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.

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.

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.

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.

Chromogenic sensing of biogenic amines using a chameleon probe and the red-green-blue readout of digital camera images.

We report on sensing spots containing an amine reactive chromogenic probe and a green fluorescent (amine insensitive) reference dye incorporated in a hydrogel matrix on a solid support. Such spots enable rapid and direct determination of primary amines and, especially, biogenic amines (BA). A distinct color change from blue to red occurs on dipping the test spots into a pH 9.0 sample containing primary amines. BAs can be determined in the concentration range from 0.01 to 10 mM within 15 min, enabling rapid, qualitative, and semiquantitative evaluation. In the "photographic" approach, the typically 4-7.5-fold increase in fluorescence intensity of the probe at 620 nm along with the constant green fluorescence at 515 nm of a reference dye are used for quantitation of BAs. The sensing spots are photoexcited with high-power 505 nm light-emitting diodes (LEDs) in a black box. A digital picture is acquired with a commercially available digital camera, and the color information is extracted via red-green-blue (RGB) readout. The ratio of the intensities of the red (signal) channel and the green (reference) channel yields pseudocolor pictures and calibration plots.

SDS-PAGE of proteins using a chameleon-type of fluorescent prestain.

A new prestaining method for protein SDS-PAGE was developed using the fluorogenic amino-reactive label Py-1. This resulted in one of the fastest, most sensitive, and environmentally friendly protocols available. It is mainly due to the unique optical properties of Py-1, which is blue and virtually nonfluorescent but turns to red and becomes much more strongly fluorescent once it is conjugated to the amino group of a protein. Staining times of 30 min are adequate to visualize subnanogram quantities of proteins because pre-electrophoretic labeling Py-1 does not require the time-consuming steps of washing or fixation of gels. LODs as low as 16 pg of protein are found which is better than the best (commercial) poststains and comparable to the best (commercial) prestains. In addition, prestaining requires marginal amounts of staining solution. The change in electrophoretic mobility and band broadening is at a low level because Py-1 causes a mass shift of 288 Da per bound molecule only. By virtue of the small mass shift it causes, this stain is compatible with mass spectrometric protein analysis even though it acts as a covalent label.