Evaluating the clinical utility of measuring levels of factor H and the related proteins.

After years of disappointing clinical results, the tide has finally changed and complement targeted-therapies have become a validated and accepted treatment option for several diseases. These accomplishments have revitalized the field and brought renewed attention to the prospects that complement therapeutics can offer. Streamlining diagnostics and therapeutics is imperative in this new era of clinical use of complement therapeutics. However, the incredible success in therapeutics has not been accompanied by the development of novel standardized tools for complement testing. Complement biomarkers can assist in the risk assessment and diagnosis of diseases as well as the prediction of disease progression and treatment response. Recently, a group of complement proteins has been suggested to be highly relevant in various complement-associated disorders, namely the human factor H (FH) protein family. This family of closely related proteins consists of FH, FH-like protein 1, and five factor H-related proteins, and they have been linked to eye, kidney, infectious, vascular, and autoimmune diseases as well as cancer. The goal of this review is to provide a comprehensive overview of the available data on circulating levels of FH and its related proteins in different pathologies. In addition, we examined the current literature to determine the clinical utility of measuring levels of the FH protein family in health and disease. Finally, we discuss future steps that are needed to make their clinical translation a reality.

Corrigendum: A Family Affair: Addressing the Challenges of Factor H and the Related Proteins.

[This corrects the article DOI: 10.3389/fimmu.2021.660194.].

Highly sensitive interleukin 6 detection by employing commercially ready liposomes in an LFA format.

Recent years have confirmed the ubiquitous applicability of lateral flow assays (LFA) in point-of-care testing (POCT). To make this technology available for low abundance analytes, strategies towards lower limits of detections (LOD), while maintaining the LFA's ease of use, are still being sought. Here, we demonstrate how liposomes can significantly improve the LOD of traditional gold nanoparticle (AuNP)-based assays while fully supporting a ready-to-use system for commercial application. We fine-tuned liposomes towards photometric and fluorescence performance on the synthesis level and applied them in an established interleukin 6 (IL-6) immunoassay normally using commercial AuNP labels. IL-6's low abundance (< 10 pg mL-1) and increasing relevance as prognostic marker for infections make it an ideal model analyte. It was found that liposomes with a high encapsulant load (150 mmol L-1 sulforhodamine B (SRB)) easily outperform AuNPs in photometric LFAs. Specifically, liposomes with 350 nm in diameter yield a lower LOD even in complex matrices such as human serum below the clinically relevant range (7 pg mL-1) beating AuNP by over an order of magnitude (81 pg mL-1). When dehydrated on the strip, liposomes maintained their signal performance for over a year even when stored at ambient temperature and indicate extraordinary stability of up to 8 years when stored as liquid. Whereas no LOD improvement was obtained by exploiting the liposomes' fluorescence, an extraordinary gain in signal intensity was achieved upon lysis which is a promising feature for high-resolution and low-cost detection devices. Minimizing the procedural steps by inherently fluorescent liposomes, however, is not feasible. Finally, liposomes are ready for commercial applications as they are easy to mass-produce and can simply be substituted for the ubiquitously used AuNPs in the POCT market.

"CHicable" and "Clickable" Copolymers for Network Formation and Surface Modification.

In this study, we present the generation of novel, multifunctional polymer networks through a combination of C,H-insertion cross-linking (CHic) and click chemistry. To this, copolymers consisting of hydrophilic N,N-dimethylacrylamide as matrix component and repeat units containing azide moieties, as well as benzophenone or anthraquinone groups, are generated. The benzophenone or anthraquinone groups allow photo-cross-linking, surface attachment or covalent immobilization of adjacent (bio)molecules through CHic reactions. The azide moieties either can react with available alkynes through conventional click reactions or can be activated to form nitrenes, which can also undergo CHic reactions. By choosing appropriate reaction conditions, the same polymer can be used to follow very different reaction paths, opening up a plethora of choices for the generation of functional polymer networks. In the exemplary presented case ("CHic-Click"), irradiation of the copolymers with UV-A light (λirr = 365 nm) leads to cross-linking (network formation) and surface attachment simultaneously. The azide units remain intact during this cross-linking step, and alkyne-modified (bio)molecules can be bound through click reactions. Biofunctionalization of the polymer network with alkynylated streptavidin, followed by application of biotin-conjugated antibody and a model analyte, highlights the potential of these surface architectures as a toolbox which can be adapted for diverse bioanalytical applications.

A Family Affair: Addressing the Challenges of Factor H and the Related Proteins.

Inflammation is a common denominator of diseases. The complement system, an intrinsic part of the innate immune system, is a key driver of inflammation in numerous disorders. Recently, a family of proteins has been suggested to be of vital importance in conditions characterized by complement dysregulation: the human Factor H (FH) family. This group of proteins consists of FH, Factor H-like protein 1 and five Factor H-related proteins. The FH family has been linked to infectious, vascular, eye, kidney and autoimmune diseases. In contrast to FH, the functions of the other highly homologous proteins are largely unknown and, hence, their role in the different disease-specific pathogenic mechanisms remains elusive. In this perspective review, we address the major challenges ahead in this emerging area, including 1) the controversies about the functional roles of the FH protein family, 2) the discrepancies in quantification of the FH protein family, 3) the unmet needs for validated tools and 4) limitations of animal models. Next, we also discuss the opportunities that exist for the immunology community. A strong multidisciplinary approach is required to solve these obstacles and is only possible through interdisciplinary collaboration between biologists, chemists, geneticists and physicians. We position this review in light of our own perspective, as principal investigators of the SciFiMed Consortium, a consortium aiming to create a comprehensive analytical system for the quantitative and functional assessment of the entire FH protein family.

Hyperflexion and Femoral Interference Screw Insertion in ACL Reconstruction.

BACKGROUND: In anatomic anterior cruciate ligament (ACL) reconstructions produced with flexible reamers and no knee hyperflexion, it is unknown whether knee hyperflexion is necessary for femoral interference screw insertion. PURPOSE: To compare femoral screw-graft divergence in anatomic ACL reconstructions with endoscopic interference screws placed without knee hyperflexion and with the use of flexible versus rigid screwdrivers. STUDY DESIGN: Controlled laboratory study. METHODS: Ten matched pairs of cadaveric knees had bone-tendon-bone graft ACL reconstructions with anatomic femoral tunnels. The knees were flexed to 90°. Femoral interference screws (7 × 20 mm) were placed in pairs of knees: in 1 knee with a flexible screwdriver and in the opposite knee with a rigid screwdriver. Graft-screw divergence was imaged with computed tomography scans and tested with cyclic and static biomechanical tests. RESULTS: The mean screw-graft divergence was 12.07° ± 4.04° with the rigid screwdriver and 10.68° ± 3.23° with the flexible screwdriver (P = .35). The cyclic tests with screws placed by a rigid screwdriver had a mean increase in displacement of 0.56 ± 0.20 mm. For screws placed with the flexible screwdriver, the mean increase in displacement was 0.58 ± 0.32 mm (P = .66). Yield load was 393.3 ± 95.1 N for screws placed by a rigid screwdriver and 408.2 ± 119.0 N for screws inserted with the flexible screwdriver (P = .78). Maximum load was 523.1 ± 88.7 N for screws placed by a rigid screwdriver and 467.1 ± 107.3 N for screws inserted with the flexible screwdriver (P = .09). CONCLUSION: With either a rigid or a flexible screwdriver, there were no significant effects on screw divergence or fixation strength. CLINICAL RELEVANCE: Knees can be kept at 90° during endoscopic femoral interference screw insertion. The use of a traditional rigid or flexible screwdriver will not affect screw-graft divergence or fixation strength.

Editorial Commentary: Size Does Matter-Anterior Cruciate Ligament Graft Diameter Affects Biomechanical and Clinical Outcomes.

Anterior cruciate ligament (ACL) graft strength is related to graft diameter and how ACL grafts heal. All grafts appear to lose strength during healing. Clinical studies have documented that hamstring grafts less than 8 mm wide are more vulnerable to failure. Tripling the semitendinosus allows to increase the graft diameter and strength. A recent study documents a semitendinosus tripling technique with excellent clinical results.

Validation of a Fluorescence Sensor Microtiterplate for Biogenic Amines in Meat and Cheese.

An optical sensor microtiterplate for quantitative analysis of the total content of biogenic amines (TAC) in meat and cheese was developed and validated for the first time. In the plate, a chameleon dye (Py-1) is embedded in a polymeric cocktail which is deposited on the bottom of the wells in a common microtiterplate. On reaction with biogenic amines (BAs), the fluorescence of Py-1 at 620 nm rapidly delivers a precise TAC. After 10 min incubation at 25 °C the determination of the TAC in various (real) samples is possible in high-throughput with a standard microplate reader. The optimized fluorescence method was validated for linearity, sensitivity, accuracy, precision (intraday and inter day repeatability) and recovery using histamine (HIS) as a representative BA. The sensor microtiterplate was successfully applied to quantitatively analyze the TAC in 10 real samples of cheese and meat obtained from various Egyptian markets. The TAC of these real samples obtained by the sensor microtiterplate was validated against the contents of BAs obtained by GC-MS at various times of storage. The data of the sensor microtiterplate agreed well with those of GC-MS. This demonstrates that the sensor microtiterplate is a reliable screening tool for the degradation status of food samples.

Enzyme-Based Test Strips for Visual or Photographic Detection and Quantitation of Gaseous Sulfur Mustard.

Sulfur mustard is a chemical agent of high military and terroristic significance. No effective antidote exists, and sulfur mustard can be fairly easily produced in large quantity. Rapid field testing of sulfur mustard is highly desirable. Existing analytical devices for its detection are available but can suffer from low selectivity, laborious sample preparation, and/or the need for complex instrumentation. We describe a new kind of test strip for rapid detection of gaseous sulfur mustard that is based on its degradation by the enzyme haloalkane dehalogenase that is accompanied by a change of local pH. This change can be detected using pH indicators contained in the strips whose color changes from blue-green to yellow within 10 min. In addition to visual read-out, we also demonstrate quantitative reflectometric readout by using a conventional digital camera based on red-green-blue data acquisition. Organic haloalkanes, such as 1,2-dichloroethane, have a negligible interfering effect. The visual limit of detection is 20 µg/L, and the one for red-green-blue read-out is as low as 3 µg/L. The assays have good reproducibility ±6% and ±2% for interday assays and intraday assays, respectively. The strips can be stored for at least 6 months without loss of function. They are disposable and can be produced fairly rapidly and at low costs. Hence, they represent a promising tool for in-field detection of sulfur mustard.

Height and Depth Guidelines for Anatomic Femoral Tunnels in Anterior Cruciate Ligament Reconstruction: A Cadaveric Study.

PURPOSE: To develop guidelines for femoral tunnel placement based on height and depth on the lateral wall of the notch and to apply these guidelines arthroscopically to show tunnel placements within the anterior cruciate ligament (ACL) femoral insertion site. METHODS: Twelve cadaveric knees were dissected to define the centers of the femoral ACL attachment and its anteromedial (AM) and posterolateral (PL) bundles. In 90° of flexion, the height and depth of each center were determined relative to the low point on the lateral intercondylar notch. Radiographic grid measurements were made to validate these measurements. Subsequently, the measurement guidelines were applied arthroscopically in 10 new cadaveric knees to evaluate their accuracy for an anatomic single-bundle femoral tunnel. Interobserver reliability analysis was evaluated with the intraclass correlation coefficient. RESULTS: In 90° of flexion, the height of the ACL center was 8.7 ± 0.6 mm from the low point of the lateral notch; PL center, 7.2 ± 1.2 mm; and AM center, 9.6 ± 1.1 mm. Relative to the low point, the ACL center was 1.7 ± 1.7 mm posterior, the PL center was 3.4 ± 1.5 mm anterior, and the AM center was 4.9 ± 1.7 mm posterior (intraclass correlation coefficient, 0.859). Radiographic grid measurements were consistent with the direct measurements. Application of the guidelines arthroscopically with or without the assistance of a 7-mm offset aimer placed all guide pins for tunnels within the femoral ACL footprint, with 90% within 4 mm of the ACL center. CONCLUSIONS: This study showed in cadaveric knees in 90° of flexion that the center of the ACL can be located on the lateral notch at a height of 8.7 ± 0.6 mm from the lowest point and anterior 11.5 ± 1.3 mm from the deepest point. How anatomic tunnels can be placed using these measurements was also shown in cadaveric knees. CLINICAL RELEVANCE: An anatomic femoral tunnel for ACL reconstruction can be placed using height and depth guidelines.

Full-Contact Practice and Injuries in College Football.

BACKGROUND: Despite recent restrictions being placed on practice in college football, there are little data to correlate such changes with injuries. HYPOTHESIS: Football injuries will correlate with a team's exposure to full-contact practice, total practice, and total games. STUDY DESIGN: Descriptive epidemiological study. METHODS: All injuries and athlete injury exposures (AE × Min = athletes exposed × activity duration in minutes) were recorded for an intercollegiate football team over 4 consecutive fall seasons. Weekly injuries and injury rates (injuries per athletic injury exposure) were correlated with the weekly exposures to full-contact practices, total practices, formal scrimmages, and games. RESULTS: The preseason practice injury rate was over twice the in-season practice injury rate ( P < 0.001). For preseason, injury exposures were higher for full-contact practice ( P = 0.0166), total practices ( P = 0.015), and scrimmages/games ( P = 0.034) compared with in-season. Preseason and in-season practice injuries correlated with exposure to full-contact practice combined with scrimmages for preseason ( P < 0.008) and full-contact practice combined with games for in-season ( P = 0.0325). The game injury rate was over 6 times greater than the practice injury rate ( P < 0.0001). Concussions constituted 14.5% of all injuries, and the incidence of concussions correlated with the incidence of all injuries ( P = 0.0001). Strength training did not correlate with injuries. CONCLUSION: Decreased exposure to full-contact practice may decrease the incidence of practice injuries and practice concussions. However, the game injury rate was over 6 times greater than the practice injury rate and had an inverse correlation with full-contact practice.

Superparamagnetic iron oxide nanoparticles for direct labeling of stem cells and in vivo MRI tracking.

To develop effective stem cell therapies, it is important to track therapeutic cells non-invasively and monitor homing to areas of pathology. The purpose of this study was to design and evaluate the labeling efficiency of commercially available dextran-coated superparamagnetic iron oxide nanoparticles, FeraTrack Direct (FTD), in various stem and immune cells; assess the cytotoxicity and tolerability of the FTD in stem cells; and monitor stem cell homing using FTD-labeled bone-marrow-derived mesenchymal stromal cells (BMSCs) and neural stem cells (NSCs) in a tumor model by in vivo MRI. BMSCs, NSCs, hematopoietic stem cells (HSCs), T-lymphocytes, and monocytes were labeled effectively with FTD without the need for transfection agents, and Prussian blue (PB) staining and transmission electron microscopy (TEM) confirmed intracellular uptake of the agent. The viability, proliferation, and functionality of the labeled cells were minimally or not affected after labeling. When 10(6) FTD-labeled BMSCs or NSCs were injected into C6 glioma bearing nude mice, the cells homing to the tumors were detected as hypointense regions within the tumor using 3 T clinical MRI up to 10 days post injection. Histological analysis confirmed the homing of injected cells to the tumor by the presence of PB positive cells that are not macrophages. Labeling of stem cells or immune cells with FTD was non-toxic, and should facilitate the translation of this agent to clinical trials for evaluation of trafficking of cells by MRI.

Novel bimodal iron oxide particles for efficient tracking of human neural stem cells in vivo.

AIMS: We validated novel bimodal iron oxide particles as substitute of ferumoxides for efficient labeling of human neural stem cells (NSCs). The dextrane-coated FeraTrack Direct (FTD)-Vio particles have additional far-red fluorophores for microscopic cell analysis. METHODS: MR relaxometry, spectrophotometric iron determination and microscopy are used for characterization in vitro and in vivo. RESULTS: Efficient uptake is not transfection agent-dependent. FTD-Vio594 labeling had no influence on viability, proliferation, migration and differentiation capacity. It allows MRI-based tracking of engrafted NSCs in mouse brain up to 11 days, complemented by bioluminescence imaging of firefly luciferase expressed by the engrafted cells. CONCLUSION: Our results highlight the FTD-Vio594 particles as safe and sensitive substitute of ferumoxides for longitudinal tracking of NSCs in preclinical studies.

Flexible instruments outperform rigid instruments to place anatomic anterior cruciate ligament femoral tunnels without hyperflexion.

PURPOSE: This study evaluated the ability of flexible instruments compared with rigid instruments to place anatomic femoral tunnels in anterior cruciate ligament reconstructions by use of both transtibial drilling and anteromedial drilling without hyperflexion. METHODS: Rigid and flexible pins were placed in 12 matched pairs of cadaveric knees with transtibial drilling (6 pairs) and anteromedial drilling (6 pairs) at 110° of flexion. Intraosseous pin lengths, femoral exit locations, and tunnel alignment were measured. RESULTS: Transtibial drilling with rigid pins placed relatively vertical femoral tunnels 5.8 ± 1.0 mm superior to the central anterior cruciate ligament insertion. Transtibial drilling with flexible pins placed tunnels in the center of the femoral attachment, but the tunnels were relatively close to the posterior femoral cortex, with a mean distance of 8.0 ± 5.9 mm (P < .05), compared with transtibial drilling with rigid pins. Anteromedial drilling resulted in central anatomic pin placements with rigid and flexible instruments. Tunnel lengths with flexible pins were longer (42.0 ± 7.2 mm) compared with tunnel lengths with rigid pins (32.5 ± 7.1 mm) (P < .01). Flexible pins exited farther from the posterior cortex compared with rigid pins (P < .01). In 3 of 6 knees with rigid pins, the exit point was at the posterior border of the femoral cortex. All flexible pins exited a safe distance from the posterior femoral cortex. CONCLUSIONS: Transtibial drilling with rigid instruments did not produce anatomic femoral tunnels. Transtibial drilling with flexible pins produced anatomic tunnels, but the tunnels were close to the posterior femoral cortex. Anteromedial drilling without hyperflexion produced anatomic tunnels by use of rigid and flexible instruments, but with flexible instruments, the tunnels were longer and were farther from the posterior femoral cortex. Anteromedial drilling with flexible pins produced tunnels with good length and the best position. CLINICAL RELEVANCE: Flexible instruments compared with rigid instruments can facilitate the creation of anatomic femoral tunnels by use of anteromedial drilling without hyperflexion.

High-throughput sensing microtiter plate for determination of biogenic amines in seafood using fluorescence or eye-vision.

A new optical sensing microplate was developed for rapid screening for the presence of biogenic amines (BAs) in seafood samples with high sensitivity. The deposition of a sensing spot (containing a chameleon dye (Py-1) in a polymeric cocktail) on the bottom of the wells of a standard microplate renders the plate a new sensing tool for a rapid and parallel detection of up to 96 (real) samples. This sensing microplate enables (1) a semi-quantitative readout of analyte concentration by eye-vision, (2) a rapid fluorescence readout of 96 samples with standard instrumentation in less than two minutes (unlike chromatographic and electrophoretic methods), (3) a statistically robust data evaluation (with 8-12 replicates) and (4) a rapid parallel sample preparation with standard 8 or 12-channel micropipettes. On reaction with biogenic amines, the dye shows a significant visible color change from blue over green to red color. The appearance of red color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. The linear ranges of fluorescence calibration data for six biogenic amines cover the clinical toxicological relevant range of BAs that is too low to be detected by the human nose. The LODs range from 0.16 to 0.56 µg mL(-1), with correlation coefficients (r(2)) between 0.985 and 0.999. Finally, the evolution of spoilage of four fish samples (monitored by determination of their BA status) and the increase of their total amine content were found to agree well with previous data on time-dependent evolution of BAs in fish.

Optical methods for sensing glucose.

This critical review covers the present state of the art in optical sensing of glucose. Following an introduction into the significance of (continuous) sensing of glucose and a brief look back, we discuss methods based on (a) monitoring the optical properties of intrinsically fluorescent or labeled enzymes, their co-enzymes and co-substrates; (b) the measurement of the products of enzymatic oxidation of glucose by glucose oxidase; (c) the use of synthetic boronic acids; (d) the use of Concanavalin A; and (e) the application of other glucose-binding proteins. We finally present an assessment in terms of the advantages and disadvantages of the various methods (237 references).

Luminescent ruthenium probe for the determination of acetyl phosphate in complex biological matrices.

The first probe for the fluorogenic determination of acetyl phosphate (AcP), (bpy)(2)Ru(1,10-phenanthroline-5,6-dione dioxime) (RuPDO), was prepared and its reaction with AcP was studied in detail. The emission of the weakly luminescent RuPDO is red shifted and strongly enhanced upon reaction with AcP in the presence of metal cations like Zn(2+) or Cu(2+). The reaction occurs within 60 min incubation time under highly biocompatible conditions (aqueous buffer of pH 7, 37 °C). A linear dynamic range from 10 to 200 µmol L(-1) is observed with an LOD of AcP of 3.4 µmol L(-1) (for RuPDO-Zn). Other bio-phosphates studied show only weak interference. Furthermore, the applicability of the probe in complex biological matrices was evaluated.