A multidisciplinary combinatorial approach for tuning promising hydrogen storage materials towards automotive applications.

HyStorM is a multidisciplinary hydrogen-storage project aiming to synthesise and tune materials hydrogen storage properties for automotive applications. Firstly, unique high-throughput combinatorial thin-film technologies are used to screen materials' hydrogen storage properties. Then promising thin-film candidate compositions are synthesised and examined in the bulk. In this paper, we report on our results within the ternary compositions Mg-Ti-B and Ca-Ti-B. Primary screening of the Mg-Ti-B ternary identified a high capacity hotspot corresponding to Mg0.36Ti0.06B0.58, with 10.6 wt% H2 capacity. Partial reversibility has been observed for this material in the thin-film. Bulk Ti-doped Mg(BH4)2 composites show rehydrogenation to MgH2 under the conditions used. The synthesised thin-film Ca-Ti-B ternary showed only low hydrogen storage capacities. In the bulk, Ti-doping experiments on Ca(BH4)2 demonstrated reversible storage capacities up to 5.9 wt% H2. Further characterisation experiments are required to decipher the role of the Ti-dopant in these systems in both films and in the bulk.

Increased platelet count and leucocyte-platelet complex formation in acute symptomatic compared with asymptomatic severe carotid stenosis.

OBJECTIVE: The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference. METHODS: A full blood count was done and whole blood flow cytometry used to measure platelet surface expression of CD62P, CD63, and PAC1 binding and the percentage of leucocyte-platelet complexes in patients with acute (0-21 days, n = 19) and convalescent (79-365 days) symptomatic (n = 16) and asymptomatic (n = 16) severe (> or =70%) carotid stenosis. Most patients were treated with aspirin (37.5-300 mg daily) although alternative antithrombotic regimens were more commonly used in the symptomatic group. RESULTS: The mean platelet count was higher in patients with acute and convalescent symptomatic compared with asymptomatic carotid stenosis. There were no significant differences in the median percentage expression of CD62P and CD63, or PAC1 binding between the acute or convalescent symptomatic and asymptomatic patients. The median percentages of neutrophil-platelet (p = 0.004), monocyte-platelet (p = 0.046), and lymphocyte-platelet complexes (p = 0.02) were higher in acute symptomatic than in asymptomatic patients. In patients on aspirin monotherapy, the percentages of neutrophil-platelet and monocyte-platelet complexes (p = 0.03) were higher in acute symptomatic (n = 11) than asymptomatic patients (n = 14). In the convalescent phase, the median percentages of all leucocyte-platelet complexes in the symptomatic group dropped to levels similar to those found in the asymptomatic group. CONCLUSION: Increased platelet count and leucocyte-platelet complex formation may contribute to the early excess risk of stroke in patients with recently symptomatic carotid stenosis.

Comparison of von Willebrand factor antigen, von Willebrand factor-cleaving protease and protein S in blood components used for treatment of thrombotic thrombocytopenic purpura.

Replacement of normal levels of von Willebrand factor-cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD- and MB-treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.

A performance evaluation of commercial fibrinogen reference preparations and assays for Clauss and PT-derived fibrinogen.

The wide availability of fibrinogen estimations based on the prothrombin time (PT-Fg) has caused concern about the variability and clinical utility of fibrinogen assays. In a multi-centre study, we investigated fibrinogen assays using various reagents and analysers. Clauss assays generally gave good agreement, although one reagent gave 15-30% higher values in DIC and thrombolysis. Two commercial reference preparations had much lower potencies than the manufacturers declared, and plasma turbidity influenced parallelism in some Clauss assays. PT-Fg assays gave higher values than Clauss and showed calibrant dependent effects, the degree of disparity correlating with calibrant and test sample turbidity. Analyser and thromboplastin dependent differences were noted. The relationship between Clauss and PT-Fg assays was sigmoid, and the plateau of maximal PT-Fg differed by about 2 g/l between reagents. ELISA and immunonephelometric assays correlated well, but with a high degree of scatter. Antigen levels were higher than Clauss, but slightly lower than PT-Fg assays, which appeared to be influenced by degraded fibrinogen. Clauss assays are generally reproducible between centres, analysers and reagents, but PT-Fg assays are not reliable in clinical settings.

Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.

Dramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 +/- 2.0 mg versus 17.1 +/- 1.4 mg, P < 0.01) was preceded by significant elevations in alpha1(I) procollagen and CTGF mRNA levels (3.0 +/- 0.4-fold and 6.3 +/- 0.4-fold respectively, (P < 0.01), and total inflammatory cell number. UK-156406, administered at an anticoagulant dose, attenuated lung collagen accumulation in response to bleomycin by 35 +/- 12% (P < 0.05), inhibited alpha1(I) procollagen and CTGF mRNA levels by 50% and 35%, respectively (P < 0.05), but had no effect on inflammatory cell recruitment. This is the first report showing that direct thrombin inhibition abrogates lung collagen accumulation in bleomycin-induced pulmonary fibrosis.

The SHI-3 iron transport island of Shigella boydii 0-1392 carries the genes for aerobactin synthesis and transport.

In Shigella boydii 0-1392, genes encoding the synthesis and transport of the hydroxamate siderophore aerobactin are located within a 21-kb iron transport island between lysU and the pheU tRNA gene. DNA sequence analysis of the S. boydii 0-1392 island, designated SHI-3 for Shigella island 3, revealed a conserved aerobactin operon associated with a P4 prophage-like integrase gene and numerous insertion sequences (IS). SHI-3 is present at the pheU tRNA locus in some S. boydii isolates but not in others. The map locations of the aerobactin genes vary among closely related species. The association of the aerobactin operon with phage genes and mobile elements and its presence at different locations within the genomes of enteric pathogens suggest that these virulence-enhancing genes may have been acquired by bacteriophage integration or IS element-mediated transposition. An S. boydii aerobactin synthesis mutant, 0-1392 iucB, was constructed and was similar to the wild type in tissue culture assays of invasion and intercellular spread.

The sensitivity and specificity of commercial reagents for the detection of lupus anticoagulant show marked differences in performance between photo-optical and mechanical coagulometers.

This study was undertaken to appraise the application of those reagents most widely used in the UK for the detection and confirmation of lupus anticoagulant (LA) on an Amelung KC4A and a Sysmex CA-6000 coagulometer. Five sets of dilute Russell's viper venom time (DRVVT) reagents were assessed as well as the Textarin-PL/ Ecarin ratio. Each DRVVT method comprised both LA detection and confirmation reagents provided by the same manufacturer. Samples were obtained from 20 normal healthy subjects, 10 LA-positive patients, 10 patients receiving oral anticoagulant therapy (OAT) who had previously been documented as LA-positive, a further 10 LA-negative patients receiving OAT and 10 LA-negative patients receiving unfractionated heparin therapy. The sensitivity and specificity of the reagents exhibited considerable variation not only between reagents, but also when the same reagent was used on the two analysers. Sensitivity ranged from 62 to 97% (all reagents both analysers), specificity went as low as 23% (Gradipore reagent on the CA-6000) and as high as 100% (American Diagnostica Inc on both KC4A and CA-6000). On the KC4A instrument, Unicorn Diagnostics' lupus anticoagulant kit offered the best compromise of sensitivity and specificity (sensitivity 83% and specificity 81%). On the CA-6000 the reagents supplied by American Diagnostica Inc exhibited optimal performance (sensitivity 90% and specificity 100%). The results indicate a need to optimise test reagents for specific analyser types, a procedure which can only be undertaken with preparations such as the proposed NIBSC reference plasmas for the detection of lupus anticoagulant.

Maternal cardiolipin, beta 2-glycoprotein-I and prothrombin antibody expression in high-risk pregnancies with bilateral abnormal uterine artery Doppler waveforms.

OBJECTIVE: To compare the frequency of maternal serum antiphospholipid antibodies (to cardiolipin, beta 2-glycoprotein I and prothrombin) in pregnancies presenting with bilateral abnormal uterine artery Doppler waveforms. DESIGN: Retrospective analysis of stored serum. SUBJECTS: Cases comprised 47 singleton pregnancies with bilateral abnormal uterine artery Doppler waveforms at 24 weeks of gestation, followed from 20 weeks, and controls were 100 healthy pregnancies with normal uterine artery Doppler waveforms. METHODS: Ultrasound examination utilized a 5-MHz curvilinear transabdominal transducer with pulsed and color Doppler facilities. Antiphospholipid antibodies were analyzed by ELISA methodology, and reference ranges were established using the geometric mean +/- 2 SD of healthy non-pregnant adults. Human chorionic gonadotropin (hCG) levels were obtained from patient notes. RESULTS: Anticardiolipin antibodies were detected in 11 (23%) of the cases (IgG, n = 7; IgM, n = 6) compared with ten (10%) of the controls (p < 0.05). Low titer anticardiolipin IgG (range, 5.5-35.3; median, 6.3 GPL units) and anticardiolipin IgM (range, 3.4-14.7; median, 5.3 MPL units) were detected in cases. Amongst the cases, adverse perinatal outcomes were more common in the presence of raised levels of anticardiolipin antibodies. Anti-beta 2-glycoprotein I IgG was not detected in any of the cases. Antiprothrombin IgG was not detected, but antiprothrombin IgM occurred in 10.6% of cases compared with 2% of controls. CONCLUSIONS: Women with persistent bilateral abnormal uterine artery. Doppler waveforms in mid-gestation were more likely to express raised levels of anticardiolipin antibodies than healthy controls with normal uteroplacental perfusion. Anticardiolipin antibodies without anti-beta 2-glycoprotein I binding may be involved in the pathogenesis of uteroplacental ischemia in a proportion of high-risk pregnancies.

Prothrombin time derived fibrinogen determination on Sysmex CA-6000.

AIM: To evaluate PT derived fibrinogen determinations with reference to the Clauss fibrinogen assay using a Sysmex CA-6000 random access coagulation analyser. METHODS: Samples were analysed from normal subjects (n = 20), patients with renal or liver dysfunction (n = 25), critically ill patients (n = 25), patients receiving oral anticoagulant treatment (n = 50), and patients with a haemoglobinopathy (n = 127). Prothrombin times were performed using two thromboplastins: one derived from rabbit brain (Dade: Thromboplastin IS) and the other from recombinant human tissue factor (Dade: Innovin). Fibrinogen was assayed by the Clauss method using a commercial kit (Dade: Fibrinogen). RESULTS: The relation between Clauss fibrinogen and PT derived fibrinogen was found to be dependent on the patient's clinical group and source of the thromboplastin used. When the data from the above sample groups were pooled there was still a significant difference (p < 0.001) between Clauss fibrinogen and PT derived fibrinogen, irrespective of thromboplastin used. CONCLUSIONS: It is unsafe to use the PT derived fibrinogen for patient monitoring owing to non-uniform variability in response to clinical status and reagent employed; however, it may prove to be a useful screening test in a research environment for estimating fibrinogen levels among defined patient groups.

The association of factor VIIa, factor XIIa and beta2-glycoprotein-1 with triglyceride-rich lipoproteins in normolipidaemic subjects.

The activation of factors XII (FXII) and VII (FVII) has been shown to occur on the surface of lipoproteins in the presence of lipoprotein lipase and may be modulated by beta2glycoprotein-1 (beta2GP1). In the postprandial state FVII is activated without apparent activation of FXII in plasma. We investigated whether beta2GP1, FXIIa and FVIIa are associated with triglyceride-rich lipoproteins in the fasting and postprandial state. Six normal subjects were studied while fasting and 1, 2 and 4 h after ingestion of 100 g fat. We confirmed that plasma FVIIa activity, but not FXIIa antigen, was increased in the postprandial period. FXIIa, FVIIa and beta2GP1 were associated with chylomicra-rich lipoproteins, and lipase or Triton X-100 treatment caused an increase in FXIIa in plasma and chylomicra without an increase in FVIIa. This suggests that FXIIa may be formed in the postprandial period, but its antigenic determinants are masked by the association with lipoprotein particles, although it could still express proteolytic activity. Alternatively a FXII-independent mechanism or surface other than triglyceride-rich lipoproteins may be responsible for FVII activation in the postprandial state.

Assessment of Actin FS and Actin FSL sensitivity to specific clotting factor deficiencies.

We present a two centre study designed to assess the sensitivity of Actin FS and Actin FSL to deficiencies of factor VIII, IX, XI or XII. The study was undertaken at two centres to avoid bias due to the investigations being undertaken on one analyser. Samples from patients with a factor VIII (n = 36, F VIII = < 1.0-50 iu/dl), factor IX (n = 22, F IX = 2-48 iu/dl), factor XI (n = 23, F XI = 5-50 u/dl) or a factor XII (n = 18, F XII = 1-50 u/dl) deficient state were studied. Activated partial thromboplastin times (APTT) were determined using two batches of Actin FS and of Actin FSL; comparison of APTT results between centres was facilitated by the conversion of clotting times to ratios (test divided by geometric mean normal clotting time). APTT ratios were considered to be elevated if greater than two standard deviations above the mean normal. The factor deficient status of each sample was verified by assaying all samples for factors VIII, IX, XI and XII. Clotting factor assays were performed on a Sysmex CA-1000 fitted with research software, which permitted the auto-dilution and testing of three serial dilution of both a reference preparation and each patient's sample. Assay results were calculated using parallel-line Bioassay principles. This procedure allowed for variation in clotting times due to the effect of temporal drift of any of the reagents within the assay system. Actin FS and Actin FSL demonstrate acceptable sensitivity to factor VIII deficiency, however, both reagents failed to detect a large proportion of factor XI (17.4% and 30.4% of samples, respectively) and factor XII (66.7% and 72.2%, respectively) deficiencies. The detection rate with Actin FSL for factor IX deficiency was also poor (36.4% not detected). As factor IX and XI deficiencies are both associated with haemorrhagic disorders, the inability of these reagents to detect such abnormalities gave cause for concern.

Intracellular K+ suppresses the activation of apoptosis in lymphocytes.

Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K+ regulates the apoptotic process by controlling the activity of death enzymes. We show that K+, at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. Direct measurement of K+ by inductively coupled plasma/mass spectrometry and flow cytometry indicates a major decrease in intracellular K+ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K+. Disruption of the natural K+ electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K+ concentration is a necessary, early event in programmed cell death.

Relationship between preoperative endotoxin immune status, gut perfusion, and outcome from cardiac valve replacement surgery.

STUDY OBJECTIVE: Endotoxin is a powerful trigger of systemic inflammation. Since cardiac surgery exposes patients to endotoxemia, this study was set up to define the relationship between preoperative endogenous endotoxin immune status, gut perfusion, and outcome following cardiac valve replacement surgery. DESIGN: Observational study. SETTING: University hospital. PATIENTS: Fifty-nine consecutive patients undergoing cardiac valve replacement. MEASUREMENTS AND MAIN RESULTS: Blood was assayed for IgG and IgM endotoxin core antibody (EndoCAb) levels preoperatively, immediately postoperatively, and at 4 h and 24 h postoperatively. Intraoperative gut mucosal perfusion was assessed using gastric tonometry. Complications were assessed for groups above and below the median EndoCAb value of a healthy population (100 median units micro/mL). Of the 59 patients, 12 developed at least one of a set of predefined complications. Of these 12, all had preoperative levels of IgM EndoCAb below 100 MU/mL (p<0.025). Eleven had IgG EndoCAb levels below 100 MU/mL (0.05

Monitoring of oral anticoagulant therapy in lupus anticoagulant positive patients with the anti-phospholipid syndrome.

Introduction of the International Normalized Ratio (INR) has improved the standardization of laboratory control of oral anticoagulant therapy (OAT). However, it has been reported that misleading INR results can be obtained from OAT patients with lupus anticoagulant (LA). To investigate this claim, we studied 35 OAT patients, 14 of whom had anti-phospholipid syndrome (APS) with a documented LA. Attainment of anticoagulation was confirmed by chromogenic assay of factor VII and factor X. Prothrombin times were performed using eight thromboplastins (five derived from rabbit brain, two recombinant human tissue factor and one made from human placenta) with an International Sensitivity Index (ISI) of <1.40. When using the thromboplastin manufacturers' ISI there was a significant difference (ANOVA, P<0.0001) between INR results obtained with the eight reagents for both APS (average CV = 12.4%) and non-APS (average CV = 12.5%) patient groups. Variation using the eight thromboplastins was assessed by calculating the CV for each sample; these values were then pooled for each patient group to give the average CV for all samples with all reagents for the two patient groups. Results for both patient groups exhibited markedly reduced variation (APS group average CV = 6.5%, non-APS group average CV = 5.8%) when locally assigned ISI values were employed in the calculation of INRs. Our data does not support the suggestion that the INR may not reflect the true level of anticoagulation in the long-term warfarin-treated patient, in whom lupus anticoagulant was detected. However, there was strong evidence that thromboplastin use should be restricted to those clot detection systems for which the reagent's manufacturer has assigned an ISI, or local ISI assignment must be undertaken. The inappropriate use of a generic (i.e. optical or mechanical clot detection system without regard to specific analyser type) ISI value can lead to ambiguous results.

Haemostatic changes in the pulmonary blood during cardiopulmonary bypass.

Pulmonary injury may result from the use of cardiopulmonary bypass (CPB). We investigated changes in the haemostatic system in the pulmonary vein during CPB compared with blood that circulated through the bypass circuit. Paired samples were taken from the pulmonary vein and central venous pressure (CVP) line during the peri-operative period from ten patients. Plasma levels of factor VII (P < 0.001), prekallikrein (P < 0.05), antithrombin III (P < 0.001) and heparin cofactor II (P < 0.005) were decreased in the pulmonary vein after 20 min of bypass compared with pre-operative levels. In the pulmonary vein there was a significant increase in neutrophil expressed CD11b (P < 0.001), neutrophil elastase: alpha 1-antitrypsin complexes (P < 0.001), endothelin-1(P < 0.001) and thrombin-antithrombin complexes (P < 0.001) by the end of bypass compared with pre-operative levels. There was no significant change in monocyte expressed CD11b, factor XII or C1-esterase inhibitor in the pulmonary vein for the study period. None of these variables were significantly different in the pulmonary vein compared with CVP line. In the pulmonary vein plasma levels of activated factor VII decreased following heparin administration (P < 0.001) in the majority of patients which was coincidental to an increase (P < 0.001) in tissue factor pathway inhibitor (TFPI). This increase in TFPI was significantly higher in the pulmonary vein compared with CVP line (P < 0.05) There was a decrease in neutrophil count by 20 min on CPB in both the pulmonary vein and CVP line (P < 0.001) and this did not return to pre-operative levels in the pulmonary vein. Soluble thrombomodulin levels decreased by 20 min on CPB in the CVP line (P < 0.05) but tended to increase in the pulmonary vein, although this was not statistically significant. In conclusion we found evidence of thrombin generation and possible endothelial damage together with increased neutrophil activation and adhesion molecule expression in the pulmonary vein during CPB which may play an important role in the development of post-CPB pulmonary injury.

Molecular characterization of newly recognized rodent parvoviruses.

Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).

Expression of recombinant parvovirus NS1 protein by a baculovirus and application to serologic testing of rodents.

A recombinant baculovirus containing the NS1 gene of minute virus of mice was constructed. Optimal expression of the recombinant NS1 protein (rNS1) was achieved by infecting Trichoplusa ni High Five cells at a multiplicity of 10 and incubating them for 72 h postinfection. An enzyme-linked immunosorbent assay (ELISA) with rNS1 as the antigen was evaluated for serologic testing of laboratory rodents. The rNS1 ELISA proved to be a more sensitive method for the detection of antibodies to recently recognized rodent parvovirus species (mouse orphan parvovirus and rat orphan parvovirus) and prototypic parvovirus species (minute virus of mice, Kilham's rat virus, and H-1) than were conventional parvovirus ELISAs that use whole parvovirus virions.