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Aims Apolipoprotein C-II (ApoC-II) is thought to activate lipoprotein lipase (LPL) and is therefore a possible target for treating hypertriglyceridemia. Its relationship with cardiovascular risk has not been investigated in large-scale epidemiologic studies, particularly allowing for apolipoprotein C-III (ApoC-III), an LPL antagonist. Furthermore, the exact mechanism of ApoC-II–mediated LPL activation is unclear. Methods and results ApoC-II was measured in 3141 LURIC participants of which 590 died from cardiovascular diseases during a median (inter-quartile range) follow-up of 9.9 (8.7–10.7) years. Apolipoprotein C-II–mediated activation of the glycosylphosphatidylinositol high-density lipoprotein binding protein 1 (GPIHBP1)–LPL complex was studied using enzymatic activity assays with fluorometric lipase and very low-density lipoprotein (VLDL) substrates. The mean ApoC-II concentration was 4.5 (2.4) mg/dL. The relationship of ApoC-II quintiles with cardiovascular mortality exhibited a trend toward an inverse J-shape, with the highest risk in the first (lowest) quintile and lowest risk in the middle quintile. Compared with the first quintile, all other quintiles were associated with decreased cardiovascular mortality after multivariate adjustments including ApoC-III as a covariate (all P < 0.05). In experiments using fluorometric substrate-based lipase assays, there was a bell-shaped relationship for the effect of ApoC-II on GPIHBP1–LPL activity when exogenous ApoC-II was added. In ApoC-II-containing VLDL substrate-based lipase assays, GPIHBP1–LPL enzymatic activity was almost completely blocked by a neutralizing anti-ApoC-II antibody. Conclusion The present epidemiologic data suggest that increasing low circulating ApoC-II levels may reduce cardiovascular risk. This conclusion is supported by the observation that optimal ApoC-II concentrations are required for maximal GPIHBP1–LPL enzymatic activity.

Triglyceride (TG) metabolism is highly regulated by angiopoietin-like protein (ANGPTL) family members [Y. Q. Chen et al. , J. Lipid Res. 61 , 1203–1220 (2020)]. During feeding, ANGPTL8 forms complexes with the fibrinogen-like domain-containing protein ANGPTL4 in adipose tissue to decrease ANGPTL3/8- and ANGPTL4-mediated lipoprotein lipase (LPL)-inhibitory activity and promote TG hydrolysis and fatty acid (FA) uptake. The ANGPTL4/8 complex, however, tightly binds LPL and partially inhibits it in vitro. To try to reconcile the in vivo and in vitro data on ANGPTL4/8, we aimed to find novel binding partners of ANGPTL4/8. To that end, we performed pulldown experiments and found that ANGPTL4/8 bound both tissue plasminogen activator (tPA) and plasminogen, the precursor of the fibrinolytic enzyme plasmin. Remarkably, ANGPTL4/8 enhanced tPA activation of plasminogen to generate plasmin in a manner like that observed with fibrin, while minimal plasmin generation was observed with ANGPTL4 alone. The addition of tPA and plasminogen to LPL-bound ANGPTL4/8 caused rapid, complete ANGPTL4/8 cleavage and increased LPL activity. Restoration of LPL activity in the presence of ANGPTL4/8 was also achieved with plasmin but was blocked when catalytically inactive plasminogen (S760A) was added to tPA or when plasminogen activator inhibitor-1 was added to tPA + plasminogen, indicating that conversion of plasminogen to plasmin was essential. Together, these results suggest that LPL-bound ANGPTL4/8 mimics fibrin to recruit tPA and plasminogen to generate plasmin, which then cleaves ANGPTL4/8, enabling LPL activity to be increased. Our observations thus reveal a unique link between the ANGPTL4/8 complex and plasmin generation.

PURPOSE OF REVIEW Over the last two decades, evolving discoveries around angiopoietin-like (ANGPTL) proteins, particularly ANGPTL3, ANGPTL4, and ANGPTL8, have generated significant interest in understanding their roles in fatty acid (FA) metabolism. Until recently, exactly how this protein family regulates lipoprotein lipase (LPL) in a tissue-specific manner to control FA partitioning has remained elusive. This review summarizes the latest insights into mechanisms by which ANGPTL3/4/8 proteins regulate postprandial FA partitioning. RECENT FINDINGS Accumulating evidence suggests that ANGPTL8 is an insulin-responsive protein that regulates ANGPTL3 and ANGPTL4 by forming complexes with them to increase or decrease markedly their respective LPL-inhibitory activities. After feeding, when insulin levels are high, ANGPTL3/8 secreted by hepatocytes acts in an endocrine manner to inhibit LPL in skeletal muscle, whereas ANGPTL4/8 secreted by adipocytes acts locally to preserve adipose tissue LPL activity, thus shifting FA toward the fat for storage. Insulin also decreases hepatic secretion of the endogenous ANGPTL3/8 inhibitor, apolipoprotein A5 (ApoA5), to accentuate ANGPTL3/8-mediated LPL inhibition in skeletal muscle. SUMMARY The ANGPTL3/4/8 protein family and ApoA5 play critical roles in directing FA toward adipose tissue postprandially. Selective targeting of these proteins holds significant promise for the treatment of dyslipidemias, metabolic syndrome, and their related comorbidities.

Background & Aim: Oxyntomodulin (Oxm) is a proglucagon-derived peptide agonist of both the GLP-1 and glucagon receptors and is a key regulator of gastric acid secretion and energy expenditure. Differential processing from proglucagon hinders assay immunoassay selectivity. Method & results: Antibody engineering was used to develop a sandwich immunoassay that selectively measures endogenous Oxm. The pre- and postprandial levels of Oxm from 19 healthy individuals over the course of 2 h were measured. Postprandial increases in Oxm occurred within minutes and levels significantly correlated with those obtained using previously published mass spectrometry assays. Conclusion: This sandwich immunoassay is appropriately sensitive and selective and is also amenable to high-throughput application for the reliable determination of endogenous levels of intact Oxm from human samples.

Lay abstract Measuring immunogenicity, an immune response to a drug, is important in understanding the benefits and risks associated with a drug. Immunogenicity is measured by specific tests within a laboratory; however, these tests and laboratories may change over time. This paper proposes a method to determine if a change in test and laboratory will impact the interpretation of immunogenicity for a drug. Blood samples from clinical trial patients were combined in order to provide representative samples for the immunogenicity tests. The samples were tested at two laboratories with two tests to measure if any interpretation of immunogenicity results would change due to the different tests and laboratories. Laboratories and tests demonstrated similar and reliable results of the samples. This study has established a method which allows for the comparison of immunogenicity results when tests and/or laboratories are changed.

Triacylglycerol (TG) metabolism is tightly regulated to maintain a pool of TG within circulating lipoproteins that can be hydrolyzed in a tissue-specific manner by lipoprotein lipase (LPL) to enable the delivery of fatty acids to adipose or oxidative tissues as needed. Elevated serum TG concentrations, which result from a deficiency of LPL activity or, more commonly, an imbalance in the regulation of tissue-specific LPL activities, have been associated with an increased risk of atherosclerotic cardiovascular disease through multiple studies. Among the most critical LPL regulators are the angiopoietin-like (ANGPTL) proteins ANGPTL3, ANGPTL4, and ANGPTL8, and a number of different apolipoproteins including apolipoprotein A5 (ApoA5), apolipoprotein C2 (ApoC2), and apolipoprotein C3 (ApoC3). These ANGPTLs and apolipoproteins work together to orchestrate LPL activity and therefore play pivotal roles in TG partitioning, hydrolysis, and utilization. This review summarizes the mechanisms of action, epidemiological findings, and genetic data most relevant to these ANGPTLs and apolipoproteins. The interplay between these important regulators of TG metabolism in both fasted and fed states is highlighted with a holistic view toward understanding key concepts and interactions. Strategies for developing safe and effective therapeutics to reduce circulating TG by selectively targeting these ANGPTLs and apolipoproteins are also discussed.

Background Physicians treating COVID-19 patients increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. Objective To identify and characterize the host inflammatory response to SARS-CoV-2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. Methods Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. Results Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within COVID-19 patients, and strong biomarker association with patient response as measured by Ordinal scale. As patients progress, we observe statistically significant dysregulation of IFNγ, IL-1RA, IL-6, IL-10, IL-19, MCP-1, -2, -3, CXCL9, CXCL10, CXCL5, ENRAGE and PARP-1. Furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. Conclusion These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of SARS-CoV-2 pathogenesis and the host response., Capsule Summary: This detailed analysis of plasma biomarkers in COVID-19 patients provides definitive understanding of the cytokine storm and changes in vascular endothelial markers that may inform us of the changes in the Ordinal scale for COVID-19.

We previously demonstrated that angiopoietin-like 8 (ANGPTL8) forms a localized complex with ANGPTL4 to reduce its lipoprotein lipase (LPL)-inhibitory activity and enable increased postprandial uptake of fatty acids (FA) into adipose tissue. Because prolonged cold exposure may increase adipose tissue FA uptake and decrease circulating triglycerides (TG) by reducing ANGPTL4 expression and inducing ANGPTL8 expression (and thus ANGPTL4/8 expression), we investigated the effect of temperature on ANGPTL4 and ANGPTL4/8 LPL-inhibitory activities in vitro. As the ANGPTL4(E40K) mutation results in decreased TG, we also characterized ANGPTL4(E40K) and ANGPTL4(E40K)/8 complex LPL-inhibitory activities. Interestingly, while ANGPTL3, ANGPTL3/8, and ANGPTL4 showed similar LPL inhibition at 37 °C and 22 °C, the already reduced LPL-inhibitory activity of ANGPTL4/8 at 37 °C was even more decreased at 22 °C. At 37 °C, ANGPTL4(E40K) manifested decreased LPL-inhibitory activity compared to ANGPTL4/8, while ANGPTL4(E40K)/8 had even further reduced potency. Remarkably, ANGPTL4/8, ANGPTL4(E40K), and ANGPTL4(E40K)/8 were each actually capable of stimulating LPL activity at 22 °C. Together, these results indicate that ANGPTL4/8 stimulation of LPL activity at low temperatures may represent an additional mechanism for further increasing adipose tissue FA uptake during cold exposure, beyond that already occurring due to decreased ANGPTL4 expression and increased ANGPTL8 expression. In addition, because ANGPTL4(E40K) has decreased LPL-inhibitory activity compared to ANGPTL4/8, our findings also suggest why ANGPTL4(E40K) carriers have decreased circulating TG levels.

Over the developmental lifetime of a therapeutic protein, the immunogenicity assay validation history can become substantial, frustrating review of clinical immunogenicity within the biologics license application. In our experience, this can lead to questions by regulators, resulting in numerous information requests during the review process. To address this, we propose a new document, the method history report (MHR), which can comprehensively present the history of the immunogenicity assay for regulators, including assay development and validation. The flexibility of the MHR allows for adaptation to the specific needs of each therapeutic program, while maintaining a consistent template. Here, we detail the rationale, general outline and template for the MHR and recommend others consider adopting it for their biologics license application-related activities.

Angiopoietin-like protein (ANGPTL)8 has been implicated in metabolic syndrome and reported to regulate adipose FA uptake through unknown mechanisms. Here, we studied how complex formation of ANGPTL8 with ANGPTL3 or ANGPTL4 varies with feeding to regulate LPL. In human serum, ANGPTL3/8 and ANGPTL4/8 complexes both increased postprandially, correlated negatively with HDL, and correlated positively with all other metabolic syndrome markers. ANGPTL3/8 also correlated positively with LDL-C and blocked LPL-facilitated hepatocyte VLDL-C uptake. LPL-inhibitory activity of ANGPTL3/8 was >100-fold more potent than that of ANGPTL3, and LPL-inhibitory activity of ANGPTL4/8 was >100-fold less potent than that of ANGPTL4. Quantitative analyses of inhibitory activities and competition experiments among the complexes suggested a model in which localized ANGPTL4/8 blocks the LPL-inhibitory activity of both circulating ANGPTL3/8 and localized ANGPTL4, allowing lipid sequestration into fat rather than muscle during the fed state. Supporting this model, insulin increased ANGPTL3/8 secretion from hepatocytes and ANGPTL4/8 secretion from adipocytes. These results suggest that low ANGPTL8 levels during fasting enable ANGPTL4-mediated LPL inhibition in fat tissue to minimize adipose FA uptake. During feeding, increased ANGPTL8 increases ANGPTL3 inhibition of LPL in muscle via circulating ANGPTL3/8, while decreasing ANGPTL4 inhibition of LPL in adipose tissue through localized ANGPTL4/8, thereby increasing FA uptake into adipose tissue. Excessive caloric intake may shift this system toward the latter conditions, possibly predisposing to metabolic syndrome.

Adeno-associated virus (AAV) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, a yet unsolved concern for using AAV as a vector is the high potential to elicit humoral and cellular responses, which are often exacerbated by pre-existing immunity due to exposure to wild type AAV. Therefore, characterization of pre-existing and treatment emergent anti-AAV antibodies is of great importance to the development of AAV based gene therapies. In this project, a sensitive and drug tolerant total antibody (TAb) assay was developed using recombinant AAV9-GFP (green fluorescent protein) as a surrogate AAV9. The assay format was affinity capture and elution (ACE) with ruthenium labeled AAV9-GFP as detection. Upon evaluation, three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) were chosen and mixed at equal concentrations as positive control material. The assay sensitivity was estimated to be 11.2 ng/mL. Drug tolerance was estimated to be 5.4 × 10E10 DRP/mL AAV9-GFP at 100 ng/mL anti-AAV9 antibodies and to be at least 1 × 10E11 DRP/mL at 500 ng/mL and 250 ng/mL anti-AAV9 antibodies. The assay showed desirable specificity and precision. Using this TAb assay, significant pre-existing antibodies were detected from normal human sera.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

Despite recent advances in the development of tools to predict immunogenicity risk of biotherapeutic molecules, the ability of a protein to elicit the formation of anti-drug antibodies (ADA) remains one of the most common causes for termination of clinical development programs. In this study, we use ADA assays to detect and measure pre-existing reactivity or the ability of a molecule to produce an ADA-like response in serum from treatment-naïve, healthy donors. We report herein that the magnitude of pre-existing reactivity evaluated pre-clinically and expressed as the 90th percentile of Tier 2 inhibition correlates with the subsequent rate of ADA emergence in the clinic. Furthermore, a multi-domain biotherapeutic (IgG-scFv bispecific antibody) showed the highest pre-existing reactivity and incidence of treatment-emergent ADA (TE-ADA) (57% and 93%, respectively). Using the components of the multidomain molecule in the Tier 2 step of the ADA assay, we were able to identify the scFv as the target of the serum pre-existing reactivity. Most importantly, the domain specificity of pre-existing ADA was the same as that of the TE-ADA from patients treated with the molecule. Based on these data, we propose the evaluation of the magnitude and of the domain specificity of pre-existing reactivity as a powerful tool to understand the immunogenic potential of novel biotherapeutics.

AIMS: Anaesthesia safety has improved over time with severe anaesthesia-related complications and death becoming rare events. We investigated anaesthesia-related complications over a period of 17 years in this single centre retrospective observational cohort study. The main aims were to assess the temporal trends of anaesthesia-related complications, their distribution among clinics and their relation to emergency versus non-emergency interventions. METHODS: In this retrospective, observational cohort study, anaesthesia and event protocols of patients who suffered from an anaesthesia-related complication were collected in the period from 1 January 2003 to 31 December 2019. These data sets were anonymised and grouped into demographic information, ASA physical status, information about dental status and hospital department. Out of the total of 257 cases that were reported to the department of anaesthesia at the Luzerner Kantonsspital, 110 were excluded as they were not anaesthesia-related and 14 because of incomplete reporting forms. The different complications were categorised by the severity injury scale score. The total number of anaesthetics during the study period amounted to 399,583. RESULTS: 133 patients with complications were included. Six patients died owing to a partially anaesthesia-related complication, two suffered from permanent damage, 62 had temporary symptoms and in 63 patients dental damage occurred. The temporal trend showed a major decrease in severe anaesthesia complications and deaths, whiledental injuries were the main complications after 2010. Most of the complications were reported in the orthopaedic department, whereas dental injuries were predominantly reported in the otorhinolaryngology department. The majority of complications occurred during elective surgery although this was not significant (p = 0.53). CONCLUSIONS: Severe anaesthesia-related complications and death decreased dramatically during the study period. Anaesthesia complications are not more frequent in emergency surgeries. Overall, complications occurred more frequently in orthopaedics, and dental injuries predominated in otorhinolaryngology.

The precise elucidation of the antigen sequences for T-cell immunosurveillance greatly enhances our ability to both understand and modulate humoral responses to viral infection or active immunization. Mass spectrometry was used to identify 529 unique sequences from SARS-CoV-2 spike glycoprotein extracellular domain in a complex with human leukocyte antigen class II molecules on antigen presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. The identified sequences span the entire spike protein and several sequences were isolated from a majority of the donors sampled indicating promiscuous binding. Importantly, many peptides derived from the receptor binding domain used for cell entry were identified. This work represents the most precise and comprehensive immunopeptidomic investigation with SARS-CoV-2 spike glycoprotein and allows detailed analysis of features which may aid vaccine development to end the current COVID-19 pandemic. Funding: This work was supported by Eli Lilly and Company. Conflict of Interest: The authors declare no competing interests. Ethical Approval: Frozen PBMCs were obtained from 9 informed consent healthy donors (Discovery Life Sciences) according to local ethical practice.

Medikationsfehler sind ein haufiges und ernstes Sicherheitsproblem. Chlorhexidin (CHX) ist ein taglich eingesetztes Desinfektionsmittel. Die akzidentelle intravasale Injektion von CHX ist selten beschrieben. Schwerwiegende Komplikationen wie akutes Atemnotsyndrom sind moglich. Wir berichten uber die zentralvenose Injektion von 0,1 %iger CHX-Mundspullosung, deren potenzielle Komplikationen und mogliche Therapiemasnahmen. Im Vergleich zu einem anderen Fallbericht traten bei unserem Patienten keine Schaden auf. Die sofortige Hamofiltration und Reinigung der i.v.-Leitung haben moglicherweise zum guten Outcome beigetragen.

Traditional production of fermented dairy products in Montenegro is carried out without adding defined starter cultures. This way of production involves lactic acid bacteria (LAB) that are normally present in the raw milk and production environment. This autochthonous ("wild") fermentation microbiota represents a reservoir of unknown strains. In order to study the LAB diversity, 25 indigenous dairy products in Montenegro have been tested. Isolation was performed on microbial media M17 and MRS agar with or without supplementations under aerobic and anaerobic conditions at temperatures of 30°C, 37 °C and 44°C. Identification of these isolates at species level was done by species-specific PCR and gene regions sequencing of representatives of each RAPD-cluster. RAPD-PCR was used to characterize the isolates at strain level. Nine Lactobacillus species, five Leuconostoc species, four Enterococcus species as well as strains of the species Lactococcus lactis, Pediococcus pentosaceus and Streptococcus thermophilus were detected. It can be concluded that a rich lactic acid bacteria diversity existed in the analyzed Montenegrin dairy products. Further examination of the isolates could lead to the development of autochthonous starter cultures that would contribute to a product that is characteristic for the geographical area and to which the local population is accustomed.

Importance Psoriasis relapse may involve compensatory T-cell activation pathways in the presence of CD28-CD80/CD86 blockade with abatacept. Objective To determine whether costimulatory signaling blockade with abatacept prevents psoriasis relapse after ustekinumab withdrawal. Design, Setting, and Participants Psoriasis Treatment with Abatacept and Ustekinumab: a Study of Efficacy (PAUSE), a parallel-design, double-blind, placebo-controlled randomized clinical trial, was conducted at 10 sites in the US and Canada. Participant enrollment opened on March 19, 2014, and concluded on April 11, 2016. Participants were adults with moderate to severe plaque psoriasis and received ustekinumab in a lead-in phase. Those who responded to ustekinumab at week 12 were randomized 1:1 to either the continued with ustekinumab group (ustekinumab group) or the switched to abatacept group (abatacept group). Treatment was discontinued at week 39, and participants were followed up for psoriasis relapse until week 88. Statistical analyses were performed in the intention-to-treat (ITT) and safety samples from May 3, 2018, to July 6, 2021. Interventions Participants received subcutaneous ustekinumab at weeks 0 and 4 (45 mg per dose for those ≤100 kg; 90 mg per dose for those >100 kg). Participants randomized to the abatacept group at week 12 received subcutaneous abatacept, 125 mg weekly, from weeks 12 to 39 and ustekinumab placebo at weeks 16 and 28. Participants randomized to the ustekinumab group received ustekinumab at weeks 16 and 28 and abatacept placebo weekly from weeks 12 to 39. Main Outcomes and Measures The primary end point was the proportion of participants with psoriasis relapse (loss of ≥50% of the initial Psoriasis Area and Severity Index improvement) between weeks 12 and 88. Secondary end points included time to psoriasis relapse, proportion of participants with psoriasis relapse between weeks 12 and 40, and adverse events. The psoriasis transcriptome and serum cytokines were evaluated. Results A total of 108 participants (mean [SD] age, 46.1 [12.1] years; 73 [67.6%] men) were treated with open-label ustekinumab; 91 were randomized to blinded treatment. Similar proportions of participants in the abatacept group and the ustekinumab group relapsed between weeks 12 and 88 (41 of 45 [91.1%] vs 40 of 46 [87.0%];P = .41). Median time to relapse from the last dose of ustekinumab was similar between groups as well: 36 weeks (95% CI, 36-48 weeks) in the abatacept group vs 32 weeks (95% CI, 28-40 weeks) in the ustekinumab group. Similar numbers and rates of adverse events occurred. Abatacept did not maintain suppression of the pathogenic IL-23-mediated psoriasis molecular signature in lesions after ustekinumab withdrawal, and serum IL-19 levels increased. Conclusions and Relevance This parallel-design, double-blind randomized clinical trial found that abatacept did not prevent psoriasis relapse that occurred after ustekinumab withdrawal because it did not completely block the pathogenic psoriasis molecular pathways that led to relapse. Trial Registration ClinicalTrials.gov Identifier:NCT01999868

Summary Background & aims In vitro studies suggest that vitamin D may reduce hepcidin expression and pro-inflammatory cytokine release from monocytes. However, data assessing the vitamin D-mediated effects on iron recycling in healthy individuals are lacking. We aimed to examine the effect of high-dose vitamin D3 on plasma hepcidin, inflammatory cytokine, and ferritin concentrations in healthy adults. Methods This was a pilot, double-blind, placebo-controlled trial in healthy adults (N = 28) randomized to receive a one-time oral dose of 250,000 IU of vitamin D3 or placebo. Between- and within-group differences in plasma hepcidin, pro-inflammatory cytokine [interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1)], and ferritin concentrations at baseline and 1 week were determined using two-sample and paired t-tests, respectively. Results At baseline, plasma 25-hydroxyvitamin D [25(OH)D], hepcidin, pro-inflammatory cytokine, and ferritin concentrations did not differ between the two groups, and greater than 70% of subjects in both groups were vitamin D deficient (25(OH)D Conclusions High-dose vitamin D3 significantly reduced plasma hepcidin concentrations in healthy adults 1 week post-dosing, without a change in plasma pro-inflammatory cytokine or ferritin concentrations. These data suggest that vitamin D may have a role in regulating iron recycling by acting independently of changes in pro-inflammatory markers.

During the Engineering Validation and Engineering Design Activities (EVEDA) phase (2007-2014) of the International Fusion Materials Irradiation Facility (IFMIF), an advanced engineering design of the High Flux Test Module (HFTM) has been developed with the objective to facilitate the controlled irradiation of steel samples in the high flux area directly behind the IFMIF neutron source. The development process addressed included manufacturing techniques, CAD, neutronic, thermal-hydraulic and mechanical analyses complemented by a series of validation activities. Validation included manufacturing of 1:1 parts and mockups, test of prototypes in the FLEX and HELOKA-LP helium loops of KIT for verification of the thermal and mechanical properties, and irradiation of specimen filled capsule prototypes in the BR2 test reactor. The prototyping activities were backed by several R&D studies addressing focused issues like handling of liquid NaK (as filling medium) and insertion of Small Specimen Test Technique (SSTT) specimens into the irradiation capsules. This paper provides an up-todate design description of the HFTM irradiation device, and reports on the achieved performance criteria related to the requirements. Results of the validation activities are accounted for and the most important issues for further development are identified.

Triglyceride (TG) molecules represent the major storage form of fatty acids, and TG metabolism is essential to human health. However, the mechanistic details surrounding TG metabolism are complex and incompletely elucidated. Although it is known that angiopoietin-like protein 8 (ANGPTL8) increases TGs through an ANGPTL3/8 complex that inhibits LPL, the mechanism governing ApoA5, which lowers TGs, has remained elusive. Current hypotheses for how ApoA5 acts include direct stimulation of LPL, facilitation of TG-containing particle uptake, and regulation of hepatic TG secretion. Using immunoprecipitation-MS and Western blotting, biolayer interferometry, functional LPL enzymatic assays, and kinetic analyses of LPL activity, we show that ApoA5 associates with ANGPTL3/8 in human serum and most likely decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition. We also demonstrate that ApoA5 has no direct effect on LPL, nor does it suppress the LPL-inhibitory activities of ANGPTL3, ANGPTL4, or ANGPTL4/8. Importantly, ApoA5 suppression of ANGPTL3/8-mediated LPL inhibition occurred at a molar ratio consistent with the circulating concentrations of ApoA5 and ANGPTL3/8. Because liver X receptor (LXR) agonists decrease ApoA5 expression and cause hypertriglyceridemia, we investigated the effect of the prototypical LXR agonist T0901317 on human primary hepatocytes. We observed that T0901317 modestly stimulated hepatocyte ApoA5 release, but markedly stimulated ANGPTL3/8 secretion. Interestingly, the addition of insulin to T0901317 attenuated ApoA5 secretion, but further increased ANGPTL3/8 secretion. Together, these results reveal a novel intersection of ApoA5 and ANGPTL3/8 in the regulation of TG metabolism and provide a possible explanation for LXR agonist-induced hypertriglyceridemia.

Polyethylene glycol (PEG) represents an effective strategy to improve the pharmacokinetic profile of a molecule as it extends the biotherapeutic's half-life, masks immunogenic epitopes or modifies its distribution. The addition of one or multiple PEG moieties, in either linear or branched form, is known to carry the risk of potentially inducing an immunogenic response against PEG. The importance of accurately quantifying anti-PEG antibodies during a clinical study is well recognized and stems from the fact that anti-PEG antibodies have been shown to negatively impact the efficacy of the biotherapeutic that the PEG is coupled to. As a consequence, sponsors are encouraged to develop immunogenicity assays to assess appropriately the presence of anti-drug antibodies (ADA) against the protein component as well as the PEG. However, detection of anti-PEG antibodies is complicated by a number of technical challenges, including the availability of appropriate positive control material. In addition, the fact that some anti-PEG antibodies are known to circulate as low-affinity IgM, drives the need for an assay able to detect low affinity anti-PEG ADA even in the presence of high concentrations of the biotherapeutic. To address this need, we developed and validated an Affinity Capture Elution (ACE)-AGL assay to detect anti-drug and anti-PEG antibodies. In this assay, which we call ACE-AGL, ADA are captured by biotin-PEG-drug, acid eluted and re-captured on a second plate coated with protein AGL. ADA are then detected using Ruthenium-PEG-drug. The new assay format described is highly sensitive to both anti-drug and anti-PEG antibodies and very drug-tolerant. The ACE-AGL assay is easy to perform and has been successfully validated at two separate CROs. We propose the ACE-AGL format as a valid and effective alternative to the currently available assay methods.

Aim: Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody. Results: Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies. Conclusion: This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

Objective Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by intestinal L-cells which stimulates glucose-dependent insulin secretion. GLP-1 is initially secreted as the active peptide GLP-17–36/7, but rapidly undergoes cleavage by dipeptidyl peptidase 4 (DPP4) to yield the inactive form, GLP-19–36/7. Despite a reduced affinity for the GLP-1 receptor, GLP-19–36/7 may have cardioprotective properties. There is currently no described immunoassay capable of specifically measuring GLP-19–36/7. Design and methods We generated a monoclonal antibody specific for the N-terminal neoepitope of GLP-19–36/7. After affinity maturation, we paired this capture antibody with an anti-total GLP-1 monoclonal detection antibody to create a sandwich ELISA specific for GLP-19–36/7. Results The sandwich ELISA was highly specific for GLP-19–36/7 and did not recognize GLP-17–36 or GLP-17–37. The ELISA exhibited a broad dynamic range and a lower limit of detection (LLOD) of 3.17 ng/L. In healthy volunteers, concentrations of GLP-19–36/7 increased dramatically in the postprandial state compared to the fasted state and were markedly elevated at both 30 and 120-minute postprandial time points. Conclusions The optimization of an N-terminal-specific monoclonal antibody for GLP-19–36/7 enabled the development of a sensitive and specific sandwich ELISA assay capable of measuring physiological concentrations of GLP-19–36/7. This ELISA may have the potential to help expand our knowledge of GLP-1 biology.

Background— The secreted protein proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising new target for lowering plasma low-density lipoprotein cholesterol and preventing cardiovascular disease (CVD). The relationship between circulating PCSK9 and incident CVD in the general population is unknown. We investigated whether serum PCSK9 concentration is associated with incident CVD in a prospective cohort study of 4232 men and women 60 years of age at the time of recruitment. Methods and Results— Incident CVD was recorded by matching to national registries. After 15 years of follow-up, a total of 491 incident events (fatal and nonfatal myocardial infarctions, unstable angina, deaths from coronary heart disease, fatal and nonfatal ischemic strokes) were recorded. Cox proportional hazards model was used to calculate hazard ratios with 95% confidence intervals. Baseline serum PCSK9 concentration predicted incident CVD; concentration in quartile 4 compared with quartile 1 was associated with a hazard ratio of 1.69 (95% confidence interval, 1.30–2.19) after adjustment for sex. Further adjustment for low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, lipoprotein(a), triglycerides, hypertension, diabetes mellitus, smoking, overweight, obesity, physical inactivity, and statin use resulted in a decrease in the hazard ratio to 1.48 (95% confidence interval, 1.12–1.95). Conclusions— Serum PCSK9 concentration is associated with future risk of CVD even after adjustments for established CVD risk factors. Further studies are needed to confirm this observation.

Therapeutic administration of peptides may result in anti-drug antibody (ADA) formation, hypersensitivity adverse events (AEs) and reduced efficacy. As a large peptide, the immunogenicity of once-weekly glucagon-like peptide-1 (GLP-1) receptor agonist dulaglutide is of considerable interest. The present study assessed the incidence of treatment-emergent dulaglutide ADAs, hypersensitivity AEs, injection site reactions (ISRs), and glycaemic control in ADA-positive patients in nine phase II and phase III trials (dulaglutide, N = 4006; exenatide, N = 276; non-GLP-1 comparators, N = 1141). Treatment-emergent dulaglutide ADAs were detected using a solid-phase extraction acid dissociation binding assay. Neutralizing ADAs were detected using a cell-based assay derived from human endothelial kidney cells (HEK293). A total of 64 dulaglutide-treated patients (1.6% of the population) tested ADA-positive versus eight (0.7%) from the non-GLP-1 comparator group. Of these 64 patients, 34 (0.9%) had dulaglutide-neutralizing ADAs, 36 (0.9%) had native-sequence GLP-1 (nsGLP-1) cross-reactive ADAs and four (0.1%) had nsGLP-1 neutralization ADAs. The incidence of hypersensitivity AEs and ISRs was similar in the dulaglutide versus placebo groups. No dulaglutide ADA-positive patient reported hypersensitivity AEs. Because of the low incidence of ADAs, it was not possible to establish their effect on glycaemic control.

Background Transfusion guidelines have become increasingly restrictive. We investigated actual transfusion practices in geriatric hip fracture patients, why they differ from current guidelines and how this affects outcome. Objectives The primary aim was to examine transfusion timing, evaluate how many red blood cell (RBC) transfusions are in keeping with guidelines and how this affects morbidity (infection, cardiac events and delirium), mortality and length of stay (LOS). Our secondary aim was to test the hypothesis that guidelines were more likely to be deviated from shortly before discharge and with consecutive transfusions. Design A retrospective observational study. Setting The Luzerner Kantonsspital, a major trauma centre, over a 12-month period from 1 February 2015 to 31 January 2016. Patients All patients over 70 years of age admitted to the Luzerner Kantonsspital with hip fractures over a 12-month period in 2015 to 2016 were included. Results 156 patients were included, to which 141 units of RBCs were transfused. All pre and intra-operative transfusions were according to guidelines; 110 transfusions were postoperative and 37 of these were not according to guidelines. Patients who were transfused had longer LOS in hospital (P = 0.002) and an odds ratio (OR) of 2.7 of contracting an infection (P = 0.04) in comparison with patients who were not transfused. No significant differences in mortality, LOS or morbidity were found between patients transfused according to guidelines and more liberal thresholds. Guidelines were more likely to be deviated from within the last 2 days before discharge than prior to this (58 vs. 24%, P = 0.03). Furthermore, 24 stable patients received two consecutive RBC units resulting in posttransfusion haemoglobin values of between 83 and 124 g l. Conclusion Most RBC transfusions occur postoperatively, many still according to liberal transfusion thresholds, in particular shortly before discharge and as part of consecutive transfusions. Transfused patients had longer LOS and more infections than patients not transfused, but there was no difference in mortality, LOS or morbidity between patients transfused according to current guidelines and those where guidelines were deviated from.

The objective of our study was to compare the diagnostic performance of nephrographic phase only, excretory phase only, and both nephrographic and excretory phases of CT urography (CTU) for the detection of upper tract urothelial carcinoma.Forty-nine consecutive patients with pathologically proven upper tract urothelial carcinoma who underwent a single-bolus CTU examination were evaluated. Forty-nine control patients with normal findings on two CTU examinations performed at a 1-year interval were included. Two radiologists independently reviewed the 98 CTU examinations at three different sessions (nephrographic phase only, excretory phase only, and both nephrographic and excretory phases simultaneously) and rated the likelihood of the presence of a urothelial carcinoma in each segment of the renal collecting system and ureter using a 5-point scale. Sensitivity, specificity, and AUC of ROC curve were calculated per segment and per patient.A total of 314 segments, 56 of which contained tumors, were evaluated. In the per-segment analysis for reviewers 1 and 2, the sensitivity, specificity, and AUC, respectively, were as follows: 88%, 98%, and 0.95 and 84%, 97%, and 0.94 for the nephrographic phase; 79%, 98%, and 0.91 and 89%, 98%, and 0.95 for the excretory phase; and 88%, 99%, and 0.95 and 89%, 99%, and 0.96 for the combined nephrographic and excretory phases. The AUC of the combined nephrographic and excretory phases was significantly higher than that of the nephrographic phase (per-patient analysis, reviewer 2) and that of excretory phase (per-segment analysis, reviewer 1) but was not significantly different in any other comparisons.The nephrographic and excretory phases are complementary for the detection of upper tract urothelial carcinoma.

Hintergrund Der Pseudotumor cerebri ist eine idiopathische Erhohung des intrakraniellen Drucks, der haufig ubergewichtige Frauen im gebarfahigen Alter betrifft. Zu den diagnostischen Kriterien gehoren laut Leitlinien der Deutschen Gesellschaft fur Neurologie erhohter Liquordruck, Symptome erhohten Liquordrucks, normaler Liquorbefund, keine relevante Medikation sowie der Ausschluss von strukturellen und vaskularen Lasionen in der Magnetresonanztomographie.

Background: FGF21 is involved in glucose and lipid metabolism. Our objective was to develop two novel sandwich immunoassays that measure active and total levels of FGF21 in human plasma. Results: Both immunoassays utilized affinity-optimized monoclonal antibodies generated specifically for either the mid-domain or the intact C-terminus of the respective FGF21 peptides. Total FGF21 levels measured from normal human plasma samples ranged from 42 to 462 pg/ml, while active FGF21 levels ranged from 11 to 399 pg/ml. The data also suggested no significant differences in the concentrations of active FGF21 when measured from pre- and post-prandial samples. Conclusion: We have successfully developed sensitive assays to measure active and total FGF21, which show the majority of total FGF21 in plasma is active FGF21.

Object The aim of this study was to provide evidence for the effect of intrathecal morphine application after spinal cord tumor resection. Methods Twenty patients participated in a prospective open proof-of-concept study. During dural closure, morphine (7 μg/kg) was injected into the subarachnoid space. All patients were monitored in an intensive care setting postoperatively. Pain, additional opioids given, and vital parameters were recorded. Results Six patients received a mean morphine dose of 365 μg between C-3 and C-7 and 14 patients received a mean dose of 436 μg between T-2 and T-12. In the cervical and thoracic groups, the mean Numeric Rating Scale score was highest upon intensive care unit admission (1.2 and 2.5, respectively) and declined at 12 hours (0.5 and 0.8, respectively). Minimal extra morphine was required. Minor side effects occurred without consequence. Conclusions Intrathecal morphine for postoperative analgesia after resection of cervical and thoracic spinal cord tumors is effective and safe. These preliminary results require confirmation by larger comparative studies and further clinical experience.

Data on cytokeratin-18 (K-18) and enhanced liver fibrosis (ELF) score in insulin-treated diabetes patients with non-alcoholic fatty liver disease (NAFLD) are limited. This study analyzed phase III data comparing basal insulin peglispro (BIL) and insulin glargine in type 1 (T1D), and type 2 diabetes (T2D) (insulin-naïve and insulin-treated). Alanine aminotransferase (ALT), K-18, ELF scores and liver fat content (LFC), measured by MRI, were obtained longitudinally. Baseline K-18 (U/L) was higher in T2D (range: 207-247) than T1D (range: 148-183), correlated with ALT in all populations (r (range) 0.264-0.637, p

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of serum low density lipoprotein cholesterol levels. PCSK9 is secreted by the liver and binds the hepatic low density lipoprotein receptor, causing its subsequent degradation. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including very low-density lipoprotein receptor, apolipoprotein E receptor 2, and beta-site amyloid precursor protein-cleaving enzyme 1, which are highly expressed in central nervous system (CNS) and have been implicated in Alzheimer’s disease. Previous studies have demonstrated that human circulating PCSK9 displays a diurnal rhythm. Currently, little is known about PCSK9 levels in human cerebrospinal fluid (CSF). In the present study, we measured PCSK9 concentrations in both serum and CSF collected from healthy human subjects at multiple time points throughout the day. While PCSK9 in serum manifested a distinct diurnal pattern, CSF PCSK9 levels were remarkably constant throughout the course of the day and were also consistently lower than corresponding serum PCSK9 concentrations. Our results indicate that regulation of PCSK9 in human CSF may be different than for plasma PCSK9, suggesting that further study of the role of PCSK9 in the CNS is warranted.

Chronic kidney disease affects 40% of adults aged 65 and older. Anemia of CKD is present in 30% of patients with CKD and is associated with increased cardiovascular risk, decreased quality of life, and increased mortality. Hepcidin-25 (hepcidin), the key iron regulating hormone, prevents iron egress from macrophages and thus prevents normal recycling of the iron needed to support erythropoiesis. Hepcidin levels are increased in adults and children with CKD. Vitamin D insufficiency is highly prevalent in CKD and is associated with erythropoietin hyporesponsiveness. Recently, hepcidin levels were found to be inversely correlated with vitamin D status in CKD. The aim of this study was to investigate the role of vitamin D in the regulation of hepcidin expression in vitro and in vivo . This study reports that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the hormonally active form of vitamin D, is associated with decreased hepcidin and increased ferroportin expression in lipopolysaccharide (LPS) stimulated THP-1 cells. 1,25(OH) 2 D 3 also resulted in a dose-dependent decrease in pro-hepcidin cytokines, IL-6 and IL-1β, release in vitro . Further, we show that high-dose vitamin D therapy impacts systemic hepcidin levels in subjects with early stage CKD. These data suggest that improvement in vitamin D status is associated with lower systemic concentrations of hepcidin in subjects with CKD. In conclusion, vitamin D regulates the hepcidin-ferroportin axis in macrophages which may facilitate iron egress. Improvement in vitamin D status in patients with CKD may reduce systemic hepcidin levels and may ameliorate anemia of CKD.

Objective Hepcidin is regulated by anemia and inflammation. It is primarily expressed in the liver but studies have reported its expression in adipose tissue. The relationship between BMI and serum hepcidin and the relationship between liver histology and serum hepcidin in the morbidly obese was investigated. Methods Serum and liver tissue from patients undergoing bariatric surgery (bariatric cohort, n = 105) and serum from healthy blood donors (n = 60) were used to conduct this study. Serum hepcidin was measured using sandwich ELISA, highly specific for hepcidin-25. Serum ferritin, IL-6, IL-1β and liver function biochemistries were also measured. Results After controlling for covariates, BMI ≥ 35 kg/m2 was significantly associated with higher serum hepcidin level compared to individuals with lower BMI groups (17.7 ± 11.5 vs. 3.3 ± 4.7 ng/ml, P = 0.002). The presence of NAFLD was not associated with higher serum levels of hepcidin (multivariate P = 0.37). There was no association between serum hepcidin levels and liver histology (presence of steatohepatitis, advanced fibrosis, or NAFLD activity score) in the bariatric cohort. Conclusions Obesity, but not the presence of NAFLD was associated with serum hepcidin levels. There was no association between serum hepcidin and liver histology in the morbidly obese undergoing bariatric surgery.

Background Hepcidin-25 regulates iron homeostasis by binding the iron transporter ferroportin, causing its degradation. Increased hepcidin-25 causes decreased intestinal iron absorption and release from intracellular stores. Our objective in this study was to measure hepcidin-25 levels in patients with chronic kidney disease (CKD) to determine if they might contribute to the anemia of CKD. Methods We used a hepcidin-25-specific enzyme-linked immunosorbent assay to measure hepcidin-25 in 103 CKD patients and 100 healthy individuals. We assessed in CKD subjects the correlation of hepcidin-25 with creatinine, estimated glomerular filtration rate (eGFR), hemoglobin, blood urea nitrogen, serum iron, transferrin, and ferritin. Results Hepcidin-25 concentrations in CKD patients were significantly increased compared to healthy subjects (60.4 ± 6.1 μg/l vs. 3.0 ± 0.5 μg/l, P < 0.001). Hepcidin-25 concentrations were directly correlated with creatinine (R = 0.28, P = 0.004) and inversely correlated with eGFR (R = −0.32, P = 0.001). Hepcidin-25 levels were also correlated with transferrin (R = −0.28, P = 0.004) and ferritin (R = 0.80, P < 0.001). Conclusion The direct correlation of hepcidin-25 with creatinine and its inverse correlation with eGFR suggest that hepcidin-25 levels increase as renal function deteriorates, possibly due to decreased hepcidin-25 renal clearance.

The last step in sphingolipid biosynthesis is the conversion of ceramide (Cer) to sphingomyelin (SM), which is catalyzed by sphingomyelin synthase (SMS). Two isoforms of SMS have been identified with differential subcellular localizations. It is not clear whether the two isoforms have any differences in biochemical or cellular SMS activities. This report describes a mass spectrometry (MS)-based method that was used to characterize biochemical and cellular SMS activities of the two isoforms of SMS, namely SMS1 and SMS2. Cellular extracts of SMS1 or SMS2 expressed in SF9 cells displayed significant SMS activity. When these activities were measured by MS, both SMS1 and SMS2 demonstrated similar time- and substrate-dependent SMS activity. A previously reported SMS inhibitor, D609, inhibited both SMS1 and SMS2 activity. In HEK293 cells, overexpression of either SMS1 or SMS2 significantly increased SMS activity. These studies using MS methods to measure SMS activity of SMS1 and SMS2 represent the first quantitative measurement of SMS activities. The establishment of quantitative biochemical and cellular SMS assays may help to facilitate the discovery of novel SMS1- or SMS2-specific inhibitors.

Anemia and vitamin D deficiency are highly prevalent in critical illness, and vitamin D status has been associated with hemoglobin concentrations in epidemiologic studies. We examined the effect of high-dose vitamin D therapy on hemoglobin and hepcidin concentrations in critically ill adults.Mechanically ventilated critically ill adults (N = 30) enrolled in a pilot double-blind, randomized, placebo-controlled trial of high-dose vitamin DAt enrollment,75% of participants in all groups had plasma 25-hydroxyvitamin D (25(OH)D) concentrations30 ng/mL, and85% of participants across groups were anemic. In the 500,000-IU DIn these critically ill adults, treatment with 500,000 IU D

In high power density single-phase PV inverter systems, active auxiliary circuits are installed, shifting the double line-frequency power pulsation away from the dc link to a dedicated buffer capacitor. Being relieved from strict voltage ripple requirements, a larger voltage ripple is allowed at the buffer capacitor, significantly reducing the capacitance requirements and consequently the overall volume of the converter system. Since the capacitance density of electrolytic capacitors must be derated because of imposed current limitations, ceramic capacitors become the preferred choice in power pulsation buffer applications. In particular, TDK's class II X6S MLCC with BaTiO 3 ceramic and the recently launched CeraLink with PLZT ceramic are promising candidates due to their high energy density. The actual prevailing capacitance of these two types of ceramics, strongly depends on the operating point, that is, a dc bias voltage and a superimposed large-signal amplitude ac voltage. Unfortunately, the large-signal behavior and performance of these ceramic capacitors is not specified by the manufacturer. In this paper, a comprehensive characterization of the X6S MLCC and the CeraLink large-signal performance is carried out by means of experimental measurements. The acquired data enables an accurate dimensioning of the power pulsation buffer capacitor and an estimate of the capacitor losses during operation. Since the device temperature has a strong impact on the ceramic properties, results for 30 °C, 60 °C and 90 °C are presented.

The purpose of this study is to assess the complication rate and diagnostic accuracy of percutaneous image-guided core needle biopsy (CNB) of the spleen at a single center over the course of 12 years.A retrospective review of an institutionally maintained biopsy database was used to identify CNBs of the spleen performed between October 2002 and January 2015. Clinical notes were reviewed from the date of biopsy to 3 months after biopsy to ascertain whether any immediate or delayed complications had occurred. Minor complications included pain requiring analgesia and incidental asymptomatic bleeding. Major complications were those scored at or above grade 3 according to the National Institutes of Health's Common Terminology Criteria for Adverse Events, version 4.0. The pathology reports issued for each specimen were evaluated and compared with results obtained from splenectomy, biopsy performed at another anatomic site, or longitudinal clinical or imaging follow-up. Sensitivity, specificity, and accuracy were calculated.A total of 97 CNBs of the spleen were performed, 23 of which were CT guided and 74 of which were ultrasound guided. There were seven (7.2%) minor complications and a single (1.0%) major complication; the overall complication rate was 8.2% (n = 8). The diagnostic yield-defined as adequate tissue to establish a diagnosis-of CNB was 93.8%, the sensitivity was 90.7%, the specificity was 100%, and the accuracy was 94.5%.Percutaneous image-guided CNB of the spleen is safe and effective in achieving a tissue diagnosis.

Objective — Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that influences plasma low-density lipoprotein concentration and susceptibility to coronary heart disease. Circulating PCSK9 levels show considerable interindividual differences, but the factors responsible for this variation are largely unknown. Methods and Results— We analyzed circulating PCSK9 levels in 4 cohorts of healthy, middle-aged Swedes (n=5722) and found that PCSK9 levels varied over ≈50-fold range, showed a positive relationship with plasma low-density lipoprotein–cholesterol concentration, and were associated with plasma triglyceride, fibrinogen, insulin, and glucose concentrations. A genome-wide association study conducted in 2 cohorts (n=1215) failed to uncover common genetic variants robustly associated with variation in circulating PCSK9 level. As expected, the minor allele of the PCSK9 R46L variant was in all cohorts associated with reduced PCSK9 levels and decreased plasma low-density lipoprotein–cholesterol concentrations, but no relationship was observed with the plasma triglyceride concentration. Further mapping of the PCSK9 locus revealed a common polymorphism (rs2479415, minor allele frequency 43.9%), located ≈6 kb upstream from PCSK9 , which is independently associated with increased circulating PCSK9 levels. Conclusion— Common and low-frequency genetic variants in the PCSK9 locus influence the pronounced interindividual variation in circulating PCSK9 levels in healthy, middle-aged white (predominantly Swedish) subjects.

An increasing need in the development of biotherapeutic agents is the ability to monitor a potential autoimmune response to the therapeutic target of interest. Unfortunately, the presence of high concentrations of therapeutic antibody can hinder such detection, because there is competition for binding in cases where epitopes are not structurally distinct. This situation was encountered in the development of LY2062430, a therapeutic mid-domain monoclonal anti-amyloid beta peptide (Aβ) antibody undergoing clinical trials for the treatment of Alzheimer's disease. This communication reports the development and validation of a novel radioimmunoassay used to measure potential patient immune responses to Aβ in the presence of LY2062430. This assay employs a radioiodinated analog of the human amyloid beta 1-40 peptide (Aβ1-40) in which a single amino acid substitution of alanine for phenylalanine at position 19 (F19A) effectively eliminates binding by LY2062430. In contrast, F19A binding by monoclonal antibodies specific for the N- and C-termini of the human Aβ1-40 peptide was shown to be unaltered. Additional experiments involving a polyclonal rabbit antibody raised against the midregion of Aβ1-40 (residues 15-30) resulted in only a slight reduction in binding to the F19A tracer, suggesting that the modification does not affect distal epitopes in Aβ1-40 and supporting the notion that this conservative substitution produces only subtle change in the overall peptide structure. The assay is therefore believed to detect most, if not all, patient antibodies to native Aβ peptides. The assay was validated for use in clinical trials allowing detection of antibodies to Aβ in human serum in the presence of therapeutic concentrations of LY2062430.