Low Inhibitor Levels Research Articles

Clinical manifestations of hereditary angioedema and a systematic review of treatment options.

ObjectiveThis study systematically reviews the existing literature on the management of hereditary angioedema (HAE) and provides an update on the clinical presentation and specific therapies.MethodsA literature search of PubMed and Embase databases was conducted from start of the database to February 2021. Inclusion criteria included relevant systematic reviews, randomized control clinical trials, prospective and retrospective cohort studies, and outcomes research published in English and available in full‐text. Out of 310 candidate articles, a total of 55 articles were included in our study.ResultsThe most common genetic form of HAE in up to 85% of cases is caused by low levels of C1 esterase inhibitor (C1‐INH) protein, leading to a bradykinin‐mediated increase in vascular permeability. During an attack of HAE, abortive treatment with C1‐INH replacement is most commonly described, however, icatibant, ecallantide, or fresh frozen plasma are also used. Long‐term prophylaxis in the form of C1‐INH replacement (subcutaneous or intravenous), monoclonal antibodies targeting plasma kallikrein, attenuated androgens, and transexemic acid should be considered for those who suffer from frequent, severe attacks.ConclusionProgressively distal involvement of the upper airway, especially the larynx, has been shown to pose an increased risk of asphyxiation and death in the acute presentation of HAE. Evaluation by an otolaryngologist is often sought during the emergent clinical management of HAE; therefore, it is prudent that the consulting physician is well‐versed in the prompt recognition, triage of patients, and appropriate treatment modalities.Level of Evidence1A.

Ideal conditions of microwave-assisted acid pretreatment of sugarcane straw allow fermentative butyric acid production without detoxification step

Sugarcane straw (SCS) was pretreated with dilute sulfuric acid assisted by microwave to magnify fermentable sugars and to minimize the concentration of inhibitors in the hydrolysates. The optimum conditions for maximum recovery of sugars were 162 °C and 0.6% (w/v) H2SO4. The low level of inhibitors, such as acetate (2.9 g/L) and total phenolics (1.4 g/L), in the SCS slurry from the pretreatment stage allowed the enzymatic hydrolysis and fermentation steps to occur without detoxification. Besides consuming the total sugar content (31.0 g/L), Clostridium beijerinckii Br21 was able to use acetate from the SCS hydrolysate, to give butyric acid at high conversion factor (0.49 g of butyric acid /g of sugar). The optimized pretreatment conditions spared acid, time, and the detoxification stage, making bio-butyric acid production from SCS extremely attractive.

Study of thrombin generation in healthy children

Exploring coagulation in newborns and children is a challenge due to the low levels of both procoagulant factors and inhibitors. Conventional coagulation tests might be inadequate to explore all of these changes. The aim of the study is to evaluate the thrombin generation assay, a global test to explore coagulation, in a pediatric population (n=586) compared to an adult population (n=166). The thrombin generation assays were performed using Calibrated Automated Thrombography with two different tissue factor concentrations (1 and 5 pM), with and without thrombomodulin (TM). In the absence of TM, the endogenous thrombin potential (ETP) is significantly lower in the pediatric population, reflecting the decrease in procoagulant factors. In the presence of TM, ETP values in pediatric subjects are within the reference range of adult values. The thrombin generation assay demonstrates that coagulation balance is maintained in the pediatric population.

RARE GENETIC DISORDER: HEREDITARY ANGIOEDEMA TYPE 1

SESSION TITLE: Medical Student/Resident Allergy and Airway Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Hereditary Angioedema is a rare genetic disorder characterized by recurrent episodes of angioedema without pruritus or urticaria, affecting the skin or mucosal tissues of the upper respiratory and GI tract. This is caused by a mutation in the SERPING1 gene, resulting in deficiency (type I) or dysfunction (type II) of C1 inhibitor protein. CASE PRESENTATION: A 43-year-old female with PMH of recurrent episodes of angioedema requiring intubation was admitted due to chief complaint of facial angioedema, hand and feet swelling associated with dyspnea, and stridor. Due to acute respiratory failure, emergent cricothyrotomy was attempted multiple times, and eventually open tracheostomy was performed and she was placed on mechanical ventilation. Simultaneously, the patient received dexamethasone, epinephrine, and famotidine. Work up revealed low levels of C1 inhibitor and C4, consistent with bradykinin-mediated hereditary angioedema type I. Rapid weaning from mechanical ventilation to a venturi mask was achieved. Her tracheostomy tube was changed to a Montgomery long-term cannula. Later, the Montgomery tube was changed to a #6 cuffless due to hemoptysis and posterior tracheal wall irritation. Her hospital course was complicated with supraglottic edema and grade 1 subglottic stenosis treated with subglottic balloon dilation and steroid Kenalog injection. Broad-spectrum antibiotics were administered due to purulent drainage around the tracheostomy site and thick sputum concerning for tracheobronchitis. The patient had significant improvement and was successfully capped. Due to exorbitant cost and limited insurance long term prophylaxis with Takhzyro (Lanadelumab), and Icatibant was not possible. She was started on Danazol 200 mg daily with 2 rescue doses of Takhzyro SC, 6 rescue doses of icatibant 30 mg SC, and EpiPen for respiratory distress. She was counseled to avoid any NSAIDs, ACEI, tamoxifen, and was discharged to another facility for rehabilitation and decannulation. DISCUSSION: This is a case describing a rare genetic disease, with a mortality rate of 30% due to asphyxiation from laryngeal edema that accounts for less than 1% of all the angioedema episodes. Laryngeal swelling usually develops around several hours (mean of 7h). There are 3 phases: predyspnea, dyspnea, and loss of consciousness phase ending with death; with an average length of 3.7h, 41 minutes, and 9 minutes respectively. CONCLUSIONS: Early diagnosis and education are paramount in these patients, to receive long term prophylaxis as C1 inhibitor concentrate, monoclonal antibody to kallikrein (lanadelumab), attenuated androgens; avoiding exacerbating factor; and being prepared for acute attacks with the same agents. Reference #1: .https://www.haea.org/assets/img/2013Recommendations.pdf Reference #2: Uptodate hereditary angioedema DISCLOSURES: No relevant relationships by Karim Anis, source=Web Response No relevant relationships by Lady Sanchez Fernandez, source=Web Response

Deep Intronic Mutation in SERPING1 Caused Hereditary Angioedema Through Pseudoexon Activation.

Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.

Prothrombin Complex Concentrates (PCCs) Have Limited Effect on TF-Initiated Thrombin Generation in FXa Inhibitor-Anticoagulated Plasma: In Vitro Comparison between Direct Reversal By Andexanet Alfa and “Work Around” By PCCs

Introduction: Direct oral FXa inhibitors (DOACs, e.g., apixaban [Apix] and rivaroxaban [Riva]) exert their anticoagulant activity by specifically targeting FXa with subnanomolar affinities. Reversal of DOAC-induced anticoagulation can be achieved by immediate and effective sequestration of the inhibitors to relieve inhibition of FXa activity and restore normal thrombin generation (TG). Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for patients treated with Apix or Riva, when reversal of anticoagulation is needed due to life-threatening bleeding. Factor replacement therapies (e.g., 4 factor-PCCs [4F-PCCs]) have demonstrated clinical benefit for vitamin K antagonist-anticoagulated patients by replacing factors II, VII, IX, and X rendered deficient by the anticoagulant. In contrast, the effectiveness and clinical benefit of 4F-PCCs are unclear for reversing DOACs because PCCs do not affect anti-FXa activity. In the present study, we evaluated the effect of individual coagulation factors (FIX, FX, FII), as well as 4F-PCCs on TF-initiated TG (TF-TG) in human platelet poor plasma (PPP), with or without Apix or Riva, and compared it with AnXa under the same conditions. Methods: TF-TG was measured using a calibrated automated thrombogram (CAT, 5 pM TF, Diagnostica Stago). Purified plasma proteins (FIX, FX, FII), PPP, and a commercially available 4F-PCC (Kcentra/Beriplex) were used. 4F-PCC was used with or without pretreatment with the heparinase cup (Haemonetics Corporation) at room temperature for 30 minutes. TF-TG was measured in PPP spiked with purified plasma proteins, 4F-PCC (0-1.0 IU/mL), 4F-PCC + Riva or Apix (0-250 ng/mL), or PPP with AnXa (0-4 μM) + Riva or Apix (0-2000 ng/mL). For comparison, reversal of warfarin anticoagulation by 4F-PCC was performed under similar conditions using individual plasma from warfarin-treated patients (INR = 1.5-6.9) spiked with 4F-PCC (0-1.0 IU/mL). Five CAT parameters were collected, and the endogenous thrombin potential (ETP) and Peak thrombin (Peak) were used for the comparisons. Results: In normal PPP, addition of FX (up to 1.0 IU/mL) had minimal effect on TF-TG parameters, whereas FIX (1.0 IU/mL) mainly increased Peak (~60%). In contrast, FII (1.0 IU/mL) increased both the ETP (~2x) and Peak (~50%). These observations are consistent with the relative plasma concentration and affinity of these coagulation factors as the substrate for the respective enzyme complexes, demonstrating the predominant role of prothrombinase activity in the TF-TG. Addition of 4F-PCC (1.0 IU/mL, equivalent to the therapeutic dose of 50 IU/kg) to PPP caused similar increases of ETP (~2.4x) and Peak (~40%). However, individual plasma protein (FIX, FX, FII) or 4F-PCC up to 1.0 IU/mL was unable to restore normal ETP or Peak in the presence of Apix or Riva (75-250 ng/mL). ETP and Peak increased only when the inhibitor concentrations were sufficiently low (<75 ng/mL), leading to less inhibition of FXa and increased FIIa formation, likely due to an increased FII level from 4F-PCC addition. Similar results were obtained with or without preincubation of 4F-PCC with heparinase prior to spiking in plasma. In contrast, AnXa (0-4.0 μM) dose-dependently reversed Apix- and Riva- (up to 2000 ng/mL) induced inhibition of TF-TG. In the same TF-TG assay, 4F-PCC dose-dependently reversed warfarin anticoagulation in warfarin-treated patients' plasma spiked with 4F-PCC. Conclusions: Specific reversal agents are effective for restoration of TF-TG by either direct sequestration of the FXa inhibitor and restoration of FXa activity (in the case of AnXa with FXa inhibitors) or by replacement of inactive factors due to warfarin treatment (in the case of 4F-PCCs). Addition of coagulation factors or use of 4F-PCCs had limited effect on restoration of normal TG in the presence of a FXa inhibitor. These data demonstrate that AnXa can normalize TF-TG over a wide range of FXa inhibitor concentrations (0-2000 ng/mL), in particular over the range observed in bleeding patients in the ANNEXA-4 study, where 77% of bleeding patients had inhibitor levels ≥75 ng/mL at baseline. In contrast, 4F-PCCs were only able to restore normal TF-TG at low inhibitor levels (<75 ng/mL). These in vitro data are consistent with ANNEXA-4, where AnXa demonstrated >90% immediate reversal of anti-FXa activity and >80% hemostatic efficacy with a wide range of FXa inhibitor levels and multiple bleed types. Disclosures Lu: Portola Pharmaceuticals: Employment, Equity Ownership. Lin:Portola Pharmaceuticals, Inc: Employment, Equity Ownership. Bui:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Curnutte:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals: Employment, Equity Ownership.

Low levels of coagulation inhibitors: a high-risk thrombotic factor in thalassemic patients

Background and aimThe β-thalassemia major (β-TM) is defined as a hereditary red blood cell (RBC)- related disease. Thrombotic events are associated with thalassemia in adult patients. Thus, the present investigation was aimed to examine some hemostatic parameters, including anti thrombin- III (AT-III), protein-C (PRC) and protein-S (PRS) in β-TM patients. MethodsThirty B-TM patients who referred for routine follow-up admission to the thalassemia clinic of Kerman Special Disease Center alongside with 30 healthy subjects were selected and enrolled in the present study. Further registration, the peripheral blood specimens were collected after 3 weeks of last transfusion and then the plasma concentrations of AT-III, PRC and PRS were measured in them. ResultsWe have observed that the concentrations of natural coagulation inhibitors (PRC and PRS) were significantly attenuated in β-TM patients (P < 0.05), while the plasma level of AT-III was not remarkably differed in β-TM patients in compare to healthy subjects. ConclusionAccording to the findings of present work, significant changes in the PRC, PRS and AT-III which could be observed in multi transfused β-TM patients may attribute as critical risk factors for the development of upcoming thromboembolic events in their future life.

The Role of Complement in Hereditary Angioedema.

Low levels of C1 inhibitor, the main inhibitor of the classic complement system, result in paroxysmal angioedema attacks that can be incapacitating or even life-threatening in affected individuals. Molecular defects in the gene for C1 inhibitor cause hereditary angioedema. In recent years, new insights in the pathways leading to angioedema due to a deficiency of C1 inhibitor have been gathered. Bradykinin, which is formed upon activation of the kallikrein-kinin system under insufficient regulation by C1 inhibitor, plays a crucial role. Whereas C1 inhibitor also occupies a central mediatory role in other plasma systems, such as the contact activation system of coagulation and the fibrinolytic plasminogen-plasmin system, a C1 inhibitor deficiency may also cause enhanced activation of these pathways. Novel therapeutic modalities for treatment and prevention of hereditary angioedema are now available, such as different forms of C1 inhibitor concentrate and novel agents that interfere in the kallikrein-kinin system.

New insights into neonatal coagulation: normal clot formation despite lower intra-clot thrombin levels

Healthy neonates exhibit no bleeding tendencies, but exhibit longer partial thromboplastin times than adults. Lower clotting factor levels may be balanced by lower inhibitor levels, which is not reflected in routine coagulation assays, but could result in normal clot formation in vivo. The novel thrombodynamics assay simulates a damaged vessel with tissue factor immobilized to a surface. We hypothesized that intra-clot thrombin levels and spatial fibrin clot formation with this assay are comparable in neonates and adults. Coagulation was tested in plasma from venous neonatal blood (N = 12), cord blood (N = 30), and adult blood (N = 20) using thrombodynamics and calibrated automated thrombography. Neonates exhibited a higher initial rate of clot formation than adults (adult: 60.7 ± 3.9 µm/min; neonatal: 66.8 ± 3.9 µm/min; cord: 68.1 ± 3.3 µm/min; P < 0.001) and a comparable stationary rate of clot formation (adult: 35.8 ± 8.5 µm/min; neonatal: 37.0 ± 4.6 µm/min; cord: 36.0 ± 5.2 µm/min; P = 0.834). Intra-clot thrombin levels were lower in neonates (adult: 41.9 ± 11.2 AU/l; neonatal: 22.6 ± 10.2 AU/l; cord: 23.6 ± 9.7 AU/l; P < 0.001), but the longitudinal rate of thrombin propagation was comparable (adult: 27.2 ± 4.2 µm/min neonatal; 27.9 ± 2.9 µm/min; cord: 27.6 ± 3.4 µm/min; P = 0.862). Despite lower intra-clot thrombin levels, neonates exhibit normal spatial fibrin clot growth, which concurs with clinically well-functioning hemostasis in healthy neonates.

Do Immune-Mediated TTP Patients Presenting with No Detectable ADAMTS13 Antibody Represent a Unique Clinical Phenotype?

Background:Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterised by antibody mediated activity against ADAMTS13. Prompt diagnosis and treatment is critical in this rare condition which has an untreated mortality of around 90%. An ADAMTS13 activity level of <10% is consistent with a diagnosis of TTP and for a significant proportion of patients, ADAMTS13 antibodies can also be demonstrated, confirming iTTP. Recent literature suggests that the sub-group of patients with low ADAMTS13 antigen levels and high ADAMTS13 antibody have the highest mortality (Ferras et al., Blood 2017.); however, the sub-group of patients who have never had a detectable inhibitor remain poorly defined. With therapies becoming increasingly available as adjuvants to plasma exchange, in addition to elective agents for suspected relapsing disease, there is a need to better risk stratify this heterogeneous patient group and identify which patients may benefit most from additional therapies. In most literature to date, patients with low ADAMTS13 inhibitor levels have been excluded from further data analysis and therefore remain an under-represented population. Aim: To define the characteristics of the iTTP patient sub-group where an ADAMTS13 inhibitor has never been demonstrated throughout their clinical course Methods: This was retrospective study of data from a single UK TTP tertiary referral centre from 2011 to 2018. Inclusion criteria for patients with iTTP included ADAMTS13 activity ≤10% at acute presentation. Patients with congenital TTP were excluded from data analysis. A positive ADAMTS13 antibody was defined as >12 U/ml using the Technoclone ADAMTS13 inhibitor ELISA assay. Data collected included patient demographics, presenting symptoms, laboratory markers at first presentation, ADAMTS13 activity and antibody and number of relapses. Clinical relapse was defined as reappearance of clinical manifestations or laboratory parameters consistent with acute TTP and serological relapse was defined as ADAMTS13 activity of <15%. Patients with no detectable antibody were compared with a like group of TTP patients with a detectable ADAMTS13 antibody. Results: A total of 61 patients and 72 acute patient episodes were analysed, of which 7 patients had never demonstrated an ADAMTS13 inhibitor in 10 acute episodes. This sub-group was subsequently compared to an equal sample size of patients with a detectable ADAMTS13 antibody and similar demographics. The average antibody level for the antibody negative group was 5.8 U/ml (range 3 - 10 U/ml) compared to 98.7 U/ml (range 37 - 147U/ml) in the antibody positive group. The respective average ADAMTS13 activity was 4.5% (0-10%), compared with 0.3% (0-1%). 100% of the patients in the antibody positive group had neurological symptoms at presentation compared with 43% of the patients with no detectable antibody. The average presenting platelet count and Troponin T for the antibody negative cohort was 27 x 109/L and 21 ng/L respectively; compared with 10 x 109/L and 206 ng/L in the antibody positive group. 43% of patients in the antibody negative group had multiple acute clinical relapses (1-3) compared with 0% patients presenting with a detectable antibody. 14% of the antibody positive group had a serological relapse treated with elective Rituximab compared with 43% of the patients in the antibody negative group. Conclusion: This data specifically reports the apparent characteristics of patients with ADAMTS13 antibody negative iTTP. Up to 10% of patients may never demonstrate an inhibitor against ADAMTS13 and appear to have a less severe clinical presentation with fewer neurological and cardiac manifestations. This study further suggests that this sub-group may represent a more ‘grumbling‘/ relapsing phenotype with a tendency for more frequent clinical or serological relapse of TTP. Although TTP is rare, further study of this sub-population of iTTP patients is important in risk stratification, and to support treatment decisions, in particular towards preventing future relapse. DisclosuresNo relevant conflicts of interest to declare.

EXPLORATORY ANALYSIS REVEALS POSITIVE CORRELATION BETWEEN C1 ESTERASE INHIBITOR AND SERUM COMPLEMENT 4 ANTIGEN LEVELS

Introduction Low levels of C1-esterase inhibitor (C1-INH) and concomitant reduced complement 4 (C4) concentrations have been identified in patients with hereditary angioedema (HAE). C4 constitutes a promising candidate for a biomarker of disease activity. This investigation aims to determine the relationship between C1-INH functional activity levels (C1-INH[f]) and C4 antigen concentrations in patients with HAE treated with subcutaneous (SC) C1-INH, inferring significance for clinical practice. Methods C1-INH(f) and C4 antigen levels were assayed in three clinical studies (NCT01576523, NCT01912456, NCT02316353 [COMPACT Phase 2, 3 and open-label extension studies]). The relationship between these measurements was inspected in an exploratory manner. All measurements were performed at a central laboratory using validated assays and investigated with respect to time after C1-INH(SC) administration and HAE type. C4 antigen concentration over time after C1-INH(SC) administration was characterized, and the correlation with C1-INH(f) evaluated. Results C4 antigen concentration increased gradually following C1-INH(SC) administration until ∼100 hours post-administration when concentrations declined. A linear relationship was observed between C4 antigen concentrations and C1-INH(f) in patients with type I HAE, based on the Loess fit, until C1-INH(f) of ∼50%, at which point signs of saturability became apparent (example figure shown). The relationship in patients with type II HAE was not clear. Conclusions An exploratory analysis showed a linear relationship between C1-INH(f) and C4 antigen levels in patients with type I HAE, consistent with previous reports. The interplay between C1-INH(f) and C4 should be further explored to understand the potential role of C4 monitoring in treatment decision-making.

Characterization of phenology, physiology, morphology and biomass traits across a broad Euro‐Mediterranean ecotypic panel of the lignocellulosic feedstock Arundo donax

AbstractGiant reed (Arundo donax L.) is a perennial rhizomatous grass, which has attracted great attention as a potential lignocellulosic feedstock for bioethanol production due to high biomass yield in marginal land areas, high polysaccharide content and low inhibitor levels in microbial fermentations. However, little is known about the trait variation that is available across a broad ecotypic panel of A. donax nor the traits that contribute most significantly to yield and growth in drought prone environments. A collection of 82 ecotypes of A. donax sampled across the Mediterranean basin was planted in a common garden experimental field in Savigliano, Italy. We analysed the collection using 367 clumps representing replicate plantings of 82 ecotypes for variation in 21 traits important for biomass accumulation and to identify the particular set of ecotypes with the most promising potential for biomass production. We measured morpho‐physiological, phenological and biomass traits and analysed causal relationships between traits and productivity characteristics assessed at leaf and canopy levels. The results identified differences among the 82 ecotypes for all studied traits: those showing the highest level of variability included stomatal resistance, stem density (StN), stem dry mass (StDM) and total biomass production (TotDM). Multiple regression analysis revealed that leaf area index, StDM, StN, number of nodes per stem, stem height and diameter were the most significant predictors of TotDM and the most important early selection criteria for bioenergy production from A. donax. These traits were used in a hierarchical cluster analysis to identify groups of similar ecotypes, and a selection was made of promising ecotypes for multiyear and multisite testing for biomass production. Heritability estimates were significant for all traits. The potential of this ecotype collection as a resource for studies of germplasm diversity and for the analysis of traits underpinning high productivity of A. donax is highlighted.

T-Cell Receptor Diversity and the Control of T-Cell Homeostasis Mark Ebola Virus Disease Survival in Humans.

Differences in T-cell phenotype, particularly the expression of markers of T-cell homeostasis, have been observed in fatal and nonfatal Ebola virus disease (EVD). However, the relationship between these markers with T-cell function and virus clearance during EVD is poorly understood. To gain biological insight into the role of T cells during EVD, combined transcriptomics and T-cell receptor sequencing was used to profile blood samples from fatal and nonfatal EVD patients from the recent West African EVD epidemic. Fatal EVD was characterized by strong T-cell activation and increased abundance of T-cell inhibitory molecules. However, the early T-cell response was oligoclonal and did not result in viral clearance. In contrast, survivors mounted highly diverse T-cell responses, maintained low levels of T-cell inhibitors, and cleared Ebola virus. Our findings highlight the importance of T-cell immunity in surviving EVD and strengthen the foundation for further research on targeting of the dendritic cell-T cell interface for postexposure immunotherapy.

Hemostasis in the Very Young.

Hemostasis is a dynamic process that starts in utero. The coagulation system evolves with age, as evidenced by marked physiological differences in the concentration of the majority of hemostatic proteins in early life compared with adulthood. This concept, known as "developmental hemostasis," has important biological and clinical implications. Overall, impaired platelet function, along with physiologically reduced levels of vitamin K-dependent and contact coagulation factors, may cause poorer clot firmness even in healthy neonates. However, increased activity of von Willebrand factor and low levels of coagulation inhibitors that promote hemostasis counterbalance the delicate and immature hemostatic system. Since this hemostatic system has little reserve capacity, preterm neonates or sick infants are extremely vulnerable and predisposed to either hemorrhagic or thrombotic complications. This review will address the concept and manifestations of developmental hemostasis with respect to clinical disease phenotypes. It will discuss bleeding diagnosis in neonates, dealing especially with the devastating complications of intracerebral and pulmonary hemorrhage in preterm infants. Neonates, especially the sickest preterm ones, are also extremely susceptible to thrombotic complications; thus, thrombosis in neonates will be reviewed, with special focus on arterial ischemic perinatal stroke. Based on the concept of developmental hemostasis, the phenotypes of clinically relevant bleeding or thrombotic disorders among neonates may differ from those of older infants and children. Treatment options for these conditions will be suggested and reviewed.

Interferon-\u03b3 and Smac mimetics synergize to induce apoptosis of lung cancer cells in a TNF\u03b1-independent manner

BackgroundThe prognosis of lung cancer is very poor and hence new therapeutic strategies are urgently desired. In this study, we searched for efficacious Smac mimetic-based combination therapies with biomarkers to predict responses for non-small cell lung cancer (NSCLC).MethodsNSCLC cell lines and normal human alveolar epithelial cells were treated with Smac mimetics plus IFNγ or other agonists and cell viabilities were assessed by MTS assay, cell counting, flow cytometry and cell colony assay. Western blot analysis was performed to assess the cleavage (activation) of caspases and expression of signaling molecules. Caspase activity was determined to verify caspase activation. The pathways involved in NSCLC cell death were investigated using specific inhibitors.ResultsWe found that IFNγ could cooperate with various Smac mimetics to trigger a profound apoptosis in a number of NSCLC cell lines that are competent for IFNγ signaling (i.e. expressing IFNγ receptor-1 and STAT1) but have low expression levels of inhibitor of apoptosis proteins survivin and livin without harming normal human lung epithelial cells. IFNγ co-treatment with a novel class dimeric Smac mimetic AZD5582 eradicated NSCLC cell colony formation. Unlike IFNγ, IFNα, IFNλ, TNFα, or TRAIL alone or plus AZD5582 had minor effects on NSCLC cell viability. IFNγ/AZD5582-induced cell death in NSCLC cells was independent of TNFα autocrine but relied on apoptosis mediated by JAK kinase, caspase 8 and RIPK1 pathways.ConclusionOur results indicate that IFNγ and Smac mimetics can synergize to induce apoptosis of NSCLC cells and suggest that IFNγ and Smac mimetic regimen may be a novel and efficacious apoptosis targeted therapy with biomarkers to predict responses for NSCLC cells.

Sustenance of endothelial cell stability in septic mice through appropriate activation of transient receptor potential vanilloid-4

Therapeutic target transient receptor potential vanilloid-4 (TRPV-4) is frequently applied in endotoxemia research. It has been reported that HC067047, an inhibitor of TRPV-4, mitigated LPS-induced injury. However, the inhibition of TRPV-4 with HC06047 did not attenuate LPS-induced symptoms and exaggerated pathology. This study was carried with a view to unravelling the reason(s) behind these conflicting results. Different doses of the inhibitor were used in the same degree of sepsis, and their effects were determined through assays for sepsis-related physiological indicators such as endothelial injury markers, coagulation index, organ damage indicators, inflammatory factor levels, and cell apoptosis. The results showed that high or low inhibitor levels had no significant effect on sepsis-related physiological indicators. These findings suggest that proper activation of TRPV-4 in sepsis is important for maintaining normal physiological function. Thus, the degree of TRPV-4 activation should match the severity of sepsis.

Effect of an angiotensin-converting-enzyme inhibitor on the plasma concentration of cytokines and vasoactive molecules in patients with coronary heart disease and hypertension

To investigate the plasma concentrations of cytokines and vasoactive molecules in patients with coronary heart disease (CHD) in the presence of hypertension in relation to the angiotensin-converting-enzyme (ACE) inhibitor level reflecting the degree of renin-angiotensin-aldosterone system (RAAS) inhibition. 72 patients with NYHA functional class (FC) II-III angina pectoris and 40 healthy persons at the age of 47-65 years were examined in a controlled cohort study. Enzyme immunoassay was employed to determine the serum concentrations of interleukins (IL) (IL-2, IL-12, IL-17A, and IL-24), the vasoactive molecules of bradykinin, serotonin, ACE, angiotensin-II (AT-II), NO, and endothelin-1 (ET-1), and plasma renin activity. In addition, the plasma level of the tetrapeptide N-acetyl-Ser-Asp-Lys-Pro was used as a marker for ACE inhibition. The patients with CHD occurring in the presence of hypertension compared with the apparently healthy individuals displayed decreased ET-1 and NO production along with elevated levels of serotonin, AT-II, as well as IL-17A and IL-12. The found changes were accompanied by reduced renin activity. Thus, the individuals with low ACE inhibitor levels showed more pronounced production of the proinflammatory cytokine IL-17A, as well as high plasma concentrations of ACE and NO. The high ACE inhibitor level that reflects patient adherence to appropriate antihypertensive therapy is associated with the reduced production of IL-2 and with the minimum serum levels of ACE, AT-II, and NO, being characterized by the high production of IL-12 and serotonin at the same time. In patients with CHD and hypertension, the high plasma enzyme inhibitor concentration that reflects the activity of appropriate antihypertensive therapy, by contributing to the strengthening of the mechanisms of relaxation of blood vessels, is associated with the risk for proinflammatory activation of whole blood cells and platelets. The mean ACE inhibitor levels that reflect moderate RAAS suppression and are characterized by a relatively low proinflammatory activation of mononuclear cells may be more preferable than the maximum ones, from the point of view of slowing the progression of the subclinical inflammatory process of the vascular wall and preventing possible CHD exacerbations. This determines the feasibility of estimating the plasma level of an ACE inhibitor to control the depth of inhibition of RAAS activity.

IgG subclasses of FVIII inhibitors in an argentine cohort of severe hemophilia A patients: Analysis by flow cytometry.

FVIII inhibitors consist of a polyclonal population of antibodies. Previous studies have demonstrated different distribution of IgG subclasses. IgG4 was associated to high level of FVIII inhibitors and failure of immune tolerance induction (ITI) treatment. This study monitored the relative distribution of IgG subclasses of anti-FVIII in patients with severe hemophilia A (SHA). Anti-FVIII antibodies were measured employing an immunomethod, developed in our laboratory, that combines flow cytometry (FC) with microspheres coupled (FVIII-m) or not (Control-m) to FVIII. Seventy-five patients with SHA were studied, 17 without inhibitors (Group I); 58 with inhibitor history, 13 low responders: (LR: Group II), and 45 high responders (HR: Group III). Eight patients undergoing ITI were also included. We found anti-FVIII antibodies in 11 of 27 patients (40%) without inhibitors and in 45 of 48 with inhibitors at the moment of the study. IgG4 was predominant only in the Group III: P=0.02 in patients with low level of inhibitors and P=0.0001 with high titer of inhibitors. Longitudinal analysis performed on patients undergoing ITI showed a gradual decrease of IgG4 values that was associated to improvement of clinical parameters during treatment. We suggest the use of the FC method to supplement functional traditional assays and to help to improve the management of patients with SHA.

Moringa frequently asked questions

The moringa tree Moringa oleifera is increasingly being studied for numerous properties of applied interest. In the course of work on these properties, many questions arise from both scientists and potential consumers. Here I address some of the most common such questions. I explain that moringa's correct scientific name is Moringa oleifera Lam., and that Moringa pterygosperma is not a synonym but an illegitimate name. The wild range of Moringa oleifera is unknown but it might be native of lowland northwestern India. It is cultivated in all tropical countries, but it is probably best to avoid saying that it is “naturalized” because some uses of this word imply that the plant has become invasive. There are thirteen described species in the genus Moringa, but additional new species probably await description, especially in northeast Africa. Traditionally, leaves of Moringa oleifera, M. concanensis, and M. stenopetala are eaten, and the tubers of young M. peregrina are sometimes eaten roasted. All other species have local medicinal uses. Current commercial use so far emphasizes M. oleifera dried leaf meal in capsules, often touting protein content. Simple calculations show that capsules have negligible protein nutritional value. Such use in pill form rather than as a food leads to the frequent question of whether moringa has “side effects”. A review of studies shows that moringa has low levels of trypsin inhibitors, tannins, saponins, and lectins, meaning that there is no reason to expect that normal levels of consumption would lead to discomfort from these compounds. Nearly 40% of moringa calcium may be present in the form of oxalates, but current data suggest that these are insoluble, excreted, and do not contribute to the formation of kidney stones. Goitrogenic glucosinolates are probably absent, and if present are probably in very low concentrations. Moringa might have abortifacient potential, especially in concentrates. Given its usefulness and relative lack of antinutritional factors, there is global interest in growing moringa, so I examine its climate preferences. Moringa oleifera is a plant of the lowland dry tropics, growing best in places where annual low temperatures do not dip below 15°C, rainfall is less than 1500 mm and distributed in one or two rainy seasons, and below 500 m a.s.l. I conclude by underscoring the need for studies of all of these aspects across the entire diversity within M. oleifera and across the genus.

A mild thermomechanical process for the enzymatic conversion of radiata pine into fermentable sugars and lignin

BackgroundConversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route.ResultsWe summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine (Pinus radiata) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3–72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained β-ether interunit linkages, providing an opportunity for conversion into saleable co-products.ConclusionsThis process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up.