Assessment of HMGB1 Release During Experimentally Induced Pyroptosis.

Inflammasomes are multiprotein complexes that can be activated by a wide array of infectious and inflammatory agents. Inflammasome activation culminates in the maturation and secretion of pro-inflammatory cytokines, as well as lytic cell death, known as pyroptosis. During pyroptosis, the entire contents of a cell are released into the extracellular space, propagating the local innate immune response. One component of particular interest is the alarmin high mobility group box-1 (HMGB1). Extracellular HMGB1 is a potent inflammatory stimulus, acting upon multiple receptors to drive inflammation. In this series of protocols, we will outline how to trigger and assess pyroptosis in primary macrophages, with a focus on the assessment of HMGB1 release.

Role of pulsatile growth hormone (GH) secretion in the regulation of lipolysis in fasting humans.

BACKGROUND: The increase in growth hormone (GH) secretion during a prolonged fast stimulates lipolytic rate, thereby augmenting the mobilization of endogenous energy at a time when fuel availability is very low. STUDY AIM: To identify the specific component of GH secretory pattern responsible for the stimulation of lipolytic rate during fasting in humans. STUDY PROTOCOL: We measured lipolytic rate (using stable isotope dilution technique) after an overnight fast in 15 young, healthy, non-obese subjects (11 men and 4 women), and again on four separate occasions after a 59 h fast. These four prolonged fasting trials differed only by the contents of an infusion solution provided throughout the 59 h fasting period. Subjects were infused either with normal saline ("Control"; n = 15) or with graded doses of a GH Releasing Hormone Receptor Antagonist (GHRHa):10 µg/kg/h ("High"; n = 15), 1 µg /kg/h ("Medium"; n = 8), or 0.5 µg /kg/h ("Low"; n = 6). RESULTS: As expected, the 59 h fast completely suppressed plasma insulin levels and markedly increased endogenous GH concentrations (12 h vs 59 h Fast; p = 0.0044). Administration of GHRHa induced dose-dependent reduction in GH concentrations in response to the 59 h fast (p < 0.05). We found a strong correlation between the rate of lipolysis and GH mean peak amplitude (R = 0.471; p = 0.0019), and total GH pulse area under the curve (AUC) (R = 0.49; p = 0.0015), but not the GH peak frequency (R = 0.044; p = 0.8) or interpulse GH concentrations (R = 0.25; p = 0.115). CONCLUSION: During prolonged fasting (i.e., 2-3 days), when insulin secretion is abolished, the pulsatile component of GH secretion becomes a key metabolic regulator of the increase in lipolytic rate.

Results of a multinational survey of diagnostic and management practices of thromboembolic pulmonary embolism in children.

INTRODUCTION: The incidence of thromboembolic (TE)-pediatric pulmonary embolism (PPE) is increasing. We sought to evaluate current practice patterns and gaps in the management of TE-PPE. MATERIALS AND METHODS: After Institutional Review Board approval, SurveyMonkey® questions were sent to members of the Pediatric/Neonatal Thrombosis and Hemostasis Subcommittee, of the International Society on Thrombosis and Haemostasis and the Hemostasis and Thrombosis Research Society. RESULTS: Of 442 members of the two groups, 134 (30%) responded, and 125 (28%) complete responses were analyzed. Eighty percent practiced at a pediatric facility, 88% at academic centers, and 59% in the USA. Computed tomography pulmonary angiography (CTPA) was the preferred diagnostic modality (89%). D-dimer testing was variably used; 22% used clinical diagnostic prediction models and 8% had specific clinical care pathways for TE-PPE management. Prognostic stratification models were used to guide therapy by 4%. Indications for thrombolytic therapy varied considerably; 40% had a standardized protocol for thrombolysis, employing various modalities (45% systemic, 25% catheter-directed, 19% pharmaco-mechanical) and tissue plasminogen activator dose intensities. Duration of anticoagulation was variable with 58% prescribing anticoagulation for duration of >3 months-6 months; 61% followed for long-term adverse outcomes. CONCLUSION: This multinational survey of thrombosis/hemostasis specialists mainly based at pediatric academic centers demonstrates that antithrombotic management of TE-PPE (including duration of anticoagulation and use/non-use of thrombolysis) varies considerably. Furthermore, standardized care pathways to facilitate acute evaluation and management decisions are in place in a minority of centers. These findings help to inform the design of future clinical trials in TE-PPE.

Probiotics administration following sleeve gastrectomy surgery: a randomized double-blind trial.

BACKGROUND: Probiotics are commonly used after bariatric surgery; however, uncertainty remains regarding their efficacy. Our aim was to compare the effect of probiotics vs placebo on hepatic, inflammatory and clinical outcomes following laparoscopic sleeve gastrectomy (LSG). METHODS: This randomized, double-blind, placebo-controlled, trial of 6-month treatment with probiotics (Bio-25; Supherb) vs placebo and 6 months of additional follow-up was conducted among 100 morbidly obese nonalcoholic fatty liver disease (NAFLD) patients who underwent LSG surgery. The primary outcome was a reduction in liver fat content, measured by abdominal ultrasound, and secondary outcomes were improvement of fibrosis, measured by shear-wave elastography, metabolic and inflammatory parameters, anthropometrics and quality of life (QOL). Fecal samples were collected and analyzed for microbial composition. RESULTS: One hundred patients (60% women, mean age of 41.9±9.8 years and body mass index of 42.3±4.7 kg m-2) were randomized, 80% attended the 6-month visit and 77% completed the 12-month follow-up. Fat content and NAFLD remission rate were similarly reduced in the probiotics and placebo groups at 6 months postsurgery (-0.9±0.5 vs -0.7±0.4 score; P=0.059 and 52.5 vs 40%; P=0.262, respectively) and at 12 months postsurgery. Fibrosis, liver-enzymes, C-reactive protein (CRP), leptin and cytokeratin-18 levels were significantly reduced and QOL significantly improved within groups (P⩽0.014 for all), but not between groups (P⩾0.173 for all) at 6 and 12 months postsurgery. Within-sample microbiota diversity (alpha-diversity) increased at 6-month postsurgery compared with baseline in both study arms (P⩽0.008) and decreased again at 12 months postsurgery compared with 6 months postsurgery (P⩽0.004) but did not reach baseline values. CONCLUSIONS: Probiotics administration does not improve hepatic, inflammatory and clinical outcomes 6- and 12 months post-LSG.

UNBLOCK: an open-label, dose-finding, pharmacokinetic and safety study of bivalirudin in children with deep vein thrombosis.

BACKGROUND: Direct thrombin inhibitors offer potential advantages over unfractionated heparin but have been poorly studied in children. OBJECTIVES: To determine appropriate dosing of bivalirudin in children and adolescents and the relationship between activated partial thromboplastin time (APTT) and plasma bivalirudin concentration. PATIENTS/METHODS: The UNBLOCK (UtilizatioN of BivaLirudin On Clots in Kids) study was an open-label, single-arm, dose-finding, pharmacokinetic, safety and efficacy study of bivalirudin for the acute treatment of deep vein thrombosis (DVT) in children aged 6 months to 18 years. Drug initiation consisted of a bolus dose (0.125 mg kg(-1) ) followed by continuous infusion (0.125 mg kg h(-1) ). Dose adjustments were based on the APTT, targeting a range of 1.5-2.5 times each patient's baseline APTT. Safety was assessed by specific bleeding endpoints and efficacy by repeat imaging at 48-72 h and 25-35 days. RESULTS: Eighteen patients completed the study. Following the bolus dose and the initial infusion rate, most patients' APTT values were within the target range. The infusion rate bivalirudin correlated more closely with drug concentration than the APTT. At 48-72 h, nine (50%) patients had complete or partial thrombus resolution, increasing to 16 (89%) at 25-35 days. No major and one minor bleeding event occurred. CONCLUSIONS: Bivalirudin demonstrated reassuring safety and noteworthy efficacy in terms of early clot resolution in children and adolescents with DVT. Although a widely available and familiar monitoring tool, the APTT correlates poorly with plasma bivalirudin concentration, possibly limiting its utility in managing pediatric patients receiving bivalirudin for DVT.

Multicenter randomized controlled trial on Duration of Therapy for Thrombosis in Children and Young Adults (the Kids-DOTT trial): pilot/feasibility phase findings.

BACKGROUND: Randomized controlled trials (RCTs) on pediatric venous thromboembolism (VTE) treatment have been challenged by unsubstantiated design assumptions and/or poor accrual. Pilot/feasibility (P/F) studies are critical to future RCT success. METHODS: The Kids-DOTT trial is a multicenter RCT investigating non-inferiority of a 6-week (shortened) versus 3-month (conventional) duration of anticoagulation in patients aged < 21 years with provoked venous thrombosis. Primary efficacy and safety endpoints are symptomatic recurrent VTE at 1 year and anticoagulant-related, clinically relevant bleeding. In the P/F phase, 100 participants were enrolled in an open, blinded-endpoint, parallel-cohort RCT design. RESULTS: No eligibility violations or randomization errors occurred. Of the enrolled patients, 69% were randomized, 3% missed the randomization window, and 28% were followed in prespecified observational cohorts for completely occlusive thrombosis or persistent antiphospholipid antibodies. Retention at 1 year was 82%. Interobserver agreement between local and blinded central determination of venous occlusion by imaging at 6 weeks after diagnosis was strong (k-statistic = 0.75; 95% confidence interval [CI] 0.48-1.0). The primary efficacy and safety event rates were 3.3% (95% CI 0.3-11.5%) and 1.4% (95% CI 0.03-7.4%). CONCLUSIONS: The P/F phase of the Kids-DOTT trial has demonstrated the validity of vascular imaging findings of occlusion as a randomization criterion, and defined randomization, retention and endpoint rates to inform the fully powered RCT.

Multicenter dose-finding and efficacy and safety outcomes in neonates and children treated with dalteparin for acute venous thromboembolism.

BACKGROUND: Low molecular weight heparins (LMWHs) constitute the mainstay of anticoagulant therapy for pediatric venous thromboembolism (VTE). The safety and effectiveness of dalteparin, an LMWH, has not been established in children, and pediatric data on dalteparin for VTE are limited to one single-center experience. OBJECTIVE: To establish dose-finding (primary endpoint) and efficacy/safety outcomes (secondary endpoints) in children treated with dalteparin in a substudy of the Kids-DOTT trial. PATIENTS AND METHODS: A prospective multicenter trial using dalteparin subcutaneously twice daily for acute VTE in children aged ≤ 21 years was conducted under an investigator-held Investigational New Drug application registered with the US Food and Drug Administration. Initial weight-based dosing per protocol was as follows: infants (< 12 months), 150 IU kg(-1) ; children (1-12 years), 125 IU kg(-1) ; and adolescents (13-18 years), 100 IU kg(-1) . Bleeding events were categorized according to ISTH criteria. Descriptive non-parametric statistics were employed for all analyses. RESULTS: Eighteen patients (67% male) were enrolled from January 2010 to October 2013 across four centers. No supratherapeutic levels were observed. Median (range) therapeutic doses by age group were as follows: infants (n = 3), 180 IU kg(-1) (146-181 IU kg(-1) ); children (n = 7), 125 IU kg(-1) (101-175 IU kg(-1) ); and adolescents (n = 8), 100 IU kg(-1) (91-163 IU kg(-1) ). The median duration of dalteparin use was 48 days (range: 2-169 days), and the median follow-up was 10.5 months (range: 2-35 months). There were no related serious adverse events, no clinically relevant bleeding events, and no symptomatic recurrent VTEs. CONCLUSION: Dalteparin successfully achieved targeted anti-factor Xa levels in 18 children and young adults with acute VTE with a standardized age-based dosing regimen, with a favorable safety and efficacy profile.

Balancing risk and benefit in venous thromboembolism trials: concept for a bivariate endpoint trial design and analytic approach.

Antithrombotic trials in venous thromboembolism treatment and prevention, including those evaluating the new oral anticoagulants, have typically evaluated thromboembolism risk as an efficacy endpoint and bleeding risk as a separate safety endpoint. Findings often occur in opposition (i.e. decreased thromboembolism accompanied by increased bleeding, or vice-versa), leading to variable interpretation of the results, which may ultimately be judged as equivocal. In this paper, we offer an alternative to traditional designs based on the concept of a bivariate primary endpoint that accounts for simultaneous effects on antithrombotic efficacy and harm due to bleeding. We suggest a bivariate endpoint as a general approach to the assessment of 'net clinical benefit' in recently published trials and to the design of future trials. Lastly, we illustrate the bivariate endpoint design using two examples: a recently published superiority trial of rivaroxaban (RECORD1) and an ongoing non-inferiority trial of the duration of anticoagulant therapy in children with venous thrombosis (Kids-DOTT).

Use of global assays to understand clinical phenotype in congenital factor VII deficiency.

Congenital factor VII (FVII) deficiency is characterized by genotypic variability and phenotypic heterogeneity. Traditional screening and factor assays are unable to reliably predict clinical bleeding phenotype and guide haemorrhage prevention strategy. Global assays of coagulation and fibrinolysis may better characterize overall haemostatic balance and aid in haemorrhagic risk assessment. We evaluated the ability of novel global assays to better understand clinical bleeding severity in congenital FVII deficiency. Subjects underwent central determination of factor VII activity (FVII:C) as well as clot formation and lysis (CloFAL) and simultaneous thrombin and plasmin generation (STP) global assay analysis. A bleeding score was assigned to each subject through medical chart review. Global assay parameters were analysed with respect to bleeding score and FVII:C. Subgroup analyses were performed on paediatric subjects and subjects with FVII ≥ 1 IU dL(-1). CloFAL fibrinolytic index (FI2 ) inversely correlated with FVII:C while CloFAL maximum amplitude (MA) and STP maximum velocity of thrombin generation (VT max) varied directly with FVII:C. CloFAL FI2 directly correlated with bleeding score among subjects in both the total cohort and paediatric subcohort, but not among subjects with FVII ≥ 1 IU dL(-1) . Among subjects with FVII ≥ 1 IU dL(-1), STP time to maximum velocity of thrombin generation and time to maximum velocity of plasmin generation inversely correlated with bleeding score. These preliminary findings suggest a novel potential link between a hyperfibrinolytic state in bleeding severity and congenital FVII deficiency, an observation that should be further explored.

Peri-procedural bridging with low molecular weight heparin in patients receiving warfarin for venous thromboembolism: a pediatric experience.

INTRODUCTION: The incidence of venous thromboembolism (VTE) in children appears to be increasing, and warfarin remains one of the few standard anticoagulants used for secondary VTE prevention. When invasive procedures are required in adults with high TE risk who are receiving warfarin, low-molecular weight heparin (LMWH) bridging is recommended, based mainly upon observational evidence; in children, no such studies have been published. We sought to determine the risks of recurrent TE (both VTE and arterial TE [ATE]) and major bleeding with peri-procedural LMWH bridging in children receiving warfarin for VTE. METHODS: Children (age≤21years of age at the time of bridge) receiving warfarin for VTE and undergoing a standardized clinical care protocol for peri-procedural LMWH bridging were enrolled and followed in an institution-based prospective inception cohort study at Children's Hospital Colorado between March 2006 and February 2012. Outcomes were assessed at 30days post-procedure, and followed International Society on Thrombosis and Haemostasis guidelines. RESULTS: Seventeen children comprised the cohort, with a total of 23 bridging episodes. Median age at bridging episode was 17.5years (range, 12 to 21years). In 22% of bridging episodes, indication was for major surgery. Median duration of LMWH administration prior to procedure was 6days (range, 4-10days); median duration off anticoagulation peri-procedurally was 1.5days (range: 1-2days). The risks of major bleeding, recurrent VTE, and ATE at 30days post-procedure were 4.3% (1/23), 0% and 0%, respectively. CONCLUSIONS: This study provides important preliminary data on safety and efficacy of perioperative LMWH bridging for adolescent VTE patients receiving warfarin. Larger collaborative pediatric studies are warranted to substantiate these findings and to investigate prognostic factors of bleeding and recurrent TE in this setting.

Outcomes of mentored, grant-funded fellowship training in haemostasis /thrombosis: findings from a nested case-control survey study.

Successful strategies by which to effectively recruit and retain academic subspecialists in benign haematology have not been established. To evaluate the effectiveness of a grant-funded, mentored fellowship with respect to retention and early career goals in haemostasis/thrombosis, we sought to compare outcomes for graduates of a grant-funded, mentored fellowship training programme in haemostasis/thrombosis [the National Hemophilia Foundation (NHF)-Baxter Clinical Fellowship Award] during conventional haematology/oncology fellowship training (cases), vs. their training peers who were graduates of conventional haematology/oncology fellowship training alone (controls), via a nested case-control survey study. Survey response rate was 85% (11/13) for cases and 90% (9/10) for controls. All respondents had pursued careers in academic haematology/oncology. Median (range) percent time spent in benign haematology postfellowship was 98% (70-100%) for cases vs. 0% (0-20%) for controls. Time spent in research was significantly greater among cases than controls (median 80% [range: 42-90%] vs. 55% [10-80%], respectively; P = 0.01). By years 3-4 postfellowship, median annual number of peer-reviewed publications was higher for cases than controls (3.5 vs. 1.0; P = 0.01). Cases were also more successful in grant funding (including K-awards). These data suggest that a grant-funded, mentored fellowship training programme in haemostasis/thrombosis may be superior to conventional haematology/oncology fellowship training alone with respect to outcomes of retention in clinical care/research, early-career grant funding and publication productivity.

Do viral infections mimic bacterial sepsis? The role of microvascular permeability: A review of mechanisms and methods.

A dysregulated immune response and functional immunosuppression have been considered the major mechanisms of the bacterial sepsis syndrome. More recently, the loss of endothelial barrier function and resultant microvascular leak have been found to be a key determinant of the pathogenesis of bacterial sepsis. Whether a similar paradigm applies to systemic viral syndromes is not known. Answering this question has far-reaching implications for the development of future anti-viral therapeutic strategies. In this review, we provide an overview of the structure and function of the endothelium and how its barrier integrity is compromised in bacterial sepsis. The various in vitro and in vivo methodologies available to investigate vascular leak are reviewed. Emphasis is placed on the advantages and limitations of cell culture techniques, which represent the most commonly used methods. Within this context, we appraise recent studies of three viruses - hantavirus, human herpes virus 8 and dengue virus - that suggest microvascular leak may play a role in the pathogenesis of these viral infections. We conclude with a discussion of how endothelial barrier breakdown may occur in other viral infections such as H5N1 avian influenza virus.

Rate of inhibitor development in previously untreated hemophilia A patients treated with plasma-derived or recombinant factor VIII concentrates: a systematic review.

BACKGROUND: Different rates of inhibitor development after either plasma-derived (pdFVIII) or recombinant (rFVIII) FVIII have been suggested. However, conflicting results are reported in the literature. OBJECTIVES: To systematically review the incidence rates of inhibitor development in previously untreated patients (PUPs) with hemophilia A treated with either pdFVIII or rFVIII and to explore the influence of both study and patient characteristics. METHODS: Summary incidence rates (95% confidence interval) from all included studies for both pdFVIII and rFVIII results were recalculated and pooled. Sensitivity analysis was used to investigate the effect of study design, severity of disease and inhibitor characteristics. Meta-regression and analysis-of-variance were used to investigate the effect of covariates (testing frequency, follow-up duration and intensity of treatment). RESULTS: Two thousand and ninety-four patients (1965 treated with pdFVIII, 887 with rFVIII; median age, 9.6 months) from 24 studies were investigated and 420 patients were observed to develop inhibitors. Pooled incidence rate was 14.3% (10.4-19.4) for pdFVIII and 27.4% (23.6-31.5) for rFVIII; high responding inhibitor incidence rate was 9.3% (6.2-13.7) for pdFVIII and 17.4% (14.2-21.2) for rFVIII. In the multi-way anova study design, study period, testing frequency and median follow-up explained most of the variability, while the source of concentrate lost statistical significance. It was not possible to analyse the effect of intensity of treatment or trigger events such as surgery, and to completely exclude multiple reports of the same patient or changes of concentrate. CONCLUSIONS: These findings underscore the need for randomized controlled trials to address whether or not the risk of inhibitor in PUPs with hemophilia A differs between rFVIII and pdFVIII.

Influence of the factor II G20210A variant or the factor V G1691A mutation on symptomatic recurrent venous thromboembolism in children: an international multicenter cohort study.

OBJECTIVE: To determine the relative importance of the factor (F) II G20210A or FV G1691A mutations as risk factors or predictors for fatal/non-fatal recurrent venous thromboembolism (VTE) in children. METHODS: In the present cohort, the rate of VTE recurrence and the time to recurrence in relation to FII, FV, age, and sex was determined in consecutively enrolled patients with VTE aged newborn to