Large Molecule Specific Assay Operation: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

The L2 Global Harmonization Team on large molecule specific assay operation for protein bioanalysis in support of pharmacokinetics focused on the following topics: setting up a balanced validation design, specificity testing, selectivity testing, dilutional linearity, hook effect, parallelism, and testing of robustness and ruggedness. The team additionally considered the impact of lipemia, hemolysis, and the presence of endogenous analyte on selectivity assessments as well as the occurrence of hook effect in study samples when no hook effect had been observed during pre-study validation.     

Treatment with lecinoxoids attenuates focal and segmental glomerulosclerosis development in nephrectomized rats

Focal segmental glomerulosclerosis (FSGS) is a scarring process associated with chronic low‐grade inflammation ascribed to toll‐like receptor (TLR) activation and monocyte migration. We developed synthetic, small‐molecule lecinoxoids, VB‐201 and VB‐703, that differentially inhibit TLR‐2‐ and TLR‐4‐mediated activation and monocyte migration. The efficacy of anti‐inflammatory lecinoxoid treatment on FSGS development was explored using a 5/6 nephrectomy rat model. Five‐sixths of nephrectomized rats were treated with lecinoxoids VB‐201, VB‐703 or PBS, for 7 weeks. Upon sacrifice, albumin/creatinine ratio, glomerulosclerosis, fibrosis‐related gene expression and the number of glomerular and interstitial monocyte were evaluated. Treatment of nephrectomized rats with lecinoxoids ameliorated glomerulosclerosis. The percentage of damaged glomeruli, glomerular sclerosis and glomeruli fibrotic score was significantly reduced following VB‐201 and VB‐703 treatment. VB‐703 attenuated the expression of fibrosis hallmark genes collagen, fibronectin (FN) and transforming growth factor β (TGF‐β) in kidneys and improved albumin/creatinine ratio with higher efficacy than did VB‐201, but only VB‐201 significantly reduced the number of glomerular and interstitial monocytes. These results indicate that treatment with TLR‐2, and more prominently, TLR‐4 antagonizing lecinoxioids, is sufficient to significantly inhibit FSGS. Moreover, inhibiting monocyte migration can also contribute to treatment of FSGS. Our data demonstrate that targeting TLR‐2‐TLR‐4 and/or monocyte migration directly affects the priming phase of fibrosis and may consequently perturb disease parthogenesis.     

Transcirculation Silk Vista Baby-assisted coiling in half-T configuration for the treatment of posterior communicating artery aneurysms associated with a fetal posterior circulation: An alternative flow diversion strategy

Flow diverter devices have become a routine first-line option for treatment of an increasing population of intracranial aneurysms at many neurovascular centers. Despite the promising results of flow diverter stents on anterior circulation, incomplete occlusion on the presence of fetal posterior circulation has been described on several reports. Here we describe a novel technical alternative to conventional flow diversion approach for this specific subgroup of aneurysms using the low-profile flow diverter, Silk Vista Baby. The device was selectively placed into the fetal type posterior cerebral artery in half-T configuration for the treatment of a posterior communicating aneurysm using a transcirculation approach through the anterior communicating artery. This represents a useful and effective technique and should be considered when encountering the above-described situation.     

Comparison of faculty versus structured peer-feedback for acquisitions of basic and intermediate-level surgical skills

Purpose

Video feedback and faculty feedback has been shown to improve surgical performance; however, consistent access to faculty is challenging. We studied the utility of structured peer-feedback (PF) compared to faculty-feedback (FF) during acquisition of basic and intermediate surgical skills.

Serum-Mediated Inhibition of Enzyme Replacement Therapy in Fabry Disease

Fabry disease (FD) is a progressive multisystemic disorder, treatable with recombinant enzyme replacement therapy (agalsidase). However, recent studies suggest an endogenous inhibition of agalsidase in patients with FD, as reported for other lysosomal storage diseases. To assess the clinical consequences of serum-mediated agalsidase inhibition in affected patients, we determined the agalsidase inhibition status of 168 patients (68 male) with FD and compared outcomes of inhibition-positive patients with those of inhibition-negative patients. The assessment included clinical events during time on agalsidase, determination of renal and cardiac function, and evaluation of FD-related symptoms. The frequency of serum-mediated agalsidase inhibition was 40% in agalsidase-treated males. Inhibition did not depend on the compound initially used (agalsidase-α or -β). Agalsidase inhibition was associated with higher lyso-globotriaosylceramide levels and worse disease severity scores in patients. Compared with agalsidase inhibition-negative men, agalsidase inhibition-positive men showed greater left ventricular mass (P=0.02) and substantially lower renal function (difference in eGFR of about –30 ml/min per 1.73 m²; P=0.04), which was confirmed by a longitudinal 5-year retrospective analysis. Additionally, affected patients presented more often with FD-typical symptoms, such as diarrhea, fatigue, and neuropathic pain, among others. Therefore, patients with poor clinical outcome on agalsidase should be tested for agalsidase inhibition. Future studies are warranted to determine if affected patients with FD benefit from acute reduction of anti-agalsidase antibodies or long-term immune modulation therapies to suppress agalsidase inhibition and to identify mechanisms that minimize antibody generation against agalsidase.     

Velocity measurement accuracy in optical microhemodynamics: experiment and simulation

Micro particle image velocimetry (μPIV) is a common method to assess flow behavior in blood microvessels in vitro as well as in vivo. The use of red blood cells (RBCs) as tracer particles, as generally considered in vivo, creates a large depth of correlation (DOC), even as large as the vessel itself, which decreases the accuracy of the method. The limitations of μPIV for blood flow measurements based on RBC tracking still have to be evaluated. In this study, in vitro and in silico models were used to understand the effect of the DOC on blood flow measurements using μPIV RBC tracer particles. We therefore employed a μPIV technique to assess blood flow in a 15?μm radius glass tube with a high-speed CMOS camera. The tube was perfused with a sample of 40% hematocrit blood. The flow measured by a cross-correlating speckle tracking technique was compared to the flow rate of the pump. In addition, a three-dimensional mechanical RBC-flow model was used to simulate optical moving speckle at 20% and 40% hematocrits, in 15 and 20?μm radius circular tubes, at different focus planes, flow rates and for various velocity profile shapes. The velocity profiles extracted from the simulated pictures were compared with good agreement with the corresponding velocity profiles implemented in the mechanical model. The flow rates from both the in vitro flow phantom and the mathematical model were accurately measured with less than 10% errors. Simulation results demonstrated that the hematocrit (paired t tests, p = 0.5) and the tube radius (p = 0.1) do not influence the precision of the measured flow rate, whereas the shape of the velocity profile (p < 0.001) and the location of the focus plane (p < 0.001) do, as indicated by measured errors ranging from 3% to 97%. In conclusion, the use of RBCs as tracer particles makes a large DOC and affects the image processing required to estimate the flow velocities. We found that the current μPIV method is acceptable to estimate the flow rate on the condition that the measurement takes place at the equatorial plane of the vessel. Otherwise, it is not an appropriate method to estimate the shape of the velocity profile.