Acteoside isolated from Colebrookea oppositifolia attenuates I/R brain injury in Wistar rats via modulation of HIF-1α, NF-κB, and VEGF pathways.

AIMS: The objective of this study was to assess the anti-stroke activity of acteoside isolated from methanolic root extract of C. oppositifolia METHODS: Ischemia-reperfusion(I/R) brain injury was induced in Wistar rats to assess the anti-stroke activity of acteoside. Rats were pretreated with acteoside (10, 25 & 50 mg/kg, p.o.) before the induction of I/R injury. Parameters such as neurological, motor-cognitive functions were evaluated along with morphological (brain volume, infarct size), biochemical (SOD, Catalase, GSH, lipid peroxidation, TNF-α, IL-6, IL-10, ICAM-1, HIF-1α, VEGF, and NF-κB), histopathological, and gene expression studies (HIF-1α, VEGF) were performed to study the protective effect of acteoside against I/R induced brain injury. RESULTS: I/R injury caused significant deterioration of neurological (p < 0.01), motor (p < 0.01) and cognitive (p < 0.01) functions, associated with increase in the brain volume (p < 0.01), and infarct size (p < 0.01); increase in the levels of MDA, TNF-α, IL-6, ICAM-1, HIF-1α, VEGF, and NF-κB along with significant decrease in SOD, catalase, GSH, and IL-10 (p < 0.01 for all parameters) compared to Sham control group. Histology of brain tissue of disease control group exhibited significant vascular changes, neutrophil infiltration, cerebral oedema, and necrosis of the neuronal cells. Further, the gene-expression studies showed significant increase in the HIF-1α (p < 0.01) and VEGF (p < 0.01) mRNA levels in the I/R control compared to Sham control. Interestingly, the acteoside (10, 25 & 50 mg/kg) has prevented the neurological, motor and cognitive dysfunctions, along with inhibiting the morphological, biochemical, histological and gene expression changes induced by I/R-injury (p < 0.05 for 10 mg; p < 0.01 for 25 & 50 mg/kg of acteoside for all the parameters). CONCLUSION: These findings suggest that acteoside possess potent anti-stroke activity through modulation of HIF-1α, NF-κB, and VEGF pathway along with its potent antioxidant activity.

The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development.

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov #NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products ("true PUPs") and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.

Arteriovenous shunts as venous access in children with haemophilia.

INTRODUCTION: Venous access is essential in patients with haemophilia for administration of factor concentrates. Peripheral venipuncture may be challenging, particularly in young children or during immune tolerance induction (ITI). Central venous access devices (CVADs) carry a significant risk for complications. An alternative for venous access is peripheral arteriovenous shunts (AVSs), but there is sparse documentation in the literature. The aim of this study was to document our experience with AVS over 12 years in 27 boys with severe haemophilia. METHODS: For AVS creation, a subcutaneous vein is connected end-to-side with an artery at the wrist (Cimino) or at the forearm (Gracz shunt). Factor concentrates were substituted as for intermediate size surgery. To prevent shunt occlusion, heparin (5 units/kg/h) was given during the first 3 days. RESULTS: Indications for AVS creation were prophylaxis start (n = 20) and ITI (n = 7). Age at shunt insertion was median 1.5 years (minimum 8 months; maximum 11.7 years). Shunt maturation was achieved within a median of 3 weeks after surgery (1.5 weeks; 18 weeks). Age when home treatment was established was median 2.1 years (9 months; 11.7 years). Four patients required AVS revisions due to stenosis, but 26 of 27 patients (96%) achieved good long-term shunt function. There were few other complications. CONCLUSION: Arteriovenous shunts provide a good alternative to CVAD and carry a lower risk of complications. AVSs allow earlier start of prophylaxis and home therapy with an improved quality of life for patients and families.

Anticoagulant prophylaxis and therapy in children: current challenges and emerging issues.

This review is aimed at describing the unique challenges of anticoagulant prophylaxis and treatment in children, and highlighting areas for research for improving clinical outcomes of children with thromboembolic disease. The evidence presented demonstrates the challenges of advancing the evidence base informing optimal management of thromboembolic disease in children. Recent observational studies have identified risk factors for venous thromboembolism in children, but there are few interventional studies assessing the benefit-risk balance of using thromboprophylaxis in risk-stratified clinical subgroups. A risk level-based framework is proposed for administering mechanical and pharmacological thromboprophylaxis. More research is required to refine the assignment of risk levels. The anticoagulants currently used predominantly in children are unfractionated heparin, low molecular weight heparin, and vitamin K antagonists. There is a paucity of robust evidence on the age-specific pharmacology of these agents, and their efficacy and safety for prevention and treatment of thrombosis in children. The available literature is heterogeneous, reflecting age-specific differences, and the various clinical settings for anticoagulation in children. Monitoring assays and target ranges are not well established. Nevertheless, weight-based dosing appears to achieve acceptable outcomes in most indications. Given the limitations of the classical anticoagulants for children, there is great interest in the direct oral anticoagulants (DOACs), whose properties appear to be particularly suitable for children. All DOACs currently approved for adults have Pediatric Investigation Plans ongoing or planned. These are generating age-specific formulations and systematic dosing information. The ongoing pediatric studies still have to establish whether DOACs have a positive benefit-risk balance in the various pediatric indications and age groups.

Incidence of infective endocarditis and its thromboembolic complications in a pediatric population over 30years.

BACKGROUND: Pediatric infective endocarditis (IE) has been associated with high morbidity and mortality, mostly related to thromboembolic complications (TEC). The objective of our study was to describe the experience in children with IE and to review the changes over a thirty-year period, regarding origin of IE, incidence of vegetations, TEC and their respective morbidity and mortality rates. METHODS: A retrospective chart review of children aged 0-18years with IE defined by the Duke Criteria and admitted to The Hospital for Sick Children, was conducted. Data were divided into three periods (P); P1 (1979-1988); P2 (1989-1998); and P3 (1999-2008). RESULTS: The study included 113 patients, median age 7yrs.; females: 46 (41%), congenital heart defects 95 (84%), comparable in all periods. Overall, cardiac vegetations were found in 68/113 patients (60%); large vegetations (≥1cm) in 32 patients (28%). Fourty-five (45/133 [40%]) TEC were documented, 22 patients (20%) developed cerebrovascular events (CVE) and 23 patients (20%) had non-CVE. Patients diagnosed during P3 were older, had more vegetations (p<0.05), and a higher incidence of community acquired-IE (p<0.05). Overall, mortality was 15%, comparable in all periods. Significant risk factors for mortality were vegetations (HR 6.44; 95% CI: 2.07-20.01, p=0.002) and heart failure (HR 28.39; 95% CI: 10.49-76.85, p<0.001). CONCLUSIONS: Over the study period, we report a growing incidence of community acquired pediatric IE in older children accompanied by an increasing rate of TEC. Heart failure and vegetations were associated with an increased mortality. These preliminary data need to be confirmed by prospective data.

Intensity of factor VIII treatment and the development of inhibitors in non-severe hemophilia A patients: results of the INSIGHT case-control study.

Essentials Research suggests that intensive treatment episodes may increase the risk to develop inhibitors. We performed an international nested case-control study with 298 non-severe hemophilia A patients. Surgery and a high dose of factor VIII concentrate were associated with increased inhibitor risk. Physicians need to review arguments for factor VIII dose and elective surgery extra critically. SUMMARY: Background Inhibitor development is a major complication of treatment with factor VIII concentrates in hemophilia. Findings from studies among severe hemophilia A patients suggest that intensive treatment episodes increase the risk of developing inhibitors. Objectives We set out to assess whether intensive treatment is also associated with an increased risk of inhibitor development among non-severe hemophilia A patients. Patients/Methods We performed a nested case-control study. A total of 75 inhibitor patients (cases) and 223 control patients were selected from 2709 non-severe hemophilia A patients (FVIII:C, 2-40%) of the INSIGHT cohort study. Cases and controls were matched for date of birth and cumulative number of exposure days (EDs) to FVIII concentrates. Conditional logistic regression was used to calculate both unadjusted and adjusted odds ratios (aOR); the latter were adjusted for a priori specified confounders. Results Peak treatment of 5 or 10 consecutive EDs did not increase inhibitor risk (aOR, 1.0; 95% confidence interval (CI), 0.4-2.5; and aOR, 1.8; CI, 0.6-5.5, respectively). Both surgical intervention (aOR, 4.2; CI, 1.7-10.3) and a high mean dose (> 45 IU kg-1 /ED) of FVIII concentrate (aOR, 7.5; CI, 1.6-35.6) were associated with an increased inhibitor risk. Conclusions Our findings suggest that high-dose FVIII treatment and surgery increase the risk of inhibitor development in non-severe hemophilia A. Together with the notion that non-severe hemophilia A patients are at a lifelong risk of inhibitor development, we suggest that in the future physicians will review the arguments for the FVIII dose and elective surgery extra critically.

Anti-activated factor II assay for monitoring unfractionated heparin in children: results of the HEARTCAT study.

Essentials Unfractionated heparin has variable effects in children and therefore, monitoring is essential. A randomized controlled trial substudy investigating an anti-IIa assay in children was conducted. Anti-IIa values are lower in younger children, an effect more pronounced at low-dose heparin. Heparin effect on Xa and IIa is not equal, particularly in infants and after high-dose heparin. SUMMARY: Background Unfractionated heparin (UFH) is used for the prophylaxis and treatment of thrombosis in children. Laboratory monitoring of UFH is needed to prevent over-anticoagulation or under-anticoagulation. Objectives To investigate the association between UFH dose and UFH effect as monitored with the anti-activated factor II (FIIa) assay, the relationship between anti-FIIa and anti-activated factor X (FXa) effects, and the influence of patient age and other factors on UFH effect. Patients and methods This was a randomized controlled trial in children during cardiac catheterization, comparing high-dose UFH (100 units kg-1 bolus) with low-dose UFH (50 units kg-1 bolus). Blood samples were drawn at baseline, and after 30 min, 60 min, and 90 min. For the purpose of this study, 49 children and 117 blood samples were evaluated. Results The anti-FIIa assay discriminated well between high-dose and low-dose UFH. Multiple regression demonstrated significant influences of UFH dose and age on anti-FIIa levels. Younger children had lower anti-FIIa levels than older children, an effect that was more pronounced with low-dose UFH. Anti-FXa/anti-FIIa ratios were equal with low-dose UFH. However, anti-FXa levels were relatively increased over anti-FIIa levels in infants and after high-dose UFH bolus administration. Conclusion The UFH effect on anti-FIIa levels is lower in infants than in older children. This influence of age appears to be dose-dependent, being more pronounced with low-dose UFH. Anti-FXa and anti-FIIa levels are not equal, particularly in infants and after high-dose UFH. Monitoring UFH solely with anti-FXa assays may not be sufficient in children, and the anti-FIIa assay may provide important complementary information.

No impact of endogenous prothrombotic conditions on the risk of central venous line-related thrombotic events in children: results of the KIDCAT study (KIDs with Catheter Associated Thrombosis).

BACKGROUND AND OBJECTIVE: Central venous lines (CVLs) are the major exogenous risk factor for deep venous thrombosis (DVT) in children. The study objective was to assess whether endogenous prothrombotic conditions contribute to the risk of CVL-related DVT in children. METHODS: This was a cohort study of consecutive children with heart disease requiring CVLs for perioperative care. CVLs were inserted percutaneously in the upper venous system and patients received prophylaxis with continuous unfractionated heparin (50 u kg(-1)  d(-1) ). Blood samples to test for prothrombotic conditions were collected prospectively and assayed in a blinded fashion. Outcome assessment was by screening for DVT by venography, venous ultrasound and echocardiography. RESULTS: The study population consisted of 90 children, median age 2.7 years (0 months-18 years). Prevalence rates of antithrombin deficiency, protein C deficiency, protein S deficiency, heterozygous factor V Leiden, prothrombin G20210A mutation, methylentetrahydrofolate C677TT genotype, hyperhomocysteinemia, lupus anticoagulant, anticardiolipin antibodies and increased levels of lipoprotein (a) were within the range reported for the general population. At least one prothrombotic condition was present in 38% of children and combined abnormalities in 8%. The incidence of DVT was 28% (25/90), and most DVTs were asymptomatic. None of the prothrombotic conditions showed a significant association with DVT. The population attributable risk (i.e. the risk of DVT in the overall population attributable to a specific condition) did not exceed 2.2%. CONCLUSION: Prothrombotic conditions did not have an important impact on the risk of DVT in children with short-term CVLs. The results of the study suggest that screening for prothrombotic conditions is not justified in this setting.

Inhibition of tissue factor pathway inhibitor by the aptamer BAX499 improves clotting of hemophilic blood and plasma.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the major inhibitor of tissue factor-initiated coagulation, making it an interesting and novel therapeutic target in hemophilia treatment. The aptamer BAX499 (formerly ARC19499) is designed to improve hemostasis by specifically inhibiting TFPI. OBJECTIVES: The aim of the study was to examine the concentration-dependent augmentation of clotting by BAX499. METHODS: Whole blood clot formation was quantified by rotational thromboelastometry and thromboelastography, and thrombin generation in platelet-poor plasma was assessed with the calibrated automated thrombogram, in samples from patients with congenital hemophilia A (N=55) and B (N=11), patients with acquired hemophilia A (N=1), and healthy controls (N=37). RESULTS: BAX499 significantly improved clotting of samples from hemophilic patients in a concentration-dependent manner, resulting in clotting profiles in samples from patients with severe hemophilia that were similar to those of healthy controls. CONCLUSION: BAX499 improved ex vivo clotting parameters in blood and plasma from patients with hemophilia A and B with different severity of disease, and also in a patient with acquired hemophilia. These results further support the contention that anti TFPI strategies may be an effective treatment for hemophilic patients.

A prospective registry of European haemophilia B patients receiving nonacog alfa, recombinant human factor IX, for usual use.

Nonacog alfa, a recombinant factor IX (FIX) used for the treatment of haemophilia B, was approved in Europe in 1998. In accordance with European Medicines Agency requirements, a registry study was conducted from 2002 to 2009. A reformulated iso-osmotic version was approved for European use in 2007. This study was conducted to evaluate the safety of nonacog alfa in a usual care setting, and provide clinical trial and postmarketing surveillance data support. This open-label, non-interventional, prospective observational cohort study (registry) comprised 52 sites in nine European countries. Patients with haemophilia B receiving nonacog alfa in either formulation for prevention or treatment were followed on a usual care schedule. A total of 218 patients were enrolled, of whom 66 (30.3%) were <18 years of age. Haemophilia severity was evenly distributed, with baseline FIX activity of <1%, 1-5% and >5% in 33.3%, 36.6% and 30.1% of patients, respectively. One hundred thirty-eight patients received the original formulation alone; 80 switched to or received only the new formulation. There was a low incidence of events of special interest (ESIs), with less-than-expected therapeutic effect in five patients (2.2%), inhibitor development in two (0.9%), thrombosis in one (0.5%) and allergic events in eight (3.7%). These accounted for the majority of the 15 serious AEs reported in six patients. Six patients discontinued because of AEs, primarily related to hypersensitivity. Nonacog alfa was shown to be safe for the treatment of haemophilia B, with a low incidence of serious AEs and ESIs.

Incidence of thrombotic and bleeding complications during cardiac catheterization in children: comparison of high-dose vs. low-dose heparin protocols.

BACKGROUND: During cardiac catheterization (CC) in children, unfractionated heparin (UFH) is used for primary prophylaxis of thrombotic events (TE). However, the optimal UFH dose to minimize TE and bleeding in children has yet to be established. OBJECTIVES: To (i) objectively assess the incidence of TE and bleeding during pediatric CC using clinical assessment and ultrasound; and (ii) compare a high-dose vs. low-dose UFH protocol for thromboprophylaxis. METHODS: A randomized controlled trial (RCT) comparing high-dose UFH (100 units kg(-1) bolus, followed by 20 units kg h(-1) continuous infusion) vs. low-dose UFH (50 units kg(-1) bolus) during CC. Outcome assessment was by clinical examination and vascular ultrasound, performed by blinded examiners before and within 48 h after CC. Children with no consent for randomization were followed in a cohort receiving standard-of-care UFH (parallel-cohort RCT). RESULTS: A total of 227 children were included; 137 were randomized and 90 followed in the cohort study. The overall incidence of TE was 4.6% and bleeding 6.6%. The RCT was stopped early for futility as there were no differences between the high-dose and the low-dose UFH in TE (5% vs. 3%; risk ratios [RR] 1.5, 95% confidence interval [CI] 0.3; 9) and bleeding (7% vs. 12%, RR 0.6, 95% CI 0.2; 2). There were also no differences when RCT and cohort study populations were combined. CONCLUSIONS: The incidences of TE and bleeding during CC in children were low. There were no differences between the high-dose and the low-dose UFH protocols studied. Although Heparin Anticoagulation Randomized Trial in Cardiac Catheterization (HEARTCAT) was not designed as non-inferiority trial, low-dose UFH (50 units kg(-1) bolus) appears sufficient for thromboprophylaxis during CC.

Population pharmacokinetics of enoxaparin in infants, children and adolescents during secondary thromboembolic prophylaxis: a cohort study.

BACKGROUND: Enoxaparin has been extensively studied in adults on its safety and efficacy during prevention of symptomatic thromboembolism when acute anticoagulation or secondary prevention is required as a result of venous thrombosis or stroke. In children, it is still used off-label and little is known about the pharmacokinetics in children. OBJECTIVES: The aim of the present study was to evaluate whether a once- or twice-daily dosing regimen would be feasible in children to achieve appropriate plasma levels of enoxaparin. PATIENTS/METHODS: A population pharmacokinetic model was developed using anti-factor (F)Xa activity data from 126 children (median age: 5.9 years) receiving enoxaparin either as a once- or twice-daily dosing regimen. RESULTS: A two-compartment model was adequate for describing the enoxaparin kinetics. Body weight proved to be the most predictive covariate for clearance and central volume of distribution: clearance 15 mL h⁻¹ kg⁻¹, central volume of distribution 169 mL kg⁻¹, intercompartmental clearance 58 mL h⁻¹, peripheral volume of distribution 10 L and absorption rate 0.414 h⁻¹. Interindividual variability was found to be 54% for clearance and 42% for volume of distribution. CONCLUSION: The model is capable of describing all age groups and dosing levels of our population and predicts 12 h and 24 h enoxaparin activities sufficiently. According to our results, a once-daily enoxaparin dosing regimen with frequent monitoring is feasible. In 53.2% of the patients the median 24 h trough level was above the desired range of 0.1 IU mL⁻¹ anti-FXa activity for prophylaxis therapy.

Determinants of factor VIII plasma levels in carriers of haemophilia A and in control women.

Factor VIII (FVIII) levels show a considerable variability in female carriers of haemophilia A. Presently, the reasons for this are poorly understood. The aim of the study was to elucidate the influence of genetic and non-genetic parameters on FVIII plasma levels in carriers (n = 42). Results were compared with age-matched healthy women without carriership of haemophilia A (n = 42). Each carrier was tested for the family-specific mutation, ABO blood group, FVIII level, von Willebrand factor (VWF) antigen and activity and C-reactive protein (CRP). FVIII levels were lower in carriers compared to non-carriers [74% (51-103) vs. 142% (109-169), P < 0.001]. No statistically significant differences were observed between the two groups with respect to VWF activity, prothrombin-time, hs-CRP, fibrinogen, body mass index (BMI), age and smoking status as well as the distribution of ABO blood groups. In non-carriers, FVIII was statistically significantly correlated with BMI, activated partial thromboplastin time (APTT), VWF antigen, hs-CRP and fibrinogen. In carriers, significant correlations between FVIII and APTT, VWF antigen and activity were found, whereas BMI, hs-CRP or fibrinogen did not correlate with FVIII. In non-carriers, the association of FVIII with ABO blood groups was statistically significant (P = 0.006), but not in carriers of haemophilia A (P = 0.234). The type of FVIII gene mutation did not influence FVIII levels. Carrier status is the major determinant of a carrier;s FVIII plasma level. Factors known to influence FVIII levels in the general population do not significantly affect FVIII activity in carriers, neither does the type of mutation influence FVIII levels.

[Austrian haemophilia registry: up-date 2008 ]. / Osterreichisches Hämophilie Register: Update 2008.

The treatment of haemophilia requires continuous development of knowledge related to various aspects of diagnosis and therapy. It is, therefore, essential to collect valid and representative data, which are comparable on an international level. The Austrian Haemophilia Registry was set up by the Scientific Advisory Panel of the Austrian Haemophilia Society and by the patient organisation. For the design, it was decided to divide the registry into three sections, two concerning quality control and a third concerning scientific questions, the latter requiring written informed consent. A web-based software is used to collect data. Transfer and storage of data are secured and the server is situated in a computer center with video and access control. Data entry was initiated early 2008. Currently, only preliminary data are available. Our further focus is on continued data entry, which will further enable us to provide information concerning the characteristics of the haemophilia patient population in Austria and the actual treatment modalities used.

1/falpha noise in the fluctuations of the spectra of tridiagonal random matrices from the beta-Hermite ensemble.

The 1/falpha noise displayed by the fluctuation of the n th unfolded eigenvalue, where n plays the role of a discrete time, was recently characterized for the classical Gaussian ensembles of NxN random matrices. It is investigated here for the beta-Hermite ensemble by wavelet analysis of Monte Carlo simulated series both as a function of beta and of N. When beta decreases from 1 to 0, for a given and large enough N, the evolution from a 1/f noise at beta=1 Gaussian orthogonal ensemble (GOE) to a 1/f2 noise at beta=0 Gaussian diagonal ensemble (GDE) is heterogeneous with a approximately 1/f2 noise at the finest scales and an approximately 1/f noise at the coarsest ones.