Tumor Necrosis Factor: Function, Release and Clearance.

Tumor Necrosis Factor (TNF) is a multifunctional cytokine. It plays an important role in the pathophysiology of several diseases. Recently, it has been discovered that TNF is circulating in two different forms, a bioactive form and an immunologically detectable form. These two forms of TNF show different clearance kinetics. The immunological form is supposed to be an inactivated TNF protein. For this inactivation, proteolytic degradation or TNF binding by inactivating proteins is necessary. In this review we have focused on TNF inactivation by TNF binding proteins. Recent data show that there are soluble TNF receptors circulating which can bind and inactivate TNF. These receptors are membrane-bound TNF receptors which have been proteolytically cleaved from the cell membrane. Two TNF receptors are circulating, the soluble TNF receptor of 55 kDa (P55) and the receptor of 75 kDa (P75). The receptors are held responsible not only for inactivation of the TNF, but also for the clearance of TNF. Recent data show that the kidney is the most important organ for TNF clearance, followed by the liver. All other organs are of less importance. In this review, function, release, and clearance of TNF are discussed.

Autoantibodies against oxidized low-density lipoprotein and lipid profile in patients with chronic periaortitis: case-control study.

Chronic periaortitis is thought to result from an autoallergic reaction to oxidized low-density lipoprotein (OxLDL). No data exist on lipid profile and atherosclerotic biomarkers. We investigated circulating levels of OxLDL and of anti-OxLDL (aOxLDL) antibodies in patients with chronic periaortitis using the cross-sectional case-control study on 20 patients with chronic periaortitis. Patients were compared to 20 age- and sex-matched controls. aOxLDL antibodies were measured by ELISA and expressed as mean optical density values at 450 nm from duplicate measurements (OD(450)). aOxLDL antibody titers (median [interquartile range]) did not differ significantly between patients and controls (aOxLDL-IgM: 0.70 [0.24-1.08] vs. 0.54 [0.25-0.73] OD(450); aOxLDL-IgG: 0.59 [0.38-0.75] vs. 0.41[0.33-0.63]OD(450)). Female patients had higher aOxLDL-IgM levels than male patients (1.02 [0.46-1.38] vs. 0.29 [0.22-0.84] OD(450); P = 0.05). aOxLDL-IgM titers were lower in patients with cardiovascular disease (CVD) than in patients without CVD (0.22 [0.16-0.37] vs. 0.92 [0.70-1.30] OD(450); P = 0.003) and correlated positively with HDL-cholesterol (r = 0.47, 95% CI 0.02-0.69; P = 0.03) and inversely with diastolic blood pressure (r = -0.46, 95% CI -0.75 to -0.01; P = 0.03) and OxLDL/apoB ratio (r = -0.41, 95% CI -0.73 to 0.04; P = 0.06). No differences or associations were found between aOxLDL-IgG titers and other variables between or within patients and/or controls. In patients OxLDL levels correlated with smoking pack-years (r = 0.58, 95% CI 0.17-0.81; P = 0.007). Data suggest a differing innate immune response to OxLDL in patients with chronic periaortitis compared to controls. Whether this response is causally related to chronic periaortitis development remains to be clarified.

Oxidized LDL enhances pro-inflammatory responses of alternatively activated M2 macrophages: a crucial role for Krüppel-like factor 2.

OBJECTIVE: Macrophages are key players in atherogenesis because of their properties to form foam cells that produce a large variety of pro-inflammatory mediators. We addressed the potency of phenotypic different macrophages to accumulate oxidized LDL. METHODS AND RESULTS: Surprisingly, anti-inflammatory M2 macrophages but not pro-inflammatory M1 macrophages rapidly accumulated oxidized LDL. Simultaneously, expression of Krüppel-like factor 2, a nuclear transcription factor known to suppress inflammation in endothelial cells and monocytes, decreased and the functional phenotype of M2 macrophages shifted towards a pro-inflammatory profile, characterized by higher production of IL-6, IL-8 and MCP-1 and lower expression of IL-10 upon stimulation with LPS. In contrast, Krüppel-like factor 2 expression and the phenotype of M1 macrophages remained largely unchanged upon oxidized LDL exposure. Downregulation of Krüppel-like factor 2 expression of M2 macrophages using siRNA technology led to a significant increase of LPS-induced MCP-1 secretion. CONCLUSIONS: We show that (1) anti-inflammatory M2 macrophages are more susceptible to foam cell formation than pro-inflammatory M1 macrophages, (2) exposure to oxidized LDL renders M2 macrophages pro-inflammatory, and (3) Krüppel-like factor 2 is involved in the enhanced secretion of MCP-1 by M2 macrophages loaded with oxidized LDL. The phenotype switch of M2 macrophages from an anti- to a pro-inflammatory profile may play an important role in pathogenesis of atherosclerosis, and could represent a novel therapeutic target.

Impact of waist circumference versus adiponectin level on subclinical atherosclerosis: a cross-sectional analysis in a sample from the general population.

OBJECTIVE: Waist circumference is a clinical marker of obesity and an established risk factor for cardiovascular (CV) disease. Adiponectin, an adipocyte-derived hormone and new biomarker of obesity, was recently proposed as the missing link between obesity and increased cardiovascular risk. We evaluated waist and adiponectin in a middle-aged population-based cohort to compare the impact of both obesity-markers on subclinical atherosclerosis, in relation to other CV risk factors. DESIGN, SETTING & SUBJECTS: Seven noninvasive measurements of atherosclerosis (NIMA), as surrogate markers of (subclinical) atherosclerosis, were determined in 1517 participants of the Nijmegen Biomedical Study, aged 50-70 years, who were drawn from the Dutch community. RESULTS: Both men and women with a high waist (M >104 cm; F >95 cm) showed increased pulse wave velocity (PWV) (M: +9.4%; F: +8.3%) and thicker intima-media thickness (IMT) (M: +7.3%; F: +4.3%) and women also showed increased plaque thickness (+16.6%). After adjustment for other CV risk factors both men and women showed increased IMT (M: +4.8%; F: +2.8%) and men also showed increased PWV (+9.6%). Both men and women with a low adiponectin level (M <2.2 mg L(-1); F <3.5 mg L(-1)) showed a decreased ankle-brachial index after exercise (M: -9.5%; F: -3.9%) and increased IMT (M: +3.7%; F: +3.6%) and women also showed increased PWV (+6.8%), but after adjustment for other CV risk factors low adiponectin level was no longer associated with deteriorated outcomes of NIMA. CONCLUSIONS: Waist circumference showed independent associations with noninvasive measurements of subclinical atherosclerosis, whereas the association of adiponectin level with subclinical atherosclerosis was not independent of other CV risk factors. Prospective studies are needed to elucidate, if the atherogenic effect of a low adiponectin level is mediated by other CV risk factors and not by low adiponectin level intrinsically.

Scavenger receptor class A type I/II determines matrix metalloproteinase-mediated cartilage destruction and chondrocyte death in antigen-induced arthritis.

OBJECTIVE: Scavenger receptor class A type I (SR-AI) and SR-AII are expressed by macrophages in particular and bind and internalize a broad range of molecules (including endotoxins, apoptotic bodies, and oxidized low-density lipoprotein). This study was undertaken to investigate the role of SR-AI/II in mediating severe cartilage destruction in antigen-induced arthritis (AIA). METHODS: AIA was induced in the knee joints of SR-AI/II(-/-) mice and wild-type (WT) controls. Joint inflammation and cartilage destruction (chondrocyte death) were measured by examining the histology of total knee joints. Matrix metalloproteinase (MMP)-mediated neoepitopes were measured by immunolocalization using anti-VDIPEN antibodies and chondrocyte activation with anti-S100A8 antibodies. Messenger RNA (mRNA) levels were determined in inflamed synovium using microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. In synovial washouts, cytokines (interleukin-1beta [IL-1beta], IL-10, and tumor necrosis factor alpha) and S100A8/S100A9 were measured using Luminex and enzyme-linked immunosorbent assay. RESULTS: Levels of SR-AI/II mRNA were strongly elevated in inflamed synovium in AIA. On days 2, 8, and 14 after AIA induction, joint inflammation (exudates/infiltrate) was similar between the 2 groups. In WT mice, severe cartilage destruction was found in multiple cartilage surfaces of the inflamed knee joint on day 14 after AIA induction. MMP-mediated matrix destruction ranged between 40% and 60%, and chondrocyte death was prominent in 40-75% of the cartilage surfaces. In striking contrast, in SR-AI/II(-/-) mice, despite comparable joint inflammation, pronounced cartilage destruction was almost completely absent. Levels of IL-1beta and S100A8/S100A9 were significantly lower on days 7 and 14 after AIA induction, but levels of mRNA for various MMPs (MMP-2, MMP-3, MMP-9, and MMP-13) were comparable. CONCLUSION: Our findings indicate that SR-AI and SR-AII are crucial receptors involved in mediating severe cartilage destruction in AIA.

Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis.

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles (MP) plays an important role in etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells and platelets, exhibits high affinity for PS and has been shown to inhibit several of these PS-mediated pathophysiological processes. We determined plasma annexin A5 levels and MP-associated procoagulant activity, a measure of MP-PS exposure, in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty-five HbAA blood donors served as controls. Both annexin A5 and MP-PS were highest in HbSS patients (5.7 ng/mL, IQR 3.7-7.6 and 37.9 nM, IQR 31.9-69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7-7.6 and 20.9 nM, IQR 10.9-29.6) and healthy controls (2.5 ng/mL, IQR 1.4-4.4 and 13.1 nM, IQR 9.5-18.5) (p=0.01 and p<0.001, respectively). At presentation with a painful crisis, annexin A5 and MP-PS had increased in 16 of 17 patients (p=0.001 and p<0.001, respectively). Most interestingly, in 7 HbSS patients the proportional increase in MP-PS exposure was higher than the proportional increase in plasma annexin A5 concentration, leading to lower annexin A5/MP-PS ratio of HbSS patients during crisis than HbAA controls (0.0027 (0.0017-0.0049) vs 0.0048 (0.0027-0.0085), p=0.05). In conclusion, patients with SCD have elevated plasma levels of annexin A5- and PS-exposing MP. During crisis both levels increase, but in most HbSS patients MP-PS exposure increases more than annexin A5. Future studies must address a potential role of annexin A5 in modulating PS-related pathophysiological processes in SCD.

Anti-inflammatory therapy with tumour necrosis factor alpha inhibitors improves high-density lipoprotein cholesterol antioxidative capacity in rheumatoid arthritis patients.

OBJECTIVE: High-density lipoprotein (HDL) antiatherogenic functions seem to be diminished during inflammatory conditions such as rheumatoid arthritis (RA). The aim of this study was to investigate the effects of tumour necrosis factor (TNF) inhibition on the antioxidative capacity of HDL in RA. METHODS: Plasma lipids and paraoxonase (PON-1) activity were investigated in 45 RA patients, before and during 6 months of anti-TNF therapy. In addition, HDL was isolated and tested for its ability to inhibit copper-induced oxidation of low-density lipoprotein in vitro. RESULTS: Plasma HDL concentrations did not change considerably after 6 months of therapy. However, stable increases of PON-1 activities were observed throughout the same period (p<0.03). The increases were more obvious when related to HDL or apolipoprotein AI concentrations. HDL total antioxidative capacity significantly improved 6 months after the initiation of anti-TNF therapy (p = 0.015). The initial improvement of PON-1 activity paralleled a decrease in the inflammatory status, whereas specific TNF blockade was likely to be responsible for the long-term effects. CONCLUSIONS: Anti-TNF therapy with infliximab has beneficial effects on lipids through changes in HDL antioxidative capacity, which might be clinically relevant and contribute to the reported protective effect of anti-TNF on cardiovascular morbidity in RA. This emphasises the importance of HDL antiatherogenic capacity for cardiovascular risk in chronic inflammatory conditions.

Cholesterol in end-stage renal disease: the good, the bad or the ugly?

The incidence of cardiovascular disease is markedly increased in patients with end-stage renal disease (ESRD). High serum cholesterol is widely recognised as a cardiovascular risk factor in the general population. However, in patients with ESRD high concentrations of cholesterol are associated with a better survival. This reverse epidemiology is, amongst others, caused by confounding due to malnutrition and chronic inflammation. In this population, treatment with statins to lower the serum cholesterol remains a matter of debate. In ESRD, LDL cholesterol is modified by increased oxidative stress. These altered LDL particles play a pivotal role in the development of atherosclerosis. Treatment with the antioxidant vitamin E has not equivocally been shown to be beneficial in this population. This review tries to put data from literature on dyslipidaemia and oxidative stress in ESRD in perspective.

Endothelial function in familial combined hyperlipidaemia.

BACKGROUND: Familial combined hyperlipidaemia (FCH) is characterized by dyslipidaemia, visceral obesity and insulin resistance, and is associated with an increased intima-media thickness (IMT) and an increased risk for cardiovascular disease. In the present study, we investigated whether FCH is associated with early functional vascular wall changes, as represented by endothelial dysfunction, and we determined whether endothelial function in FCH is related to any of the cardiovascular risk factors associated with the FCH phenotype, or to the (increased) IMT. DESIGN: In 98 patients with FCH [mean age 51 (48-54) years, 43% male] and 230 unaffected relatives [mean age 44 (42-46) years, 48% male], venous blood was drawn in the fasting state after discontinuation of lipid lowering drugs for at least 4 weeks (if used). IMT was measured by B-mode ultrasound and endothelial function was assessed by determination of flow mediated dilation (FMD) and by measurement of plasma concentrations of various soluble adhesion molecules, including soluble vascular cell adhesion molecule (sVCAM), soluble intercellular adhesion molecule (sICAM) and soluble E-selectin. RESULTS: There were no significant differences between FCH patients and their non-affected relatives in FMD [2.9 (2.3-3.6%) vs. 2.8 (2.5-3.2%)] or in the plasma concentrations of the various adhesion molecules. None of the individual clinical and biochemical cardiovascular risk factors was an independent predictor of endothelial function in patients with FCH, nor was IMT. However, subgroup analysis revealed that IMT was an independent and powerful predictor of FMD in subjects with carotid artery plaques (St. beta = 4.11, P < 0.004), whereas IMT was no significant predictor in subjects without plaques. CONCLUSIONS: FCH patients have no impaired endothelial function when compared to their unaffected relatives. IMT is an important predictor of FMD when advanced morphological wall changes are present. Our results question the value of FMD measurements for cardiovascular risk stratification in populations with an anticipated high cardiovascular risk.

Non-transferrin-bound iron is associated with plasma level of soluble intercellular adhesion molecule-1 but not with in vivo low-density lipoprotein oxidation.

BACKGROUND: Excess body iron is associated with increased cardiovascular disease risk, possibly via non-transferrin-bound iron (NTBI)-mediated enhancement of inflammation and oxidation of low-density lipoprotein (LDL). METHODS: We assessed this proposed atherosclerotic mechanism of body iron by determining the relationship of levels of serum iron parameters, including NTBI, with plasma markers of inflammation and LDL oxidation in 232 subjects who visited the outpatient clinic for hemochromatosis family screening. RESULTS: Plasma level of soluble intercellular adhesion molecule-1 (sICAM-1) was positively related to ferritin (standardized beta coefficient 0.16) and to NTBI (0.185) and negatively to total iron-binding capacity (TIBC, -0.166). Significant higher levels of sICAM-1 were found for subjects in the highest quartile of NTBI compared to the lowest quartile of NTBI (122 microg/L (107-141) and 106 microg/L (89-125), median (interquartile range), p<0.001). Odds ratio of subjects having sICAM-1 level above 134 microg/L (75th percentile) in the highest and lowest quartile of NTBI amounted 2.3. White blood cell count was positively related to ferritin (0.149). High-sensitivity C-reactive protein, interleukin-6, interleukin-8, oxidized LDL, oxidized LDL/apolipoprotein B and IgG and IgM antibodies to oxidized LDL were not related to any of the markers of iron status. CONCLUSION: Excess body iron, reflected by elevated serum ferritin and NTBI and decreased TIBC, is associated with increased plasma level of sICAM-1 but not with markers of in vivo LDL oxidation.

The story of PON1: how an organophosphate-hydrolysing enzyme is becoming a player in cardiovascular medicine.

Since the discovery of human serum paraoxonase (PON1), the enzyme has been the subject of various fields of research. Initially, PON1 was identified as an enzyme capable of hydrolysing organophosphate compounds, but there is a growing body of evidence that PON1 plays a role in lipid metabolism and the onset of cardiovascular disease. Still, the precise mechanism by which PON1 functions in vivo remains to be clarified. Here we will briefly review developments in the field of PON1 research which merit further attention.

Proportion of oxidized LDL relative to plasma apolipoprotein B does not change during statin therapy in patients with heterozygous familial hypercholesterolemia.

OBJECTIVE: Circulating oxidized low-density lipoprotein (LDL) has been shown to be a useful marker for identifying patients with coronary heart disease (CHD) and persons at high cardiovascular risk. The effect of cholesterol-lowering therapy on plasma level of oxidized LDL is not clear. METHODS AND RESULTS: We investigated effects of cholesterol lowering by therapeutic intervention (2 years) with atorvastatin (80 mg daily) and simvastatin (40 mg daily) on circulating oxidized LDL (absolute level and in proportion to plasma apolipoprotein B) in relation to atherosclerosis progression (carotid intima-media thickness, carotid IMT) and to inflammation (high-sensitivity C-reactive protein, hsCRP) in 115 stable patients with heterozygous familial hypercholesterolemia (FH). Atorvastatin and simvastatin reduced plasma-oxidized LDL (-43 and -35%, respectively) in proportion to the decrease in plasma apolipoprotein B. Neither absolute nor relative level of oxidized LDL correlated with carotid IMT or hsCRP at baseline. Also changes in levels of circulating oxidized LDL were not related to changes in carotid IMT and hsCRP. CONCLUSIONS: In familial hypercholesterolemia-oxidized LDL carried in plasma is strongly associated with apolipoprotein B but not with inflammation nor with carotid IMT, and statin treatment does not reduce oxidized LDL relative to apolipoprotein B.

The effect of statin therapy on plasma high-density lipoprotein cholesterol levels is modified by paraoxonase-1 in patients with familial hypercholesterolaemia.

OBJECTIVES: Statins reduce low-density lipoprotein cholesterol (LDL-C) and can raise high-density lipoprotein cholesterol (HDL-C). HDL-bound paraoxonase-1 (PON1) is associated with variations in plasma HDL-C, and may, therefore, contribute to changes of HDL-C during statin therapy. DESIGN: The effects of baseline PON1 status to HDL-C changes because of statin therapy were investigated. PON1 status was determined with (i) PON1 -107C>T and 192Q>R genotype, (ii) PON1 levels and (iii) PON1 paraoxonase, diazoxonase and arylesterase activity. SETTING: Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands. SUBJECTS: A total of 134 familial hypercholesterolaemia (FH) patients undergoing atorvastatin or simvastatin therapy. RESULTS: PON1 levels and activities significantly modified the HDL-C increment (P=0.002 for PON1 levels and arylesterase activity and P=0.001 for diazoxonase activity). The effects were even more evident amongst subgroup classifications based on PON1 status and baseline HDL-C concentrations: the HDL-C increment was more pronounced in subgroups of -107CT/TT or 192QR/RR genotype combined with low baseline HDL-C (+13.9%, P<0.001, respectively+15.4%, P<0.001). In contrast, the -107CC or 192QQ genotype in combination with high baseline HDL-C, did not show a significant increase of HDL-C. CONCLUSIONS: PON1 status in conjunction with baseline HDL-C levels predicts HDL-C increment during statin therapy in FH patients.

Anti-inflammatory effects of troglitazone in nondiabetic obese subjects independent of changes in insulin sensitivity.

BACKGROUND: Obesity is characterised by insulin resistance and by elevated levels of proinflammatory markers. We investigated whether, in the absence of changes in glucose, thiazolidinediones (TZDs) have anti-inflammatory effects and whether improvement of insulin sensitivity correlates with suppression of inflammatory markers. METHODS: We performed a randomised double-blind placebo-controlled crossover study with troglitazone (400 mg daily for eight weeks) in 15 normoglycaemic obese subjects. We measured plasma high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), leptin, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and tumour necrosis factor-alpha (TNF-alpha) after each of the two treatment periods and in 13 age- and sex-matched lean individuals. RESULTS: Obese subjects were insulin resistant (decreased glucose infusion rate (GIR) during euglycaemic hyperinsulinaemic clamp) and had higher plasma levels of hsCRP, IL-6, leptin, tPA, and PAI-1 compared with lean subjects. TNF-alpha also tended to be higher. Troglitazone improved insulin sensitivity (mean increase in whole body glucose uptake 23.1 +/- 10.5% (p = 0.047)) and normalised plasma concentrations of hsCRP, tPA and TNF-alpha, whereas it did not significantly change IL-6, leptin and PAI-1. Changes in GIR did not correlate with changes in inflammatory markers. CONCLUSION: Troglitazone induces suppression of some of the inflammatory markers that are elevated in normoglycaemic obese subjects. The suppression of inflammatory markers, however, does not correlate with improvement in insulin sensitivity, suggesting involvement of partially differential mechanisms in these effects of TZDs.

Effect of lowering of homocysteine levels on inflammatory markers: a randomized controlled trial.

BACKGROUND: Elevated concentrations of homocysteine and low concentrations of folate may lead to a proinflammatory state that could explain their relation to vascular disease risk. We investigated the effect of lowering homocysteine concentrations by means of folic acid supplementation on markers of inflammation. METHODS: In a double-blind, randomized, placebo-controlled trial among 530 men and postmenopausal women with homocysteine concentrations of 1.8 mg/L or higher (>/=13 micromol/L) at screening, we investigated the effect of folic acid supplementation (0.8 mg/d) vs placebo for 1 year on serum concentrations of C-reactive protein, soluble intercellular adhesion molecule-1, oxidized low-density lipoprotein, and autoantibodies against oxidized low-density lipoprotein. RESULTS: After 1 year of supplementation, concentrations of serum folate increased by 400% (95% confidence interval [CI], 362%-436%), and those of homocysteine decreased by 28% (95% CI, 24%-36%) in the folic acid group compared with the placebo group. However, no changes in plasma concentrations of the inflammatory markers were observed. CONCLUSIONS: Although homocysteine is associated with vascular disease risk in the general population, marked lowering of slightly elevated homocysteine concentrations by means of 1-year folic acid supplementation does not influence inflammatory responses involving C-reactive protein, soluble intercellular adhesion molecule-1, oxidized low-density lipoprotein, and autoantibodies against oxidized low-density lipoprotein.

Effects of atorvastatin and vitamin E on lipoproteins and oxidative stress in dialysis patients: a randomised-controlled trial.

OBJECTIVES: The objective of this study was to examine the effects of treatment with atorvastatin, alpha-tocopherol and the combination of both, on lipoproteins and oxidative stress in dialysis patients. DESIGN AND SETTING: This double-blind randomised placebo-controlled trial was performed at the dialysis department of a non-university hospital. SUBJECTS, INTERVENTION AND MEASUREMENTS: A total of 44 clinically stable, non-diabetic patients on dialysis therapy (23 on haemo- and 21 on peritoneal-dialysis) without manifest cardiovascular disease were included in this study. They were randomised for treatment during a period of 12 weeks with 40 mg atorvastatin + placebo alpha-tocopherol (group 1) once daily, 800 IU alpha-tocopherol + placebo atorvastatin once daily (group 2), 40 mg atorvastatin + 800 IU alpha-tocopherol once daily (group 3), or placebo atorvastatin + placebo alpha-tocopherol once daily (group 4). Assessment of lipid profile and oxidative stress was performed at the start of the study and after 12 weeks of treatment. RESULTS: Treatment with atorvastatin reduced total cholesterol, triglycerides (TG), low-density lipoprotein (LDL) cholesterol, apolipoprotein B (apoB) and levels of oxidised LDL (oxLDL) with 30-43%. It had no influence on LDL oxidisability. Additional supplementation with alpha-tocopherol had no effect on lipid profile and oxLDL levels but decreased in vitro LDL oxidisability. No side-effects were observed. CONCLUSIONS: Treatment with atorvastatin is effective in lowering plasma total cholesterol, TG, LDL, apoB and oxLDL in a population of stable dialysis patients and might therefore be an effective tool in improving the poor cardiovascular outcome in these patients. Supplementation of alpha-tocopherol to atorvastatin had beneficial effects on in vitro LDL oxidisability and might therefore be of additional value. Further research on the clinical effects of treatment with atorvastatin in combination with alpha-tocopherol is necessary.

Stability studies of ubiquinol in plasma.

BACKGROUND: Ubiquinol is a sensitive redox marker in the first line of the antioxidative defence mechanism and is increasingly being measured in oxidation studies. Because of its apparent instability during storage and processing, we compared various storage conditions. METHOD: Blood was collected from three volunteers into tubes containing EDTA; it was then separated at 4 degrees C and cryopreserved with saccharose (final concentration 6 g/L). Aliquots were stored with or without glutathione or butylated hydroxytoluene at -20 degrees C and -80 degrees C. RESULTS AND CONCLUSION: Ubiquinol in samples stored at -20 degrees C was not stable; however, it was stable when stored at -80 degrees C, even without addition of antioxidant. By contrast, alpha-tocopherol was stable under all conditions studied.

Long term anti-tumour necrosis factor alpha monotherapy in rheumatoid arthritis: effect on radiological course and prognostic value of markers of cartilage turnover and endothelial activation.

OBJECTIVES: To investigate the effect of prolonged neutralisation of tumour necrosis factor alpha (TNFalpha) on the radiological course in rheumatoid arthritis (RA). To assess whether the radiological course can be predicted by clinical variables or biological markers of cartilage and synovium turnover and of endothelial activation. PATIENTS AND METHODS: Forty seven patients with active RA enrolled at our centre in monotherapy trials with adalimumab (D2E7), a fully human anti-TNFalpha monoclonal antibody, were studied for two years. Radiographs of hands and feet obtained at baseline and after one and two years were scored in chronological order by a single, blinded observer using the modified Sharp method. Radiological course was classified as stable or progressive using the smallest detectable difference as cut off point. The relation between radiological course and serum markers of cartilage and synovium turnover (metalloproteinases (MMP-1 and MMP-3), cartilage oligomeric matrix protein (COMP), human cartilage glycoprotein-39 (HC gp-39)), endothelial activation (soluble E-selectin and intercellular adhesion molecule (ICAM-1)), and integrated measures of disease activity were assessed using univariate and multivariate analysis. RESULTS: Radiological evaluation was performed in 36 patients with paired sets of radiographs at baseline and two years. After two years a total of 15/36 (42%) presented no radiological progression. More patients with stable radiological course were still receiving anti-TNFalpha treatment after two years (13/15 (87%) v 11/21 (52%); p=0.03) and had lower baseline COMP and sICAM-1 levels (p=0.01 and 0.04, respectively) than those in the group with progressive disease. In a logistic regression model the combination of sustained TNF neutralisation and baseline COMP and sICAM-1 levels was predictive for radiological outcome (p=0.03). C reactive protein and disease activity score area under the curve were significantly correlated with changes in radiological scores after two years (r=0.40 and 0.37, p<0.05). Long term TNFalpha neutralisation decreased the levels of COMP, sICAM, MMPs, and HC gp-39, but not sE-selectin. CONCLUSION: The results suggest that long term monotherapy with anti-TNFalpha has a positive effect on radiological outcome and modulates cartilage and synovium turnover as measured by biological markers. Baseline serum sICAM-1 levels and COMP levels may be helpful to identify patients with progressive or non-progressive radiological outcome.

Effects of alpha-tocopherol on superoxide production and plasma intercellular adhesion molecule-1 and antibodies to oxidized LDL in chronic smokers.

Antioxidants have been postulated to exert beneficial effects in atherosclerosis. Atherosclerosis is associated with raised plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and autoantibodies against oxidized low-density lipoprotein (oxLDL). It is not known whether antioxidants affect these plasma factors in chronic smokers. In a randomized double-blind placebo-controlled study involving 128 male normolipidemic chronic smokers the effect of a 2-year alpha-tocopherol treatment (400 IU dL-alpha-tocopherol daily) on plasma levels of sICAM-1 and autoantibodies against oxLDL was evaluated. In addition, we monitored production of superoxide by leukocytes ex vivo. It was found that compared to nonsmokers (n = 33) plasma levels of IgG but not IgM autoantibodies against oxLDL and concentrations of sICAM-1 in smokers were significantly elevated (30 and 42%, respectively). After supplementation with alpha-tocopherol concentration of TBARS in plasma and in vitro oxidizability of LDL had decreased, but autoantibodies and sICAM-1 had not changed. Production of superoxide was not different between alpha-tocopherol- and placebo-treated smokers. It is concluded that in chronic smokers, long-term treatment with alpha-tocopherol does not normalize the raised levels of sICAM-1 and autoantibodies against oxLDL, both risk factors for initiation or progression of cardiovascular disease, despite a decrease in in vitro oxidizability of LDL.

Oxidized low-density lipoprotein induces calcium influx in polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMN) have been suggested to play a role in atherosclerosis, but intracellular signaling after stimulation with oxidized low-density lipoprotein (LDL) is unknown. We investigated mechanistic aspects of oxidized LDL-induced superoxide production by human PMN, with special emphasis on intracellular Ca(2+) concentration ([Ca(2+)](i)). Oxidized LDL, but not native LDL, evoked an early but sustained increase in [Ca(2+)](i) and a delayed production of superoxide. The increase in [Ca(2+)](i) could be reduced by fucoidan and completely prevented by U73122, suggesting involvement of the scavenger receptor and coupling to the phospholipase C signal transduction pathway. Furthermore, we provide evidence that the increase in [Ca(2+)](i) partly results from protein kinase C-dependent Ca(2+) influx. The relevance of this Ca(2+) entry for oxidized LDL-stimulated effects is illustrated by the finding that superoxide production was markedly reduced in the absence of external Ca(2+). Finally, inhibition of phagocytosis by cytochalasin B abolished oxidized LDL-stimulated superoxide production without affecting, however, the Ca(2+) mobilization. These effects of oxidized LDL on [Ca(2+)](i) and on respiratory burst of PMN may underlie the occurrence of elevated levels of [Ca(2+)](i) of resting PMN in hypercholesterolemia and represent a mechanism by which PMN can amplify processes in the early phase of atherosclerosis.