Performance of SARS-CoV-2 nucleic acid amplification testing in Austria as measured by external quality assessment schemes during 3 years of the COVID-19 pandemic: an observational retrospective study.

BACKGROUND: The aim of external quality assessment (EQA) schemes is to evaluate the analytical performance of laboratories and test systems in a near-to-real-life setting. This monitoring service provides feedback to participant laboratories and serves as a control measure for the epidemiological assessment of the regional incidence of a pathogen, particularly during epidemics. Using data from EQA schemes implemented as a result of the intensive effort to monitor SARS-CoV-2 infections in Austria, we aimed to identify factors that explained the variation in laboratory performance for SARS-CoV-2 detection over the course of the COVID-19 pandemic. METHODS: For this observational study, we retrospectively analysed 6308 reverse transcriptase quantitative PCR (RT-qPCR) test results reported by 191 laboratories on 71 samples during 14 rounds of three SARS-CoV-2 pathogen detection EQA schemes in Austria between May 18, 2020, and Feb 20, 2023. We calculated the overall rates of false and true-negative, false and true-positive, and inconclusive results. We then assessed laboratory performance by estimating the sensitivity by testing whether significant variation in the odds of obtaining a true-positive result could be explained by virus concentration, laboratory type, or assay format. We also assessed whether laboratory performance changed over time. FINDINGS: 4371 (93·7%) of 4663 qPCR test results were true-positive, 241 (5·2%) were false-negative, and 51 (1·1%) were inconclusive. The mean per-sample sensitivity was 99·7% in samples with high virus concentrations (1383 [99·4%] true-positive, three [0·2%] false-negative, and five [0·4%] inconclusive results for 1391 tests in which the sample cycle threshold was ≤32), whereas detection rates were lower in samples with low virus concentrations (mean per-sample sensitivity 92·5%; 2988 [91·3%] true-positive, 238 [7·3%] false-negative, and 46 [1·4%] inconclusive results for 3272 tests in which the cycle threshold was >32). Of the 1645 results expected to be negative, 1561 (94·9%) were correctly reported as negative, 10 (0·6%) were incorrectly reported as positive, and 74 (4·5%) were reported as inconclusive. Notably, the overall performance of the tests did not change significantly over time. The odds of reporting a correct result were 2·94 (95% CI 1·75-4·96) times higher for a medical laboratory than for a non-medical laboratory, and 4·60 (2·91-7·41) times greater for automated test systems than for manual test systems. Automated test systems within medical laboratories had the highest sensitivity when compared with systems requiring manual intervention in both medical and non-medical laboratories. INTERPRETATION: High rates of false-negativity in all PCR analyses evaluated in comprehensive, multiple, and repeated EQA schemes outline a clear path for improvement in the future. The performance of some laboratories (eg, non-medical laboratories or those using non-automated test systems) should receive additional scrutiny-for example, by requiring additional EQA schemes for certification or accreditation-if the aggregated data from EQA rounds suggest lower sensitivity than that recorded by others. This strategy will provide assurances that epidemiological data as a whole are reliable when testing on such a large scale. Although performance did not improve over time, we cannot exclude extenuating circumstances-such as shortages and weakened supply chains-that could have prevented laboratories from seeking alternative methods to improve performance. FUNDING: None.

Oleic acid induces the novel apolipoprotein O and reduces mitochondrial membrane potential in chicken and human hepatoma cells.

Non-alcoholic fatty liver disease (NAFLD) is marked by hepatic fat accumulation and reflects a spectrum of chronic liver diseases associated with obesity, impaired insulin sensitivity and dyslipidemia. Apolipoprotein O (ApoO) is a new member of the plasma apolipoprotein family that may play a role in lipid metabolism and electron transport activity of the mitochondrium. However, its physiological functions have not been elucidated yet. Based on our previous data in a non-mammalian experimental system [1], we hypothesized that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to increased lipoprotein-production induced by, e.g., hormones and oxidative stress. To gain insight into a mammalian experimental system, we compared the effects of lipid loading on ApoO regulation in chicken hepatoma LMH cells with those in the human hepatoma cell line HepG2. Incubation of the cells with BSA-complexed oleic acid (OA-Alb) induced triglyceride accumulation, but did not affect cell viability. qPCR using specific primer pairs and Western blot analysis with in-house produced rabbit anti-ApoO antisera demonstrated significant increase in ApoO transcript and protein levels in both cell lines. ROS formation due to OA-Alb treatment was only slightly altered in LMH cells, indicating an intact antioxidant defense system of the cells. Oxidative stress applied by addition of H2O2 revealed induction of ApoO transcript and protein level in the same or even higher extent as monitored in the presence of OA-Alb. Upon treatment with estrogen for 24 h quantitative analysis of ApoO transcript and protein revealed increases of ApoO expression supporting the assumption that estrogen affects lipoprotein metabolism at various points. Furthermore, both cell lines showed a significant decrease of the mitochondrial membrane potential upon incubation with OA-Alb. Therefore, we assume that our findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of non-alcoholic fatty liver disease.

The effect of a dual or a triple antithrombotic therapy with apixaban on thrombus formation in vivo and in an ex vivo perfusion chamber model: An open-label, controlled, sequential study.

BACKGROUND: There is a need to optimize pharmacological treatment in patients with acute coronary syndrome and concomitant atrial fibrillation, in particular with newer antithrombotic medicines. We have therefore studied if dual or triple combination of antithrombotic agents exert similar effects on coagulation activation in an in vivo model in the skin microvasculature and in an ex vivo perfusion chamber. METHODS AND RESULTS: Shed blood platelet activation (ß-thromboglobulin [ß-TG]), thrombin generation (thrombin-antithrombin complex [TAT]) and volume as well as markers of thrombus size (D-dimer) and its platelet content (P-selectin) in a perfusion chamber were studied in a sequential, open-label, parallel group trial in 40 healthy male volunteers (n = 20 per group). Subjects received ticagrelor and apixaban without or with acetylsalicylic acid (ASA). Outcome parameters were assessed at 3 hours after therapy dosing, and at steady-state trough and peak conditions.A triple or dual therapy induced a comparable decrease in shed blood ß-TG at 3 hours after therapy dosing but was more pronounced at steady-state conditions with the more intense treatment combination. During both antithrombotic regimens a similarly sustained inhibition in thrombin generation was observed which was accompanied by comparable increases in shed blood volume. In contrast, no treatment effect could be observed in the perfusion chamber experiment. CONCLUSION: Ticagrelor and apixaban with or without ASA inhibit platelet activation and thrombin formation in vivo in healthy subjects. Platelet inhibition was greater at steady-state conditions after triple therapy administration.

Vitamin D levels and comorbidities in ambulatory and hospitalized patients in Austria.

Vitamin D in its hormonal active form, 1,25-dihydroxyvitamin D (calcitriol), has a major impact on bone turnover by regulating calcium and phosphate homoeostasis. By binding the active vitamin D hormone to the vitamin D receptor (VDR), it acts as a nuclear transcription factor (Bouillon et al., Endocr Rev 29(6):726-776, 2008). The discovery that almost all tissues and cells in the body express the VDR and that several tissues possess the enzymatic capability to convert 25-hydroxyvitamin D (25(OH)-D3; cholecalciferol) to the active form, suggests that vitamin D fulfills various extra-osseous functions (Bouillon et al., Endocr Rev 29(6):726-776, 2008; Holick, N Engl J Med 357(3):266-281, 2007). For example, VDR ensures adequate intestinal calcium absorption by regulating the synthesis of several calcium transport proteins in the duodenum (Bouillon et al., Endocr Rev 29(6):726-776, 2008). Additionally, vitamin D is important for proper muscle function, and some studies suggest it may contribute to prevent type 1 diabetes mellitus, certain autoimmune diseases, hypertension, and several types of cancer (Holick, N Engl J Med 357(3):266-281, 2007).

Overlapping and continued alendronate or raloxifene administration in patients on teriparatide: effects on areal and volumetric bone mineral density--the CONFORS Study.

Nine month teriparatide (TPTD) monotherapy followed by co-administration of raloxifene (RAL) or alendronate (ALN) for another nine 9 months resulted in incremental bone mineral density (BMD) increase. The aim of this study was to investigate the effects of continued antiresorptive treatments for 12 months in the extension phase. Postmenopausal women (n = 125) with severe osteoporosis on ongoing TPTD treatment for 9 months were randomized into three open-label groups for another 9 months: ALN (70 mg/week, n = 41), RAL (60 mg/d, n = 37) in addition to TPTD or no additional medication (n = 47) except Ca and vitamin D. After discontinuation of TPTD the respective antiresorptives were continued for a further 12 months, while patients in the TPTD monotherapy group received Ca and vitamin D. Amino-terminal propeptide of type I procollagen (P1NP) and cross-linked C-telopeptide (CTX), areal and volumetric BMD at the lumbar spine (LS) and hip were assessed. ALN resulted in continued BMD increase in LS (4.3 ± 1.5%; mean ± SD), femoral neck (4.2 ± 1.6%) and total hip (4 ± 1.6%; p < 0.001 for all), while RAL was only effective at the LS (2.4 ± 1.7%, p < 0.001) but no changes at the femoral neck (0.4 ± 1.4%) or total hip (-0.8 ± 1.5%) were observed. Cortical bone only increased in the ALN group (femoral neck 6.7 ± 2.7% and -1.3 ± 2.5%; total hip 13.8 ± 2.9% and -2.3 ± 2.5% for ALN and RAL, p < 0.001 for all; respectively). Analyzing the entire 30 months of therapy, the ALN group revealed the largest BMD increase in all regions. Our results suggest that the addition of ALN to ongoing TPTD and continuing ALN after TPTD was stopped may be beneficial for patients in terms of areal and volumetric BMD increase. Further research is warranted to determine the optimal timing of the initiation of the combination treatment, the respective antiresorptive medication and the potential benefit of this BMD increase regarding fracture prevention.

Serum sclerostin levels are decreased in adult patients with different types of osteogenesis imperfecta.

CONTEXT: There are no specific biochemical bone markers available for osteogenesis imperfecta (OI), and the role of sclerostin as a key regulator of bone formation in OI is unknown. OBJECTIVES: We aimed to evaluate the role of sclerostin and its association with bone turnover markers as well as body composition parameters in adult patients with different types of OI. DESIGN, SETTING, AND PARTICIPANTS: This was a case-control study in 27 adult patients and 50 healthy age- and gender-matched controls. MAIN OUTCOME MEASURES: Serum sclerostin levels and bone turnover markers including serum osteocalcin, amino terminal propeptide of type I procollagen, and CrossLaps as well as body composition parameters were determined in mild OI stage I (OI-I) and moderate-severe OI stages III-IV (OI-III-IV), according to Sillence classification. Data were compared with healthy controls. RESULTS: Sclerostin levels were significantly lower in OI-I (19.9 ± 10.9 pmol/L; P < .001) and OI-III-IV (13.3 ± 10.0 pmol/L; P < .001) compared with healthy adults (45.3 ± 14.9 pmol/L), even after adjustment for age, sex, bone mineral content, and body mass index. CrossLaps and PTH were significantly lower in OI-I (0.197 ± 0.15 ng/L; P = .007 and 33.7 ± 19.1 pg/L; P = .033, respectively) and OI-III-IV (0.221 ± 0.18 ng/L; P = .039, and 27.9 ± 14.7 pg/L; P = .001, respectively) than in healthy controls (0.322 ± 0.15 ng/L and 45.0 ± 16.6 pg/L). Amino-terminal propeptide of type I procollagen was below the reference range for OI-I and OI-III-IV. Patients with OI were shorter and lighter and had a decreased bone mineral content (P < .001) but similar fat distribution and lean body mass, compared with controls. Serum sclerostin levels were not related to any bone marker except osteocalcin, the number of prevalent fractures, or body composition readings. CONCLUSION: Decreased sclerostin levels in OI might reflect a down-regulation or negative feedback mechanism to prevent further bone loss.

The uremic toxin indoxyl sulfate acts as a pro- or antioxidant on LDL oxidation.

Uremic toxins have been shown to play a role in chronic kidney disease (CKD) associated oxidative stress. Oxidative stress and inflammation have been associated with increased risk of cardiovascular disease in uraemia. The oxidative modification of LDL may play a role in early atherogenesis. Enhanced LDL oxidation has been found in uremic patients which may account for accelerated atherosclerosis observed in CKD. The uremic toxin indoxyl sulfate (IS) has been reported to exert oxidative and antioxidative activity. Thus, in the present study we have investigated the influence of IS on the atherogenic modifications of LDL exposed in vitro to different oxidising systems. The transition metal ion (Cu(2) ) and hemin/H2O2 induced lipid oxidation reactions monitored by conjugated diene formation, were inhibited by the presence of IS, which points to possible antioxidant effects by this uremic toxin. A protective effect of IS on LDL apoprotein modification by the exposure to the product of the myeloperoxidase/H2O2/Cl(-) system HOCl, was also observed as estimated by protein carbonyl formation. In contrast, a marked increase in conjugated dienes and lipid hydroperoxides was observed when lipid oxidation was initiated by the free radical generator AAPH in presence of IS. The GC-MS analysis revealed the formation of indole-2,3-dione and 6,12-dihydro-6,12-dioxo-indolo[2,1-b]quinazoline (tryptanthrine) in IS/AAPH reaction. A scheme for the generation of tryptanthrine from IS via indoxyl radicals is proposed, which may facilitate LDL lipid oxidation. Our observations add further insight in the Janus-faced properties of this important uremic toxin.

Carbamoylation abrogates the antioxidant potential of hydrogen sulfide.

Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

Open-label, randomized study of the effect of rivaroxaban with or without acetylsalicylic acid on thrombus formation in a perfusion chamber.

INTRODUCTION: Rivaroxaban, a direct factor Xa inhibitor, has demonstrated effectiveness for the management of both venous and arterial thrombosis. This study was designed to investigate the antithrombotic effect of rivaroxaban, with or without acetylsalicylic acid (ASA), in an ex vivo perfusion chamber at both low and high shear rates. MATERIALS AND METHODS: Healthy subjects (N=51) were enrolled in a randomized, crossover (rivaroxaban 5, 10 or 20mg with or without ASA), and parallel-group (compared with ASA plus clopidogrel) study. Thrombi formed on pig aorta strips were measured after a 5-minute perfusion at low and high shear rates with blood from the subjects by measuring D-dimer concentration (for fibrin deposition) and P-selectin content (for platelet deposition). RESULTS: ASA alone had no impact on thrombus D-dimer levels, whereas rivaroxaban alone at peak concentrations decreased D-dimer levels by 9%, 84% and 65% at low shear rate and 37%, 73% and 74% at high shear rate after doses of 5, 10 and 20mg, respectively. Steady-state ASA plus rivaroxaban 5mg caused a greater reduction in D-dimer levels (63%) than monotherapy at low shear rate. Co-administration of ASA with clopidogrel was associated with a 30% decrease in D-dimer levels at low shear rate and a 14% decrease at high shear rate. No conclusive effect on P-selectin content was observed across the treatment groups. CONCLUSIONS: Rivaroxaban dose-dependently inhibited ex vivo thrombus formation under low and high shear rates. Co-administration of ASA had an additional effect on the antithrombotic action of low-dose rivaroxaban.

The LPS-induced increase in circulating microparticles is not affected by vitamin C in humans.

OBJECTIVE: Microparticles (MP) are considered to promote coagulation. This study aimed to characterize the time course of MP levels and the effect of high-dose vitamin C on MP formation during inflammation in an in vivo Escherichia coli endotoxin (LPS) model. METHODS: Microparticle formation was studied in 14 male subjects in a cross-over trial who received either intravenous vitamin C at 320 mg/kg body weight (BW) or 480 mg/kg BW or saline solution in a random order on alternate trial days 3 h after intravenous exposure to LPS (2 ng/kg BW). Venous blood samples were taken before, 3 and 6 h after LPS. D-dimer, leucocyte count, C-reactive protein, plasma vitamin C and body temperature were assessed as inflammatory parameters. MP were detected using flow cytometric analysis and expressed in 10³ MP/mL plasma. RESULTS: Microparticles levels were decreased from baseline 848 units [range 431-1705] by 21% to 671 units [253-1586] at 3 h and increased by 32% to 1119 units [288-4443] at 6 h after LPS. This pattern was not influenced by administration of vitamin C, with a change from 730 units [399-1396] at baseline by an increase to 832 units [215-2168] at 3 h to 1055 units [350-4858] at 6 h. MP subpopulations followed similar dynamics. Alterations in inflammatory parameters were independent from vitamin C administration during endotoxemia. CONCLUSION: Microparticles are increased in acute systemic inflammation with inconsistent changes in MP subgroups in healthy subjects. Systemic vitamin C administration does not mitigate MP formation and D-dimer levels during acute systemic inflammation, suggesting that MP-induced coagulation activity is not affected by vitamin C.

Bioactivity of enoxaparin in critically ill patients with normal renal function.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Venous thromboembolism is a frequent complication in critically ill patients that has a negative impact on patient outcomes. Critically ill patients have significantly lower plasma anti-factor-Xa activity levels compared with control patients after administration of subcutaneous heparin. The clinical relevance of the different anti-factor-Xa levels after prophylactic doses of low molecular weight heparin (LMWH) in critically ill patients is not completely understood. WHAT THIS STUDY ADDS: The standard dose of 40 mg enoxaparin led to a significant increase in anti-FXa levels in this selected cohort of ICU patients with normal renal function. This study found only subtle pharmacokinetic differences, but a comparable pharmacodynamic action, after enoxaparin administration in critically ill and normal medical ward patients. Thrombin generation with TGA RC-low and TGARC-high reagents was significantly reduced in ICU and normal ward patients after receiving LMWH. Both readouts appear equally useful for estimating the pharmacodynamics of enoxaparin. The ex vivo model of thrombosis was used for the first time in patients to evaluate the anti-thrombotic activity of LMWH. This method did not show any difference in thrombus formation after administration of enoxaparin in the individual group of patients. AIM: In critically ill patients, reduced anti-FXa plasma activity following subcutaneous administration of enoxaparin or nadroparin has been described. In this study, we aimed to investigate the bioactivity of enoxaparin in critically ill patients and controls. METHODS: A prospective, controlled, open label study was performed on a medical intensive care unit (ICU) and a general medical ward. Fifteen ICU patients (male = 12, median age 52 years [IQR 40-65], with a median Simplified Acute Physiology Score of 30 [IQR 18-52]) and sex- and age-matched medical ward patients were included. The anti-FXa plasma activity was measured after a single subcutaneous dose of40 mg enoxaparin. The thrombus size of a clot formed in an ex vivo perfusion chamber and endogenous thrombin potential (ETP) were measured. RESULTS: The anti-FXa plasma activity increased significantly after enoxaparin administration, with peak levels at 3 h after treatment, but was comparable between the ICU and medical ward groups (median 0.16 IU ml-1 [IQR 0-0.22 IU ml-1] vs. 0.2 IU ml-1 [IQR 0.15-0.27 IU ml-1],respectively, P = 0.13). The area under the anti-FXa activity curve from 0­12 h was similar between the groups (median 0.97 IU ml-1 h [IQR0.59-2.1] and 1.48 IU ml-1 h1 [IQR 0.83-1.62], P = 0.42 for the ICU group compared with the control group, respectively). The ETP was lower in the ICU group (P < 0.05) at baseline, but it was comparable at 3 h between the groups. Thrombus size decreased at 3 h compared with pre-dose (P = 0.029) and was not different between the groups. CONCLUSION: Similar bioactivity was achieved with a standard dose of subcutaneous enoxaparin in this selected cohort of ICU and general ward patients with normal renal function.

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine.

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH(2)-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H(2)O(2)/Cl(-) system is known to react with the NH(2)-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH(2)Cl) was identified as a breakdown product of this reaction.

Human pharmacokinetics of intravenous recombinant human Cu/Zn superoxide dismutase.

OBJECTIVE: Oxidative stress plays an important role in human disease, but antioxidant therapies are limited. Under physiological conditions superoxide is controlled by the enzyme superoxide dismutase. A recombinant human Cu/Zn superoxide dismutase (rhSOD) might open new therapeutic possibilities. METHODS: Safety profile and pharmacokinetics in plasma and urine were assessed in an open label phase I study with dose-escalation. 18 healthy male volunteers received a single intravenous 10-minute infusion of 150, 300, or 600 mg rhSOD, respectively (n = 6 per dose group). RESULTS: rhSOD was well tolerated. Peak plasma concentrations (cmax; mean ± SD) were reached at the end of infusion, with 32.96 ± 10.31, 51.60 ± 8.23, and 103.90 ± 19.02 µg/ ml, respectively. Non-compartmental halflife was 1.06 ± 0.37, 1.59 ± 0.64, and 1.63 ± 0.28 hours. Urinary excretion (10 h) showed dose-dependent relative increases with 11.28 ± 6.46 (7.5%), 54.93 ± 15.25 (18.3%), and 191.81 ± 104.60 mg (32.0%). CONCLUSIONS: Our results show a good safety profile and predictable pharmacokinetics of rhSOD, suggesting that therapeutic exploratory studies might be safely conducted in humans.

The effect of erythropoietin on platelet and endothelial activation markers: a prospective trial in healthy volunteers.

Erythropoietin (EPO) enhances formation of red blood cells and also affects thrombopoiesis and platelet function. We hypothesized that the effect of EPO may be reflected by changes in thromboxane B2 (TXB2) and endothelial cell function. Six male and six female subjects received recombinant human epoetin alpha (Erypo®) intravenously (300 U/kg). Biomarker levels were assessed at baseline and 4, 24, 48 and 72 hours after infusion. Epoetin alpha increased TXB2 levels by 140%, which reached significance at 48 hours (6.6 ± 5 ng/ml vs. 15 ± 9 ng/ml; p = 0.044) and remained at that level at 72 hours. In line, epoetin alpha increased E-selectin levels by 25% already at 24 hours (39 ± 21 ng/ml vs. 49 ± 26 ng/ml; p < 0.001) which stayed at this level until 72 hours (p < 0.001). The raise in platelet activation markers corresponded to an 88% increase in reticulocyte count (43 ± 10 × 10(9)/l vs. 81 ± 17 × 10(9)/l; p < 0.001) and a 9% increase in platelet count at 72 hours (224 ± 45 × 10(9)/l vs. 244 ± 52 × 10(9)/l; p = 0.005). Thrombomodulin and von Willebrand factor concentrations were not significantly altered by epoetin alpha. Interestingly, gender differences in the baseline levels of E-selectin and thrombomodulin were observed. E-selectin and thrombomodulin levels were doubled in men compared to women (51 ± 24 and 28 ± 10 ng/ml; p = 0.025 and 30 ± 5 ng/ml vs. 16 ± 5 ng/ml; p = 0.002, respectively). EPO increases TXB2 serum levels and soluble E-selectin. Further studies are needed to investigate whether these markers might be useful for estimation of thromboembolic risk during EPO-therapy and whether inhibition of thromboxane formation may lower thrombotic complications during EPO treatment: NCT01392612.

Effect on perfusion chamber thrombus size in patients with atrial fibrillation during anticoagulant treatment with oral direct thrombin inhibitors, AZD0837 or ximelagatran, or with vitamin K antagonists.

INTRODUCTION: AZD0837 and ximelagatran are oral direct thrombin inhibitors that are rapidly absorbed and bioconverted to their active forms, AR-H067637 and melagatran, respectively. This study investigated the antithrombotic effect of AZD0837, compared to ximelagatran and the vitamin K antagonist (VKA) phenprocoumon (Marcoumar), in a disease model of thrombosis in patients with non-valvular atrial fibrillation (NVAF). METHODS: Open, parallel-group studies were performed in NVAF patients treated with VKA, which was stopped aiming for an international normalized ratio (INR) of ≤ 2 before randomization. Study I: 38 patients randomized to AZD0837 (150,250 or 350 mg) or ximelagatran 36 mg twice daily for 10-14 days. Study II: 27 patients randomized to AZD0837 250 mg twice daily or VKA titrated to an INR of 2-3 for 10-14 days. A control group of 20 healthy elderly subjects without NVAF or anticoagulant treatment was also studied. Size of thrombus formed on pig aorta strips was measured after a 5-minute perfusion at low shear rate with blood from the patient/control subject. RESULTS: Thrombus formation was inhibited by AZD0837 and ximelagatran. Relative to untreated patients, a 50% reduction of thrombus size was estimated at plasma concentrations of 0.6 and 0.2 µmol/L for AR-H067637 and melagatran, respectively. For patients receiving VKA treatment, the thrombus size was about 15% lower compared with healthy elderly controls. CONCLUSIONS: Effects of AZD0837 and ximelagatran on thrombus formation were similar or greater than for VKA therapy and correlated with plasma concentrations of their active forms.

Does endovenous laser ablation induce endothelial damage at the saphenofemoral junction?

BACKGROUND AND OBJECTIVE: One of the possible complications of endovenous laser ablation (EVLA) is thrombus progression into the common femoral vein or popliteal vein with the potential risk of pulmonary embolism or stroke. We set out to investigate the effect of laser energy applied under standardized treatment conditions on biomarkers of platelet and endothelial activation and on the hemostatic system. METHODS: Twenty patients with incompetence of the great saphenous vein were included in this prospective study. Blood samples of the iliofemoral and anticubital veins were collected before, during, and after EVLA. Plasma levels of soluble (s) P-selectin, soluble thrombomodulin (sTM), prothrombin fragment F1+2 (F1+2), and d-dimer were measured. (s) P-selectin and sTM were analyzed as surrogate markers of endothelial and platelet activation. F1+2 and d-dimer were monitored to quantify the degree of surgical trauma. RESULTS: Whereas there was no immediate rise of (s) P-selectin and sTM plasma concentrations in iliofemoral or anticubital blood, plasma levels of F1+2 and d-dimer increased significantly after EVLA. CONCLUSION: Pulsed mode laser ablation with an 810-nm fiber does not induce measurable platelet and endothelium activation in the iliofemoral or systemic blood. Furthermore, the immediate surgical trauma associated with EVLA appears to be modest. The authors have indicated no significant interest with commercial supporters.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia.

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.

Vitamin C inhibits NO-induced stabilization of HIF-1alpha in HUVECs.

HIF-1alpha represents the oxygen-regulated sub-unit of the transcription factor HIF-1, which regulates the transcription of numerous genes involved in cellular response to hypoxia and oxidative stress. It is shown here that nitric oxide (NO) induces HIF-1alpha stabilization in human endothelial cells from umbilical cords (HUVECs) under normoxic conditions. HIF-1alpha protein was increased approximately 36-fold after incubation with 500 microM DETA-NO, which releases a steady state NO concentration of roughly one thousandth of the initial concentration of the donor. Loading of the cells with vitamin C counteracted NO-induced HIF-1alpha accumulation. Based on the observations that oxidative and nitrosative stress can influence the activity of the proteasomal system, which is responsible for the non-lysosomal degradation of proteins, among them HIF-1alpha, it was investigated whether NO-induced stabilization of HIF-1alpha might be due to reduced 20S proteasomal activity. This hypothesis could not be proved, because NO concentrations to inhibit 20S proteasomal activity were about one order of magnitude higher than that to inhibit HIF-1alpha degradation.

Effects of endothelin-1 and phenylephrine on plasma levels of von Willebrand factor and protein S.

Endothelial dysfunction is considered a major factor in the pathogenesis of a wide array of diseases and often leads to increased production of, or increased responsiveness to endogenous vasoconstrictive mediators, such as endothelin-1 (ET-1) or adrenergic agonists. Based on previous studies in animals and in-vitro, we hypothesized that ET-1 and alpha adrenergic stimulation by phenylephrine (PE) may alter plasma levels of von Willebrand factor-Ag (vWF) and protein S in humans. Ten healthy men were studied in a prospective randomized double blind trial: we investigated the effects of infusions of ET-1 and PE on plasma levels of vWF-Ag and protein S. Aditionally, we examined the effects of these vasoconstrictors on thrombomodulin, tissue plasminogen activator and on protein C. ET-1 increased plasma levels of vWF-Ag by 19% (CI: 9-36%; p=0.008) and increased plasma levels of protein-C by 7% (CI: 0.2 -12 %; p=0.028), even at doses which produced no increase in blood pressure. Plasma levels of protein S and TPA were not significantly affected by ET-1. After PE-infusion we observed a relative increase in plasma levels of protein S by 20% (CI: 5-28%; p=0.036) and of TPA by 14 % (CI: 2-39%; p=0.036). Also, platelet counts increased by 36% (CI: 12-53%; p=0.028) which correlated (r(2)=0.86; p=0.014) with an increase in leukocytes by 49 % (CI: 18-84%; p=0.028). Plasma levels of vWF and protein C were not affected by PE and neither drug had a significant effect on plasma levels of thrombomodulin. In conclusion, our results support the study hypothesis, that ET-1 and PE increase the plasma levels of vWF and protein S, respectively. ET-1 may induce a procoagulant status in various disease states by increasing vWF-Ag levels.

Hydrogen sulfide scavenges the cytotoxic lipid oxidation product 4-HNE.

Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.