Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice

Previous studies on the effect of glycosylation on the elimination rate of antibodies have produced conflicting results. Here, we performed pharmacokinetic studies in mice with two preparations of a monoclonal IgG1 antibody enriched for complex type or high mannose type oligosaccharides at the Fc glycosylation site. No significant difference in the serum half-life was found between the two antibody glycoforms, nor was any difference observed in the serum half-lives of different complex type glycoforms. To evaluate the influence of glycosylation within the variable domain, a second monoclonal antibody, glycosylated in both the Fc and Fv domains, was separated into fractions containing different amounts of Fv-associated sialic acid and administered to mice. Again, no significant difference was found in the clearance rates of variants carrying different amounts of Fv-associated sialic acid or lacking Fv-glycosylation. These results suggest that glycosylation has little or no impact on the pharmacokinetic behavior of these two monoclonal antibodies in mice.

Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes

Accumulating evidence indicates that agrin, a heparan sulphate proteoglycan of the extracellular matrix, plays a role in the organization and maintenance of the blood-brain barrier. This evidence is based on the differential effects of agrin isoforms on the expression and distribution of the water channel protein, aquaporin-4 (AQP4), on the swelling capacity of cultured astrocytes of neonatal mice and on freeze-fracture data revealing an agrin-dependent clustering of orthogonal arrays of particles (OAPs), the structural equivalent of AQP4. Here, we show that the OAP density in agrin-null mice is dramatically decreased in comparison with wild-types, by using quantitative freeze-fracture analysis of astrocytic membranes. In contrast, anti-AQP4 immunohistochemistry has revealed that the immunoreactivity of the superficial astrocytic endfeet of the agrin-null mouse is comparable with that in wild-type mice. Moreover, in vitro, wild-type and agrin-null astrocytes cultured from mouse embryos at embryonic day 19.5 differ neither in AQP4 immunoreactivity, nor in OAP density in freeze-fracture replicas. Analyses of brain tissue samples and cultured astrocytes by reverse transcription with the polymerase chain reaction have not demonstrated any difference in the level of AQP4 mRNA between wild-type astrocytes and astrocytes from agrin-null mice. Furthermore, we have been unable to detect any difference in the swelling capacity between wild-type and agrin-null astrocytes. These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering.

Precise pose recovery of distal locking holes from single calibrated fluoroscopic image via a novel variable decomposition approach

This paper presents a novel variable decomposition approach for pose recovery of the distal locking holes using single calibrated fluoroscopic image. The problem is formulated as a model-based optimal fitting process, where the control variables are decomposed into two sets: (a) the angle between the nail axis and its projection on the imaging plane, and (b) the translation and rotation of the geometrical model of the distal locking hole around the nail axis. By using an iterative algorithm to find the optimal values of the latter set of variables for any given value of the former variable, we reduce the multiple-dimensional model-based optimal fitting problem to a one-dimensional search along a finite interval. We report the results of our in vitro experiments, which demonstrate that the accuracy of our approach is adequate for successful distal locking of intramedullary nails.

MGOF: Stata module to perform goodness-of-fit tests for multinomial data

mgof computes goodness-of-fit tests for the distribution of a discrete (categorical, multinomial) variable. The default is to perform classical large sample chi-squared approximation tests based on Pearson's X2 statistic and the log likelihood ratio (G2) statistic or a statistic from the Cressie-Read family. Alternatively, mgof computes exact tests using Monte Carlo methods or exhaustive enumeration. A Kolmogorov-Smirnov test for discrete data is also provided. The moremata package, also available from SSC, is required.

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Radiographic technique and anatomy of the equine sacroiliac region

Radiography is part of evaluating horses with poor performance and pelvic limb lameness; however, the radiographic appearance of the sacroiliac region is poorly described. The goal of the present study was to describe the use of a simple technique to obtain radiographs of the sacroiliac region in the anesthetized horse and to describe the radiographic appearance of this region. Seventy-nine horses underwent radiography of the pelvis under general anesthesia in dorsal recumbency. During a 5s exposure time the horse was actively ventilated to blur the abdominal viscera, which allowed assessment of individual bone structures in 77 horses. A large variation in the shape of the sacral wings, their articulation with the transverse processes of L6, and the relation of the sacrum to the ilium were observed. Females had significantly narrower width of the sacral wings. Broad sacral wings and bony proliferations at the caudal aspect were commonly observed features and their size was highly correlated with gender. In males, caudal osteophytes were significantly larger than in females. Five horses had transitional or hemitransitional vertebrae. Radiography with the ventilation-induced blurring technique is a simple approach that results in diagnostic quality radiographs and delineation of the highly variable bone structures of the sacroiliac region.

Loss of astrocyte polarity marks blood-brain barrier impairment during experimental autoimmune encephalomyelitis

In multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE), dysfunction of the blood-brain barrier (BBB) leads to edema formation within the central nervous system. The molecular mechanisms of edema formation in EAE/MS are poorly understood. We hypothesized that edema formation is due to imbalanced water transport across the BBB caused by a disturbed crosstalk between BBB endothelium and astrocytes. Here, we demonstrate at the light microscopic and ultrastructural level, the loss of polarized localization of the water channel protein aquaporin-4 (AQP4) in astrocytic endfeet surrounding microvessels during EAE. AQP4 was found to be redistributed over the entire astrocytic cell surface and lost its arrangement in orthogonal arrays of intramembranous particles as seen in the freeze-fracture replica. In addition, immunostaining for the astrocytic extracellular matrix receptor beta-dystroglycan disappeared from astroglial membranes in the vicinity of inflammatory cuffs, whereas immunostaining for the dystroglycan ligands agrin and laminin in the perivascular basement membrane remained unchanged. Our data suggest that during EAE, loss of beta-dystroglycan-mediated astrocyte foot process anchoring to the basement membrane leads to loss of polarized AQP4 localization in astrocytic endfeet, and thus to edema formation in EAE.

Bone Volume Fraction and Fabric Anisotropy Are Better Determinants of Trabecular Bone Stiffness than Other Morphological Variables

As our population ages, more individuals suffer from osteoporosis. This disease leads to impaired trabecular architecture and increased fracture risk. It is essential to understand how morphological and mechanical properties of the cancellous bone are related. Morphologyelasticity relationships based on bone volume fraction (BV/TV) and fabric anisotropy explain up to 98% of the variation in elastic properties. Yet, other morphological variables such as individual trabeculae segmentation (ITS) and trabecular bone score (TBS) could improve the stiffness predictions. A total of 743 micro-computed tomography reconstructions of cubic trabecular bone samples extracted from femur, radius, vertebrae and iliac crest were analysed. Their morphology was assessed via 25 variables and their stiffness tensor (inline image) was computed from six independent load cases using micro finite element analyses. Variance inflation factors were calculated to evaluate collinearity between morphological variables and decide upon their inclusion in morphology-elasticity relationships. The statistically admissible morphological variables were included in a multi-linear regression modelling the dependent variable inline image. The contribution of each independent variable was evaluated (ANOVA). Our results show that BV/TV is the best determinant of inline image (inline image=0.889), especially in combination with fabric (inline image=0.968). Including the other independent predictors hardly affected the amount of variance explained by the model (inline image=0.975). Across all anatomical sites, BV/TV explained 87% of the variance of the bone elastic properties. Fabric further described 10% of the bone stiffness, but the improvement in variance explanation by adding other independent factors was marginal (<1%). These findings confirm that BV/TV and fabric are the best determinants of trabecular bone stiffness and show, against common belief, that other morphological variables do not bring any further contribution. These overall conclusions remain to be confirmed for specific bone diseases and post-elastic properties.

Social Salience and the Sociolinguistic Monitor: a Case Study of ING and TH-fronting in Britain

This article examines the role of social salience, or the relative ability of a linguistic variable to evoke social meaning, in structuring listeners’ perceptions of quantitative sociolinguistic distributions. Building on the foundational work of Labov et al. (2006, 2011) on the “sociolinguistic monitor” (a proposed cognitive mechanism responsible for sociolinguistic perception), we examine whether listeners’ evaluative judgments of speech change as a function of the type of variable presented. We consider two variables in British English, ING and TH-fronting, which we argue differ in their relative social salience. Replicating the design of Labov et al.’s studies, we test 149 British listeners’ reactions to different quantitative distributions of these variables. Our experiments elicit a very different pattern of perceptual responses than those reported previously. In particular, our results suggest that a variable’s social salience determines both whether and how it is perceptually evaluated. We argue that this finding is crucial for understanding how sociolinguistic information is cognitively processed.

Essays on behavioral aspects of the design and disclosure of compensation contracts

This thesis consists of four essays on the design and disclosure of compensation contracts. Essays 1, 2 and 3 focus on behavioral aspects of mandatory compensation disclosure rules and of contract negotiations in agency relationships. The three experimental studies develop psychology- based theory and present results that deviate from standard economic predictions. Furthermore, the results of Essay 1 and 2 also have implications for firms’ discretion in how to communicate their top management’s incentives to the capital market. Essay 4 analyzes the role of fairness perceptions for the evaluation of executive compensation. For this purpose, two surveys targeting representative eligible voters as well as investment professionals were conducted. Essay 1 investigates the role of the detailed ‘Compensation Discussion and Analysis’, which is part of the Security and Exchange Commission’s 2006 regulation, on investors’ evaluations of executive performance. Compensation disclosure complying with this regulation clarifies the relationship between realized reported compensation and the underlying performance measures and their target achievement levels. The experimental findings suggest that the salient presentation of executives’ incentives inherent in the ‘Compensation Discussion and Analysis’ makes investors’ performance evaluations less outcome dependent. Therefore, investors’ judgment and investment decisions might be less affected by noisy environmental factors that drive financial performance. The results also suggest that fairness perceptions of compensation contracts are essential for investors’ performance evaluations in that more transparent disclosure increases the perceived fairness of compensation and the performance evaluation of managers who are not responsible for a bad financial performance. These results have important practical implications as firms might choose to communicate their top management’s incentive compensation more transparently in order to benefit from less volatile expectations about their future performance. Similar to the first experiment, the experiment described in Essay 2 addresses the question of more transparent compensation disclosure. However, other than the first experiment, the second experiment does not analyze the effect of a more salient presentation of contract information but the informational effect of contract information itself. For this purpose, the experiment tests two conditions in which the assessment of the compensation contracts’ incentive compatibility, which determines executive effort, is either possible or not. On the one hand, the results suggest that the quality of investors’ expectations about executive effort is improved, but on the other hand investors might over-adjust their prior expectations about executive effort if being confronted with an unexpected financial performance and under-adjust if the financial performance confirms their prior expectations. Therefore, in the experiment, more transparent compensation disclosure does not lead to more correct overall judgments of executive effort and to even lower processing quality of outcome information. These results add to the literature on disclosure which predominantly advocates more transparency. The findings of the experiment however, identify decreased information processing quality as a relevant disclosure cost category. Firms might therefore carefully evaluate the additional costs and benefits of more transparent compensation disclosure. Together with the results from the experiment in Essay 1, the two experiments on compensation disclosure imply that firms should rather focus on their discretion how to present their compensation disclosure to benefit from investors’ improved fairness perceptions and their spill-over on performance evaluation. Essay 3 studies the behavioral effects of contextual factors in recruitment processes that do not affect the employer’s or the applicant’s bargaining power from a standard economic perspective. In particular, the experiment studies two common characteristics of recruitment processes: Pre-contractual competition among job applicants and job applicants’ non-binding effort announcements as they might be made during job interviews. Despite the standard economic irrelevance of these factors, the experiment develops theory regarding the behavioral effects on employees’ subsequent effort provision and the employers’ contract design choices. The experimental findings largely support the predictions. More specifically, the results suggest that firms can benefit from increased effort and, therefore, may generate higher profits. Further, firms may seize a larger share of the employment relationship’s profit by highlighting the competitive aspects of the recruitment process and by requiring applicants to make announcements about their future effort. Finally, Essay 4 studies the role of fairness perceptions for the public evaluation of executive compensation. Although economic criteria for the design of incentive compensation generally do not make restrictive recommendations with regard to the amount of compensation, fairness perceptions might be relevant from the perspective of firms and standard setters. This is because behavioral theory has identified fairness as an important determinant of individuals’ judgment and decisions. However, although fairness concerns about executive compensation are often stated in the popular media and even in the literature, evidence on the meaning of fairness in the context of executive compensation is scarce and ambiguous. In order to inform practitioners and standard setters whether fairness concerns are exclusive to non-professionals or relevant for investment professionals as well, the two surveys presented in Essay 4 aim to find commonalities in the opinions of representative eligible voters and investments professionals. The results suggest that fairness is an important criterion for both groups. Especially, exposure to risk in the form of the variable compensation share is an important criterion shared by both groups. The higher the assumed variable share, the higher is the compensation amount to be perceived as fair. However, to a large extent, opinions on executive compensation depend on personality characteristics, and to some extent, investment professionals’ perceptions deviate systematically from those of non-professionals. The findings imply that firms might benefit from emphasizing the riskiness of their managers’ variable pay components and, therefore, the findings are also in line with those of Essay 1.

Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.

Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.

Time variable Earth’s gravity field from SLR satellites

The time variable Earth’s gravity field contains information about the mass transport within the system Earth, i.e., the relationship between mass variations in the atmosphere, oceans, land hydrology, and ice sheets. For many years, satellite laser ranging (SLR) observations to geodetic satellites have provided valuable information of the low-degree coefficients of the Earth’s gravity field. Today, the Gravity Recovery and Climate Experiment (GRACE) mission is the major source of information for the time variable field of a high spatial resolution. We recover the low-degree coefficients of the time variable Earth’s gravity field using SLR observations up to nine geodetic satellites: LAGEOS-1, LAGEOS-2, Starlette, Stella, AJISAI, LARES, Larets, BLITS, and Beacon-C. We estimate monthly gravity field coefficients up to degree and order 10/10 for the time span 2003–2013 and we compare the results with the GRACE-derived gravity field coefficients. We show that not only degree-2 gravity field coefficients can be well determined from SLR, but also other coefficients up to degree 10 using the combination of short 1-day arcs for low orbiting satellites and 10-day arcs for LAGEOS-1/2. In this way, LAGEOS-1/2 allow recovering zonal terms, which are associated with long-term satellite orbit perturbations, whereas the tesseral and sectorial terms benefit most from low orbiting satellites, whose orbit modeling deficiencies are minimized due to short 1-day arcs. The amplitudes of the annual signal in the low-degree gravity field coefficients derived from SLR agree with GRACE K-band results at a level of 77 %. This implies that SLR has a great potential to fill the gap between the current GRACE and the future GRACE Follow-On mission for recovering of the seasonal variations and secular trends of the longest wavelengths in gravity field, which are associated with the large-scale mass transport in the system Earth.

Prediction of Post-Weaning Fibrinogen Status during Cardiopulmonary Bypass: An Observational Study in 110 Patients.

BACKGROUND After cardiac surgery with cardiopulmonary bypass (CPB), acquired coagulopathy often leads to post-CPB bleeding. Though multifactorial in origin, this coagulopathy is often aggravated by deficient fibrinogen levels. OBJECTIVE To assess whether laboratory and thrombelastometric testing on CPB can predict plasma fibrinogen immediately after CPB weaning. PATIENTS / METHODS This prospective study in 110 patients undergoing major cardiovascular surgery at risk of post-CPB bleeding compares fibrinogen level (Clauss method) and function (fibrin-specific thrombelastometry) in order to study the predictability of their course early after termination of CPB. Linear regression analysis and receiver operating characteristics were used to determine correlations and predictive accuracy. RESULTS Quantitative estimation of post-CPB Clauss fibrinogen from on-CPB fibrinogen was feasible with small bias (+0.19 g/l), but with poor precision and a percentage of error >30%. A clinically useful alternative approach was developed by using on-CPB A10 to predict a Clauss fibrinogen range of interest instead of a discrete level. An on-CPB A10 ≤10 mm identified patients with a post-CPB Clauss fibrinogen of ≤1.5 g/l with a sensitivity of 0.99 and a positive predictive value of 0.60; it also identified those without a post-CPB Clauss fibrinogen <2.0 g/l with a specificity of 0.83. CONCLUSIONS When measured on CPB prior to weaning, a FIBTEM A10 ≤10 mm is an early alert for post-CPB fibrinogen levels below or within the substitution range (1.5-2.0 g/l) recommended in case of post-CPB coagulopathic bleeding. This helps to minimize the delay to data-based hemostatic management after weaning from CPB.

Detection of one VH antibody sequence in both healthy donors and urticaria patients.

We have previously isolated anti-FcepsilonRIalpha autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMalpha15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMalpha15 variable heavy (V(H)) chain with a slightly mutated variable light (V(L)) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcepsilonRIalpha autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMalpha15V(H) in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) TaqMan probe that specifically detected the LTMalpha15V(H). We were able to demonstrate a broad range of rearranged V(H) gene copy number without any correlation to the state of health. Monitoring LTMalpha15V(H) gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMalpha15V(H). We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level.