COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

Background: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. Methods: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples, N=116) or on convenience samples of children under 5 years of age presenting to a pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. Results: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. Conclusions: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

Bowel Obstruction and Hydronephrosis Due to an Over-the-Scope Clip.

An over-the-scope clip is a type of endoscopically placed clip used to treat gastrointestinal perforation, bleeding, or fistula. After the defect heals, the clip usually passes uneventfully through the gastrointestinal tract. An uncommon complication of over-the-scope clip placement is intestinal obstruction caused by luminal stenosis at the site of clip placement. Intestinal obstruction can rarely cause other downstream complications such as hydronephrosis from extrinsic compression of the urinary tract. We report a rare case of bilateral hydronephrosis caused by bowel obstruction from a migrated endoscopically placed clip.

A real-time, bioluminescent annexin V assay for the assessment of apoptosis.

Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pathways and molecular mechanisms. Although many assay methods exist for confirming whether an apoptotic response has occurred in vitro, most methods are destructive and involve laborious operator effort or specialized instrumentation. Here we describe a real-time, no-wash, microplate method which utilizes recombinant annexin V fusion proteins containing evolved binary subunits of NanoBiT™ luciferase. The fusion proteins, a time-released enzymatic substrate, a necrosis detection dye and exogenous calcium ions are delivered via an optimized and physiologically inert reagent directly to cells in culture at the time of treatment or dosing. Luminescent signals proportional to phosphatidylserine (PS) exposure and fluorescent signals generated as a result of loss of membrane integrity are then collected using a standard multimode plate reader at scheduled intervals over the exposure. The resulting luminescent and fluorescent data are then used to define the kinetics and magnitude of an apoptotic response. This study details our efforts to develop, characterize, and demonstrate the features of the assay by providing relevant examples from diverse cell models for programmed cell death.

Altered cytotoxicity of ROS-inducing compounds by sodium pyruvate in cell culture medium depends on the location of ROS generation.

Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol >10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra- or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.

Self-immolative bioluminogenic quinone luciferins for NAD(P)H assays and reducing capacity-based cell viability assays.

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

A high-throughput 1,536-well luminescence assay for glutathione S-transferase activity.

Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.

N-Alkylated 6'-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase: new potential scaffolds for bioluminescent assays.

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.

Bioluminescent assays for ADMET.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.

A novel bioluminescent protease assay using engineered firefly luciferase.

Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

A bioluminescent assay for monoamine oxidase activity.

This article describes a novel two-step homogeneous bioluminescent assay for monoamine oxidase (MAO) that is simple, sensitive, and amenable to high-throughput screening. In the first step, MAO reacts with an aminopropylether analog of methyl ester luciferin. In the second step, a luciferin detection reagent inactivates MAO and converts the product of the first step into a luminescent signal. The amount of light produced is proportional to the amount of MAO and the time of incubation in the first step, but the luminescent signal is stable in the second step with a half-life greater than 5h. The assay has high precision, is more sensitive than current fluorescent methods, and can accurately measure the binding constants of known substrates and inhibitors. An automated screen of the Sigma-RBI Library of Pharmacologically Active Compounds (LOPAC(1280)) revealed a surprisingly high percentage of MAO inhibitors (16%) with a low false hit rate (0.9%). This implies that a significant number of compounds interact with the MAO enzymes and suggests that it is important to include MAO assays in drug metabolism studies. Other advantages of this bioluminescent assay over comparable fluorescent assays are discussed.

Evaluation of screen-printed gold on low-temperature co-fired ceramic as a substrate for the immobilization of electrochemical immunoassays.

Screen-printed gold (SPG, Dupont gold conductor 5734) on low-temperature co-fired ceramic (LTCC) materials (Dupont dielectric tape 951, mostly composed of alumina and silica) has been demonstrated to be a substrate for electrochemical enzyme-linked immunosorbant assays. The effect of two different cleaning treatments and the extent of nonspecific adsorption on the SPG/LTCC and plain LTCC surfaces were also evaluated. LTCC materials hold promise for constructing a new generation of devices for microelectrochemical sensing and assays. Facile fabrication in three dimensions with integrated conducting elements makes them attractive. A standard sandwich immunoassay for a model analyte, mouse IgG, was used to evaluate the LTCC materials. After the assembly of components onto chips of SPG/LTCC and plain LTCC, p-aminophenol that was generated enzymatically by the enzyme label was detected electrochemically with a separate glassy carbon electrode. Cleaning SPG/LTCC with a piranha solution (7:1 vol/vol of concentrated H2SO4/30% H2O2), traditionally used for other gold surfaces prior to SAM assembly, resulted in a notable decrease in assay signal and an increase in nonspecific adsorption when compared to cleaning with water alone. Assay components assemble specifically on plain LTCC, with only a small percent attributed to NSA. Environmental scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy reveal the tremendous chemical heterogeneity and complexity of both SPG/LTCC and plain LTCC surfaces and aid in the explanation of assay results. A 10% acetate Tween bovine serum albumin solution and an ethanolic solution of 4 mM 1-butanol eliminate assay signals originating from plain LTCC. The outcomes of these studies can now be used to achieve miniaturized electrochemical immunoassays on LTCC materials where both plain and SPG surfaces are present.

Electrophilic aromatic substituted luciferins as bioluminescent probes for glutathione S-transferase assays.

New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.

Using HFMEA to assess potential for patient harm from tubing misconnections.

BACKGROUND: Reported cases of tubing misconnections and other tubing errors prompted Columbus Children's Hospital to study their potential for harm in its patient population. A Health Failure Mode and Effects Analysis (HFMEA) was conducted in October 2004 to determine the risks inherent in the use and labeling of various enteral, parenteral, and other tubing types in patient care and the potential for patient harm. METHODS: An assessment of the practice culture revealed considerable variability among nurses and respiratory therapists within and between units. Work on an HFMEA culminated in recommendations of risk reduction strategies. These included standardizing the process of labeling of tubing throughout the organization, developing an online pictorial catalog to list available tubing supplies with all aliases used by staff, and conducting an inventory of all supplies to identify products that need to be purchased or discontinued. Three groups are working on implementing each of the recommendations. RESULTS: Most of the results already realized occurred in labeling of tubing. The pediatric intensive care unit labels all tubing with infused medications 85% of the time; tubings inserted during surgery or in interventional radiology are labeled 53% and 93% of the time. Pocket-size cards with printed labels were tested in three units. DISCUSSION: This proactive risk assessment project has identified failure modes and possible causes and solutions; several recommendations have been implemented. No tubing misconnections have been reported.

New bioluminogenic substrates for monoamine oxidase assays.

Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective beta-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.

Small-scale telomere repeat sequence content assay using pyrophosphorolysis coupled with ATP detection.

Studies of telomere length have been carried out in diverse areas of research. However, current methods to measure telomeres are cumbersome and not amenable to high-throughput analyses. Using a coupled pyrophosphorolysis/trans-phosphorylation reaction, we have developed a novel assay to quantitate telomere sequence content in a single tube or 96-well format. The method uses a telomere-specific oligonucleotide probe to sample nanogram quantities of DNA without PCR amplification. Polymerase and kinase enzymes drive the production of ATP, which is then monitored with a luciferase enzyme reporter system. Using this approach, we demonstrated that the luminescent output was linear across a 100-fold range of DNA input, and the assay was sensitive to 0.4-1 ng DNA. A control probe reaction and a DNA quantitation reaction were also designed using the same pyrophosphorolysis technology to correct for background activity and normalize the signal against variations in DNA input, respectively. Finally, we show that the normalized luminescent signal generated by this new method is highly correlated to the telomere restriction fragment length for six human cell lines.