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We report the effects of oxidative stress generated by low-intensity u.v. irradiation (366 and 254 nm), dialysis against ascorbate and isolated stimulated neutrophils on some physicochemical properties of caeruloplasmin. Low-intensity u.v. irradiation resulted in a loss of ferroxidase activity and 610 nm absorption, changes previously reported to occur during storage and manipulation of caeruloplasmin. These alterations were found to correspond to aggregation of the protein, induction of visible fluorescence (excitation, 360 nm; emission, 454 nm), changes in c.d. spectra which were indicative of alterations in protein conformation, loss of half-cystine, tryptophan and tyrosine content and loss immunoreactivity. The changes in the far-u.v. c.d. spectrum of caeruloplasmin were more pronounced than those observed for u.v.-irradiated IgG. Similar c.d. changes and induction of fluorescence were observed following dialysis of caeruloplasmin against ascorbate or exposure to stimulated neutrophils. It is concluded that the lability of caeruloplasmin may arise from oxidative modification, in addition to the previously described susceptibility of this protein to proteolysis.

Ceruloplasmin (CP) is a blue copper oxidase found in human serum. It has been suggested that CP functions as an antioxidant by virtue of its ability to scavenge the superoxide anion radical1 and/or its ability to catalyse the conversion of Fe(II) to Fe(III), thereby inhibiting the Fenton reaction2. However, the true physiological role of CP is still not clear. In attempting to define such a role it is important to recognise the relative lability of this protein3. Many of the changes in the physicochemical properties of CP3 which occur during isolation and storage can be ascribed to proteolysis3. However, this does not appear to account for all of the modifications which occur during the ageing of CP preparations.

Seven patients with rheumatoid disease were given the iron-chelating drug desferrioxamine (DFX) to evaluate its possible anti-inflammatory effects. Two of these patients, who also received the anti-emetic prochlorperazine, lost consciousness for 48-72 h and then fully recovered. Electroencephalography showed abnormalities of the type associated with metabolic disturbance. One of these patients showed pyramidal features and subsequently developed an optic neuropathy and pigmentary retinopathy. Analysis of his cerebrospinal fluid showed a decrease in loosely-bound (catalytic) iron and increase in loosely-bound (catalytic) copper, total iron and products of lipid peroxidation, with values approaching normal as the symptoms resolved. Subsequent in vivo/vitro studies clearly demonstrated that the neurological effects were due to a synergistic action of desferrioxamine and prochlorperazine, probably resulting in exceptional fluxes of intra/extra cellular iron/copper disturbing noradrenergic and serotonergic systems. Two other patients who did not receive prochlorperazine, developed retinal problems which later improved, one after only 15 g of desferrioxamine. Our observations suggest a new model for metabolic encephalopathy studies and provide insight into the mechanisms of pigmentary retinopathy.

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.

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 μM) whilst this effect was inhibited by the iron chelator desferrioxamine. Ascorbate also showed an antioxidant activity at high concentrations (125–250 μM) Our results suggest that the combination of ascorbate with ferritin may supply free iron for LDL oxidation in vivo.

Revue du role des radicaux libres dans la synovite. Definition. Production de radicaux libres chez le sujet sain; role fondamental dans les reactions d'oxydation. Discussion de leur place exacte dans la genese des lesions tissulaires dans les maladies articulaires inflammatoires (modeles animaux, homme)

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.

OBJECTIVE--To assess whether the extent of LDL oxidation influences its cytotoxic effects, thus contributing to its atherogenic potential. DESIGN AND SETTING--The effects of native and modified LDL on cultured human coronary artery smooth muscle cells (SMC) and endothelial cells (ECs) were investigated. MAIN OUTCOME MEASURES--Four indices of cytotoxicity were studied: (i) chromium-51 release; (ii) 5-bromo-2'-deoxyuridine (BrDUrd) uptake; (iii) morphological appearance; and (iv) EC migration. RESULTS--(i) Minimally modified (mm) LDL (400 micrograms/ml) causes significant 51Cr release; the cytotoxic effect was significantly greater for copper oxidised (ox) LDL (400 micrograms/ml). Native LDL had no effect. (ii) BrDUrd uptake studies showed significant inhibition of cell proliferation by 100 micrograms/ml of oxLDL and to a lesser extent by mmLDL; native LDL had no effect. (iii) Morphological appearance was not altered by native LDL. Changes in cell morphology were induced by mmLDL (400 micrograms/ml), and were more pronounced with oxLDL in concentrations of > or = 200 micrograms/ml. (iv) EC migration was significantly inhibited by oxLDL (100 micrograms/ml), but not by native or mmLDL. CONCLUSION--The extent of oxidation of LDL determined its cytotoxicity to coronary artery cells. Native LDL had no cytotoxic effect. In contrast, oxLDL and to a lesser extent mmLDL caused cytotoxicity at concentrations to which cells in vivo might be exposed. This may contribute to the atherogenicity of modified LDL by enhancing cellular injury and inflammation, and by inhibiting re-endothelialisation of areas of coronary artery damaged during the atherogenic process.

Three patients with rheumatoid disease were given the 'iron chelating' drug desferrioxamine (DFX), which also has an appreciable affinity for copper. The drug was injected cautiously, in lower doses than in patients with thalassaemia, and intramuscularly to evaluate its anti-inflammatory effects. Two of the three patients developed ocular abnormalities. One patient, who also received methyldopa, developed severe but reversible visual failure associated with an abnormal electro-oculogram (EOG); another showed reversible depression of the EOG. Analysis of the cerebrospinal fluid (CSF) of this patient showed an increase in phenanthroline detectable (non-caeruloplasmin-bound) copper. Analysis of the CSF of the third patient, who did not develop any clinical or electrophysiological ocular abnormalities, was normal. Haematological assessments indicated that all three patients probably had reduced iron stores. With in-vitro systems DFX was shown to mobilise copper from albumin and to facilitate copper movement across a cell membrane model, a property that was enhanced by methyldopa. Our observations are consistent with the concept that in rheumatoid patients low iron stores may result in binding of copper by DFX and that this may be of central importance in causing the ocular toxicity of DFX.

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.

It was observed that during the storage of human extracellular fluids at -20 degrees C the azide-inhibitable ferroxidase activity of caeruloplasmin declined, whilst a new azide-resistant ferroxidase activity (ARFA) developed. The literature suggested that storage-induced ARFA might be due to either a poorly defined enzymatic activity of a low density lipoprotein (LDL) or to lipid peroxides formed within the different lipoprotein fractions. To study this further, the major lipoprotein classes were separated from human serum by density gradient centrifugation. After storage of the lipoprotein fractions, it was found that the LDL fraction had the highest specific activity of ARFA and the highest content of lipid peroxidation products, as assessed by diene conjugates. The ARFA of LDL correlated with its content of diene conjugates and TBA reactive material, which initially suggested that the Fe(II) oxidising activity of peroxidase LDL arose from the reduction of peroxides by Fe(II) in the classical reaction between the metal ion and free radical reduction of lipid peroxides. However, steady state kinetic analysis indicated an enzymic role of LDL in Fe(II) oxidation, with lipid peroxides acting as a substrate for the enzyme. These results indicate that LDL may contain a peroxidase activity, catalysing the oxidation of Fe(II) by lipid peroxides, as well as a ferrous oxidase activity where O2 is the oxidising substrate.

Introduction: Rheumatoid arthritis (RA) is considered a T cell driven autoimmune disease, therefore, the ability of B cell depleting biologics, e.g., anti-CD20 antibodies, to alleviate RA is unclear. This study examined the proportions of IL-17-secreting lymphocytes in the blood of healthy subjects and RA patients and determined if Th17 cells belong to a CD20+ subset of T cells.Methods: Fluorescence-activated cell sorting and confocal microscopy verified CD3, CD4/CD8 and CD20-staining of T cells. IL-17 secretion was determined using a commercial assay.Results: In healthy subjects and RA patients blood, the median percentage of total CD20+ lymphocytes was similar (7.5%; n = 6 and 10.3%; n = 9, respectively) and comprised predominantly of B cells (similar to 86%). However, 2-4% of CD3+ T cells from both healthy subjects (n = 7) and RA (n = 8) individuals co-expressed CD20. The peripheral blood of healthy subjects contained few IL-17-secreting CD20+ T cells (Conclusions: In the blood of RA patients, a greater proportion of Th17 cells are of a CD20+ phenotype compared to healthy individuals. These cells may represent an additional target for anti-CD20 therapies.

International audience; Furanocoumarins are important plant defense compounds, which are mainly found in four higher plant families: Apiaceae, Moraceae, Fabaceae, and Rutaceae. These molecules constitute a subclass of polyphenolic compounds for which the biogenesis in plants has remained elusive until recently at the molecular level. Over the last decade, the first genes and enzymes involved in furanocoumarin synthesis have been described. As regards the linear furanocoumarin bergapten, starting from the precursor molecule p-coumaroyl CoA, we now have three different enzymatic steps (2 cytochrome P450 enzymes and one dioxygenase) out of a total of five that have been unraveled so far. With the exception of one enzyme that was characterized in Rutaceae, all other enzymes have been isolated from Apiaceae plants. The isolation of orthologous genes from other plant families is now under way and will likely reveal whether furanocoumarins appeared in an ancestor phylum or if they emerged independently over different plant taxonomical groups.