The Contribution of Liver Sinusoidal Endothelial Cells to Clearance of Therapeutic Antibody

Many chronic inflammatory diseases are treated by administration of “biological” therapies in terms of fully human and humanized monoclonal antibodies or Fc fusion proteins. These tools have widespread efficacy and are favored because they generally exhibit high specificity for target with a low toxicity. However, the design of clinically applicable humanized antibodies is complicated by the need to circumvent normal antibody clearance mechanisms to maintain therapeutic dosing, whilst avoiding development of off target antibody dependent cellular toxicity. Classically, professional phagocytic immune cells are responsible for scavenging and clearance of antibody via interactions with the Fc portion. Immune cells such as macrophages, monocytes, and neutrophils express Fc receptor subsets, such as the FcγR that can then clear immune complexes. Another, the neonatal Fc receptor (FcRn) is key to clearance of IgG in vivo and serum half-life of antibody is explicitly linked to function of this receptor. The liver is a site of significant expression of FcRn and indeed several hepatic cell populations including Kupffer cells and liver sinusoidal endothelial cells (LSEC), play key roles in antibody clearance. This combined with the fact that the liver is a highly perfused organ with a relatively permissive microcirculation means that hepatic binding of antibody has a significant effect on pharmacokinetics of clearance. Liver disease can alter systemic distribution or pharmacokinetics of antibody-based therapies and impact on clinical effectiveness, however, few studies document the changes in key membrane receptors involved in antibody clearance across the spectrum of liver disease. Similarly, the individual contribution of LSEC scavenger receptors to antibody clearance in a healthy or chronically diseased organ is not well characterized. This is an important omission since pharmacokinetic studies of antibody distribution are often based on studies in healthy individuals and thus may not reflect the picture in an aging or chronically diseased population. Therefore, in this review we consider the expression and function of key antibody-binding receptors on LSEC, and the features of therapeutic antibodies which may accentuate clearance by the liver. We then discuss the implications of this for the design and utility of monoclonal antibody-based therapies.

Clinical prediction rules for childhood UTIs: a cross-sectional study in ambulatory care

BackgroundDiagnosing childhood urinary tract infections (UTI) is challenging.AimValidate clinical prediction rules (UTIcalc, DUTY, Gorelick) for paediatric UTIs in primary care.Design & settingPost-hoc analysis of a cross-sectional study in 39 general practices and 2 emergency departments (Belgium, March 2019 to March 2020).MethodPhysicians recruited acutely ill children ≤18 years and sampled urine systematically for culture. Per rule, we performed an apparent validation; calculated sensitivities and specificities with 95%CI per threshold in the target group. For the DUTY coefficient-based algorithm, we performed a logistic calibration and calculated the Area Under the Curve with 95%CI.ResultsOf 834 children ≤18 years recruited, there were 297 children <5 years. The UTIcalc and Gorelick score had high to moderate sensitivity and low specificity (UTIcalc ≥2%) 75%; and 16% respectively; Gorelick (≥2 variables) 91%; and 8%. In contrast, the DUTY score ≥5 points had low sensitivity (8%), but high specificity (99%). Urine samples would be obtained in 72% vs 38% (UTIcalc), 92% vs 38% (Gorelick) or 1% vs 32% (DUTY) of children, compared to routine care. The number of missed infections per score was 1/4 (UTIcalc), 2/23 (Gorelick) and 24/26 (DUTY). The UTIcalc+ dipstick model had high sensitivity and specificity (100%; and 91%); resulting in no missed cases and 59% (95%CI 49%–68%) of antibiotics prescribed inappropriately.ConclusionIn this study, the UTIcalc and Gorelick score were useful for ruling out UTI but resulted in high urine sampling rates. The DUTY score had low sensitivity, meaning that 92% of UTIs would be missed.

Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization.

A Retrospective Analysis of Serous Effusions Based on the Newly Proposed International System for Reporting Serous Fluid Cytopathology: A Report of 3633 Cases in an Oncological Center

Background: The International System for Reporting Serous Fluid Cytopathology (TIS) was recently proposed. We retrospectively applied TIS recommendations for reporting the cytological diagnosis of serous effusions and reported our experience.Methods: All the serous effusions from January 2018 to September 2021 were retrieved from the database. Recategorization was performed using the TIS classification, the risk of malignancy (ROM) was calculated for each TIS category, and the performance evaluation was carried out among different samples (pleural, peritoneal and pericardial effusions) and preparation methods (conventional smears, liquid-based preparations and cell-blocks). Results: A total of 3633 cases were studied, 17 (0.5%) were diagnosed as ND, 1100 (30.3%) as NFM, 101 (2.8%) as AUS, 677 (18.6%) as SFM, and 1738 (47.8%) as MAL. The ROM for the categories were found to be 38.5%, 28.6%, 52.1%, 99.4% and 100%, respectively. The ROM for SFM was significantly higher than that for AUS (P < 0.001), while the difference between the ROMs for the IVa and IVb was insignificant. The sensitivity, negative predictive value(NPV) and diagnostic accuracy of liquid-based preparations were all superior to conventional smears and cell-blocks in detecting abnormalities. Using three preparation methods simultaneously had the highest sensitivity, NPV and diagnostic accuracy.Conclusion: Serous effusion cytology has a high specificity and positive predictive value(PPV), and the TIS is a user-friendly reporting system. Liquid-based preparations could improve the sensitivity of diagnosis, and it is best to use three different preparation methods simultaneously for serous effusion cytologic examination.

Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection

Background The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.

A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

Comparison Accuracy in mSIRS, NEWS and qSOFA Score to Triage Sepsis Patients in The Emergency Department at a Hospital in Thailand

Objective: We aimed to apply the modified systemic inflammatory response syndrome (mSIRS), the quick sequential organ failure assessment score (qSOFA), and National Early Warning Score (NEWS) to triage suspected sepsis patients. Therefore, knowing the predictive performance of each scoring system, using given cut-points for triaging patients with suspected sepsis, could help predict the progression of sepsis.Material and Methods: This study is a single-center retrospective chart review. The study enrolled patients older than 18 years with suspected sepsis patient at the time they presented at the triage zone. The primary outcome was to determine which scoring system were the most accurate to triage sepsis patients. The secondary outcomes were predictions of mortality related to the scoring.Results: Considering the outcome to be represented by a SOFA score of ≥2, the area under the curve of the receiver operating characteristic curves for the entire range of mSIRS, qSOFA and NEWS were 0.494, 0.669 and 0.751, respectively. Using a cut point for qSOFA of ≥2 provided a low sensitivity of 36.2% and high specificity of 93.0%; whereas, using a cut point for NEWS of >4 provided a high sensitivity of 89.0% and low specificity of 33.0%.Conclusion: In summary, qSOFA is the most accurate scoring system for diagnosis sepsis which was consistent with previous study. However, qSOFA had the lowest sensitivity, so is not appropriate in a triage situation. Therefore, we decided to use NEWS as the triage tool because of its better sensitivity and acceptable specificity as we need to triage almost all possible cases.

PCR-free, label-free detection of sequence-specific DNA with single-molecule sensitivity using in vitro N-hybrid system in microfluidic drops

We propose a novel method that can detect DNA with high specificity at the single-molecule level by employing the in vitro N-hybrid strategy realized in sub-picoliter microfluidic drops. It detects target DNA based on the specific interactions of the target-encoded proteins with their partner molecules, and achieves single-molecule sensitivity via signal-transduction and signal-amplification during gene-expression processes in a sub-picoliter droplet, therefore effectively avoiding complicated procedures in labeling-based methods or biases and artifacts in PCR-based methods.

A proximity biotinylation-based approach to identify protein-E3 ligase interactions induced by PROTACs and molecular glues

Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identify biotinylated peptides of well-known neo-substrates by mass spectrometry with high specificity and selectivity. Additional analyses reveal ZMYM2 and ZMYM2-FGFR1 fusion protein—responsible for the 8p11 syndrome involved in acute myeloid leukaemia—as CRBN neo-substrates. Furthermore, AirID-DCAF15 and AirID-CRBN biotinylate neo-substrates targeted by indisulam and PROTACs, respectively, suggesting that this approach has the potential to serve as a general strategy for characterizing drug-inducible protein–protein interactions in cells.