Fe II (ferroxidase) appears to be of physiological significance both in iron metabolism and as a serum anti-oxidant. We have developed an automated assay for ferroxidase using a centrifugal analyser. The method is quick and precise, and is not subject to interference by haemolysis, jaundice or lipaemia. Ferroxidase activities correlate significantly with caeruloplasmin and copper concentrations. Normal reference ranges have been measured.

Adult↗

Oxygen free radicals denature human IgG and increase its reactivity with rheumatoid factor antibody.

Rheumatoid inflammation is characterised by the production of rheumatoid factor antibodies directed against denatured IgG. Oxygen free radicals have the potential to denature all manner of proteins and can be generated by activated phagocytic cells in the inflamed joint. By modifying routine ELISA and nephelometric procedures for measuring rheumatoid factor, (i.e. substituting free radical altered IgG for rabbit and heat aggregated IgG as antigens) we have observed that oxygen radicals, generated by (1) UV light and (2) PMA-activated neutrophils, give rise to monomeric and polymeric forms of IgG which have increased reactivity towards IgM and IgA polyclonal rheumatoid factor antibodies. We conclude that free radical alteration of IgG may be a stimulus to the formation of immune complexes with rheumatoid factor antibody, thereby promoting and amplifying tissue damage during rheumatoid inflammation.

Antibodies↗

Effect of polymorph derived oxidants on IgG in relation to rheumatoid factor binding.

UNLABELLED: Immunoglobulin G from rheumatoid patients is denatured around the hinge region. This has been proposed as an explanation for the presence of circulating autoantibodies to IgG in these patients. It has previously been suggested that oxygen radicals (OR) derived from activated polymorphs may play a role in denaturation in vivo. Using sera from rheumatoid patients and age-matched controls in a modified ELISA technique, we have investigated the potential for polyclonal rheumatoid factors (RF) to bind to OR denatured IgG. Three model systems were used to generate OR in vitro: (a) purified PMN s activated by the cell surface stimulant PMA, (b) radiolysis of IgG in solution to generate specifically the superoxide radical and, in a separate system, the hydroxyl radical, (OH.), (c) purified myeloperoxide in the presence of H2O2 and halide ions. RESULTS: 1. The binding of both IgA and IgM RF s to PMN denatured IgG increased dose dependently for seropositive sera only. 2. The OH. radical but not the superoxide radical significantly increased the binding of IgA and M RF, again only for seropositive sera. 3. The myeloperoxidase enzyme system did not increase RF binding. 4. IgG incubated with elastase was not found to be a better antigen than native IgG. These results indicate that IgG is denatured by OR released from activated PMN, thereby producing an antigen for polyclonal RF s.

Arthritis, Rheumatoid↗

Effect of gliclazide on thromboxane B2, parameters of haemostasis, fluorescent IgG and lipid peroxides in non-insulin dependent diabetes mellitus.

Abnormalities of platelet aggregation, and increased levels of lipid peroxides and denatured proteins have been implicated in the pathogenesis of diabetic microangiopathy. Seventeen non-insulin dependent diabetics (mean age 65.7) on established sulphonylurea therapy were converted to an equivalent dose of gliclazide. They were studied at baseline and intervals to 36 weeks. Thromboxane B2 (TXB2) and beta thromboglobulin (beta TG) were measured by radioimmunoassay. Lipid peroxides were measured as total diene conjugates (DC) by scanning UV spectrophotometry and thiobarbituric acid reactivity (TBA) by absorbance at 532 nm. Oxidation products of proteins were represented by fluorescence/UV ratio of immunoglobulin G (*IgG) separated from serum by HPLC. Glycaemic control remained stable throughout the study. TXB2 fell from 734 +/- 233 ng/l at 0 weeks to 488 +/- 155 at 36 weeks, p less than 0.001 and beta TG from 120 +/- 70 ng/ml to 48 +/- 18 at 24 weeks, p less than 0.005. Lipid peroxides fell between 0 and 36 weeks; DC from 0.71 +/- 0.28 to 0.32 +/- 0.09, p less than 0.001 and TBA from 29.6 +/- 22.4 to 4.0 +/- 2.2, p less than 0.001. Fluorescent IgG ratio fell from 1.44 +/- 0.36 to 0.32 +/- 0.07, p less than 0.001. This data provides evidence for a specific action of gliclazide on thromboxane synthesis and platelet aggregation. This is independent of glycaemic control and may in part be mediated by the observed fall in lipid peroxides.

Aged↗

Serum antioxidant activity in diabetes mellitus.

Free radical medicated oxidative damage has previously been implicated in the pathogenesis of diabetic microangiopathy. Caeruloplasmin and transferrin act as antioxidants in serum by oxidizing and chelating ferrous iron which could otherwise act as a catalyst in free radical reactions. We have measured the serum anti-oxidant activity in 67 diabetics, 25 of whom had retinopathy and in 37 controls. Serum iron concentrations were normal in diabetics in comparison with controls (21.5 +/- 10 v 19.5 +/- 6.7 mumol/L) although transferrin and iron binding capacity were increased in the diabetics (3.7 +/- 0.8 v 2.9 +/- 0.9 g/L, p less than 0.001 and 69.9 +/- 10.8 v 59.0 +/- 14 mumol/L, p less than 0.002 respectively). Caeruloplasmin, measured by its ferroxidase activity, was increased in both male (46 +/- 11 v 36 +/- 9 mu/mL, p less than 0.002) and female diabetics (57 +/- 13 v 47 +/- 11 mu/mL, p less than 0.002). Ferritin was also increased in both male (124 +/- 113 v 44 +/- 38 ng/mL, p less than 0.001) and female diabetics (132 +/- 118 v 25 +/- 12 ng/mL, p less than 0.001). The presence of retinopathy, the degree of glycaemic control and duration of diabetes had no effect on these findings. We conclude that increased concentrations of iron-oxidizing and iron-binding proteins occur in diabetic serum, and that the increased serum antioxidant activity may be a response to oxidative stress.

Aged↗

Evidence of enhanced lipid peroxidation in the cerebrospinal fluid of patients taking phenothiazines.

Products of lipid peroxidation in the cerebrospinal fluid are found in higher concentrations in patients receiving phenothiazines than in control subjects matched for age and sex. Especially high concentrations are found in patients experiencing ill-effects from phenothiazine therapy. Raised concentrations of cerebrospinal fluid "phenanthroline copper" in these patients suggest that metal-catalysed mechanisms may promote the lipid peroxidation. It is suggested that lipid-soluble antioxidants, such as vitamin E, may be useful for the prevention and treatment of phenothiazine side-effects.

Adult↗

Raised cerebrospinal-fluid copper concentration in Parkinson's disease.

The cerebrospinal-fluid copper concentration, measured by electrothermal atomisation/atomic absorption spectrophotometry, was significantly higher in 24 patients with untreated, idiopathic Parkinson's disease than in a control population of 34 patients (p less than 0.001). The difference in the in-vitro capacity of copper to damage DNA, measured by the phenanthroline assay was even greater. The high phenanthroline-copper concentration correlated with disease severity (p = 0.02) and with the rate of progression of disease (p less than 0.05). A possible role is suggested for copper-catalysed oxidative mechanisms in the pathogenesis of Parkinson's disease.

Adult↗

Mechanism of exacerbation of rheumatoid synovitis by total-dose iron-dextran infusion: in-vivo demonstration of iron-promoted oxidant stress.

The mechanism by which a synovial flare occurred in a patient with rheumatoid arthritis after intravenous infusion of iron-dextran was investigated. After the infusion, serum and synovial-fluid iron-binding capacity became saturated, giving rise to low-molecular-mass iron chelates with the capacity to cause oxidative damage ("bleomycin-iron"). At the same time lipid peroxidation and the concentration of oxidised ascorbic acid (dehydroascorbate) increased in both serum and synovial fluid, and red-cell glutathione fell. These changes corresponded closely to an exacerbation of rheumatoid synovitis. Hepatic function was transiently disturbed 7 days after the infusion, reflecting hepatic oxidant stress within the iron-loaded liver. Such changes provide clear evidence that iron-catalysed oxidative reactions influence the inflammatory process in human beings.

Arthritis, Rheumatoid↗

Immunogenic and antigenic epitopes of immunoglobulin--XX. Denaturation of human IgG3 by free radicals.

It has previously been demonstrated that exposure of polyclonal IgG to free radicals results in denaturation evidenced by aggregation, auto-fluorescence and destruction of cysteine, proline and aromatic amino acids. In the present study we have used a panel of monoclonal antibodies (McAb) to epitopes expressed on the IgG3 heavy chain to detect changes in antigenicity. When IgG3 was exposed to u.v. irradiation, as a source of free radicals, subclass specific epitopes were rapidly lost whilst other epitopes were unaffected. Prolonged exposure resulted in further denaturation and a progressive loss of expression of further epitopes. The IgG3 subclass specific McAb are specific to epitopes localized to the hinge region of IgG3. Thus, this exposed cysteine and proline rich region is shown to be particularly vulnerable to free radical attack; however, prolonged exposure results in structural alterations throughout the heavy chain.

Chromatography, High Pressure Liquid↗

Reactive oxygen species selectively deplete normal T lymphocytes via a hydroxyl radical dependent mechanism.

Chronic inflammatory synovitis is characterized by both lymphocytic infiltrates and persistent polymorph exudates. Activated polymorphs release reactive oxygen species (ROS) during inflammation, but the contribution that these make to the lymphocyte abnormalities associated with RA has been little studied. We therefore investigated the cytotoxic effects of the reactive oxygen species on human peripheral blood mononuclear cells (PBMC). PBMC were exposed to RPMI 1640 medium previously irradiated for up to 60 min. Consistent dose-dependent killing was observed at 24 h. Antioxidant studies indicated that H2O2 was the effective species. Catalase, which specifically degrades H2O2, gave almost total protection against cell death, while superoxide dismutase (SOD), thiourea, and mannitol were largely ineffective. Addition of exogenous H2O2 caused an identical pattern of cell death to that observed with irradiated medium. PBMC cultures supplemented with desferrioxamine (a ferric iron chelator) also gave significant protection, suggesting that H2O2 mediated its effects via OH radicals. Analysis of lymphocyte subpopulations showed that ROS caused a selective depletion, depending on the level of H2O2 present. Low levels induced a specific loss of CD8+ cells, while higher concentrations caused significant loss of CD4+ T cells as well. sIg+ B cells were unaffected at either concentration. This selective lymphotoxic effect of ROS may be of considerable importance in the pathogenesis of autoimmune inflammatory disease.

Adult↗

Effect of free radical altered IgG on allergic inflammation.

The rheumatoid synovium is capable of producing large amounts of IgG which may become modified by the actions of free radicals. A rat model of synovitis was established and challenged with both normal and free radical altered IgG. IgG was prepared from homologous pooled serum by high performance liquid chromatography, and free radical damage was induced by 15 minutes ultraviolet (UV) irradiation. The results showed a worsening in gross assessments of inflammation, increases in biochemical indices of lipid peroxidation, and also a rise in the proportion of IgG which, on reisolation, showed the characteristic fluorescence associated with free radical damage. This demonstrated how the presence of free radical altered IgG might convert an inflammatory insult to a more persistent stimulus, and the capacity of an environment subjected to continuing antigenic stimulation to induce further free radical damage to IgG.

Animals↗

Renal impairment associated with non-steroidal anti-inflammatory drugs.

We have compared the renal function on admission and discharge of 22 patients routinely admitted to our rheumatology ward. None had previously diagnosed renal failure. Of 11 patients in whom we stopped long term non-steroidal anti-inflammatory drug (NSAID) therapy, all showed a rise in creatinine clearance (Ccr) after three to 28 days. In contrast, a control group comprising 11 similar patients who continued to receive NSAIDs showed no significant change. Recent work has suggested that it is possible to identify patients at risk of developing nephrotoxic side effects with NSAIDs. Based on these criteria (but excluding age alone as a risk), six patients from the first group and 10 from the second group would have been without risk. We infer from this that asymptomatic, reversible impairment of renal function is common, and that the potential clinical benefit from the use of NSAIDs should be balanced against this predictable toxicity.

Adult↗

Macrophage ferritin and iron deposition in the rat air pouch model of inflammatory synovitis.

Using a rat air pouch model of red cell promoted allergic inflammation, we have investigated the relation between ferritin synthesis and iron deposition in pouch wall lining cells. These cells have structural and immunohistochemical similarities to human synovial intimal cells and studies of them are pertinent to the clinical situation. In control air pouch wall tissue after single or double antigenic challenge the (apo)ferritin containing macrophages are most numerous seven days after antigenic challenge when there is active connective tissue proliferation and a generalised mononuclear cell response in the pouch wall, suggesting that (apo)ferritin is produced in macrophages as part of the tissue inflammatory response. In contrast with control tissue, where there is a steady decrease in positive cells over the ensuing weeks, injection of blood into both single and double challenge air pouches produces a significant (p less than 0.001) and continuing rise in the numbers of ferritin containing macrophages after day 7. Also, after 14 days Perls' positive ferric iron is detectable in increasing numbers of ferritin containing macrophages, a trend which is more marked in double challenge, blood injected air pouches. The histological data clearly show that there is a close relation between the presence of Perls' iron and proliferation of vascular and connective tissue elements in the pouch wall. We propose that this proinflammatory role of iron is the result of its ability to promote oxidative damage in tissues, and discuss ways in which this may take place.

Animals↗

Enhanced-latex-agglutination assay for C-reactive protein in serum, with use of a centrifugal analyzer.

This is an improved assay of C-reactive protein in serum, for use with the Baker "Encore" centrifugal analyzer. Features of this assay include: 250-specimen throughput per hour, within-batch CV 2.2%, between-batch CV 2.7%, no antigen-excess problems up to 1000 mg/L, negligible interference from rheumatoid factor, and good correlation (r = 0.99) with radial immunodiffusion. The method is inexpensive and automated, involving no predilution steps. It can be adapted for use in a wide range of systems and can be used for single urgent estimations.

Autoanalysis↗

Protein fluorescence and its relationship to free radical activity.

Evidence for free radical involvement in pathological processes is often indirect and frequently depends upon the detection of characteristic changes in tissue constituents, particularly polyunsaturated lipids. Free radical attack on protein leads to specific oxidative changes in their constituent amino acids, principally cysteine, tryptophan and tyrosine. This is associated with the induction of characteristic fluorescence (excitation 360 nm, emission 454 nm) and protein aggregation. This observation leads to a suitable assay for studying protein oxidation, and such fluorescent proteins may be relevant in the pathogenesis of diabetic microangiopathy and rheumatoid arthritis.

Arthritis, Rheumatoid↗

Vitamin C metabolites and microangiopathy in diabetes mellitus.

Recent evidence has suggested that diabetic microangiopathy is associated with increased free radical induced oxidative damage. Ascorbic acid (AA) is a free radical scavenger and using a specific HPLC method we have investigated its concentration and that of its oxidized metabolite dehydroascorbic acid (DHAA) in diabetic patients and matched normal controls. The findings have been related to the presence of microangiopathy and to glycaemic control. Ascorbic acid levels were significantly lower in diabetics (mean +/- SD 42.5 +/- 26.2 mumol/l) compared with controls (58 +/- 21 mumol/l p less than 0.02). Although there was no differences in DHAA levels between the groups the ratio DHAA/AA was increased in diabetics (0.72 +/- 0.8) compared with controls (0.4 +/- 0.2 p less than 0.05). There were no significant differences between insulin and non-insulin dependent patients in these measurements and there was no association with the presence of microangiopathy or poor glycaemic control. The plasma ratio DHAA/AA may be a reflection of increased oxidative stress and our results suggest that diabetics may be less able to prevent oxidative damage occurring due to their lower AA concentrations.

Ascorbic Acid↗