Daily monitoring of vaginal interleukin 6 as a predictor of intraamniotic inflammation after preterm premature rupture of membranes - a new method of sampling studied in a prospective multicenter trial.

OBJECTIVES: (A) To introduce a new technique for vaginal fluid sampling (biocompatible synthetic fiber sponge) and (B) evaluate the collected vaginal fluid interleukine-6 (IL-6vag)-concentration as a new diagnostic tool for daily monitoring of intrauterine inflammation after preterm premature rupture of membranes (PPROM). Secondary objectives were to compare the potential to predict an intrauterine inflammation with established inflammation parameters (e.g., maternal white blood cell count). METHODS: This prospective clinical case-control diagnostic accuracy multicenter study was performed with women after PPROM (gestational age 24.0/7 - 34.0/7 weeks). Sampling of vaginal fluid was performed once daily. IL-6vag was determined by electrochemiluminescence-immunoassay-kit. Neonatal outcome and placental histology results were used to retrospectively allocate the cohort into two subgroups: 1) inflammation and 2) no inflammation (controls). RESULTS: A total of 37 cases were included in the final analysis. (A): Measurement of IL-6 was successful in 86% of 172 vaginal fluid samples. (B): Median concentration of IL-6vag in the last vaginal fluid sample before delivery was significantly higher within the inflammation group (17,085 pg/mL) compared to the controls (1,888 pg/mL; p=0.01). By Youden's index an optimal cut-off for prediction an intrauterine inflammation was: 6,417 pg/mL. Two days before delivery, in contrast to all other parameters IL-6vag remained the only parameter with a sufficient AUC of 0.877, p<0.001, 95%CI [0.670-1.000]. CONCLUSIONS: This study established a new technique for vaginal fluid sampling, which permits assessment of IL-6vag concentration noninvasively in clinical daily routine monitoring.

Comparisons of Stereological and Other Approaches for Quantifying Macrophage Aggregates in Piscine Spleens.

Macrophage aggregates (MAs) are focal accumulations of pigmented macrophages in the spleen and other tissues of fish. A central role of MAs is the clearance and destruction of degenerating cells and recycling of some cellular components. Macrophage aggregates also respond to chemical contaminants and infectious agents and may play a role in the adaptive immune response. Tissue damage or physiological stress can result in increased MA accumulation. As a result, MAs may be sensitive biomarkers of environmental stress in fish. Abundance of MAs in tissues has been reported in a variety of ways-most commonly as density, mean size, and relative area-but the utility of these estimates has not been compared. In this study, four different types of splenic MA abundance estimates (abundance score, density, relative area, and total volume) were compared in two fish populations (Striped Bass Morone saxatilis and White Perch M. americana) with a wide range in ages. Stereological estimates of total volume indicated an increase in MA abundance with spleen volume, which generally corresponded to fish age, and with splenic infections (mycobacteria or trematode parasites). Abundance scores were generally limited in the ability to detect changes in MA abundance by these factors, whereas density estimates were greatly influenced by changes in spleen volume. In some instances, densities declined while the total volume of MAs and spleen volume increased. Experimentally induced acute stress resulted in a decrease in spleen volume and an increase in MA density, although the total volume of MAs remained unchanged. Relative area estimates accounted for the size and number of MAs but not for changes in organ volume. Total volume is an absolute measure of MA abundance irrespective of changes in organ volume or patterns of accumulation and may provide an improved means of quantifying MAs in the spleens of fish.

Validation of the Global Allergy and Asthma European Network (GA2LEN) chamber for trials in allergy: Innovation of a mobile allergen exposure chamber.

BACKGROUND: Field clinical trials of pollen allergy are affected by the impossibility of predicting and determining individual allergen exposure because of many factors (eg, pollen season, atmospheric variations, pollutants, and lifestyles). Environmental exposure chambers, delivering a fixed amount of allergen in a controlled environmental setting, can overcome these limitations. Environmental exposure chambers are currently already used in phase 2, 3, and even 4 trials. Unfortunately, few chambers exist in the world, and this makes it difficult to perform large, multicenter clinical trials. The new Global Allergy and Asthma European Network (GA2LEN) mobile exposure chamber is a step forward because the mobility of the chamber makes it convenient for patients to participate in clinical testing. OBJECTIVE: This study was made to validate the reproducibility, sensitivity, and specificity of the results obtained in the new GA2LEN chamber. METHODS: Seventy-two adult patients (19-61 years old) with allergic rhinitis with or without asthma caused by grass pollen were included in different clinical validation tests. Total symptom scores and total nasal symptom scores were recorded at time zero (0) and every 10 minutes during exposures, along with nasal and respiratory parameters. RESULTS: Exposure tests confirmed the reproducibility between subsequent runs and the sensitivity (P < .00001 vs patients exposed to placebo) and specificity (very low score in nonallergic subjects) in the GA2LEN chamber. No adverse reactions were recorded during the tests. CONCLUSIONS: The mobility of the GA2LEN chamber provides a new, potentially effective, and safe way of generating reliable data in allergy multicenter clinical trials.

Quantitative evaluation of biological reaction kinetics in confined nanospaces.

Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis.

Medical devices; immunology and microbiology devices; classification of John Cunningham Virus serological reagents. Final order.

The Food and Drug Administration (FDA) is classifying John Cunningham Virus (JCV) serological reagents into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

Using fecal immunochemical tubes for the analysis of the gut microbiome has the potential to improve colorectal cancer screening.

Colorectal cancer (CRC) is a challenging public health problem which successful treatment depends on the stage at diagnosis. Recently, CRC-specific microbiome signatures have been proposed as a marker for CRC detection. Since many countries have initiated CRC screening programs, it would be useful to analyze the microbiome in the samples collected in fecal immunochemical test (FIT) tubes for fecal occult blood testing. Therefore, we investigated the impact of FIT tubes and stabilization buffer on the microbial community structure evaluated in stool samples from 30 volunteers and compared the detected communities to those of fresh-frozen samples, highlighting previously published cancer-specific communities. Altogether, 214 samples were analyzed by 16S rRNA gene sequencing, including positive and negative controls. Our results indicated that the variation between individuals was greater than the differences introduced by the collection strategy. The vast majority of the genera were stable for up to 7 days. None of the changes observed between fresh-frozen samples and FIT tube specimens were related to previously identified CRC-specific bacteria. Overall, we show that FIT tubes can be used for profiling the microbiota in CRC screening programs. This circumvents the need to collect additional samples and can possibly improve the sensitivity of CRC detection.

A T-cell-based immunogenicity protocol for evaluating human antigen-specific responses.

Determining the antigen specificities of the endogenous T-cell repertoire is important for screening naturally occurring or therapy-induced T-cell immunity and may help identify novel targets for T-cell-based therapies. Here, we describe a rapid, sensitive, and high-throughput protocol for expanding antigen-specific T cells from human peripheral blood mononuclear cells in vitro following peptide stimulation and detecting antigen-specific effector cytokine formation by flow cytometry. Our approach can be applied to examining specific T-cell subsets from various tissues. For complete details on the use and execution of this protocol, please refer to Roudko et al. (2020) and Cimen Bozkus et al. (2019).

Shedding Structured Light on Molecular Immunity: The Past, Present and Future of Immune Cell Super Resolution Microscopy.

In the two decades since the invention of laser-based super resolution microscopy this family of technologies has revolutionised the way life is viewed and understood. Its unparalleled resolution, speed, and accessibility makes super resolution imaging particularly useful in examining the highly complex and dynamic immune system. Here we introduce the super resolution technologies and studies that have already fundamentally changed our understanding of a number of central immunological processes and highlight other immunological puzzles only addressable in super resolution.

Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper.

With its emphasis on minimally invasive high-speed imaging of intact tissues at depth, video-rate two-photon microscopy has revolutionized cell biology. This is particularly true in immunology, where the orchestration of cell migration, cell-cell interactions and intracellular signalling events in multiple distinct anatomical compartments within secondary lymphoid organs is fundamental for achieving an effective immune response. Until recently, access to this powerful tool has been limited to a handful of laboratories with the necessary skills and resources to either custom-build or purchase a commercial two-photon microscope. However, with the entry of more commercial vendors into the market and availability of turnkey solutions, two-photon microscopy is now becoming more accessible. Here, we discuss the practical aspects of establishing a basic intravital two-photon microscopy facility specifically for immunological research and how recent advances in ultrafast lasers, non-linear optics and localized photochemistry can be used to build more sophisticated instruments to support applications such as photoactivation and photobleaching, spectral fingerprinting and automated single-cell tracking. In addition, we discuss the next generation of fluorescent dyes and reporter mice and some of the microsurgical principles required to expose the relevant biology to interrogation by two-photon excitation.

Label-free chemiresistive immunosensors for viruses.

We report development, characterization, and testing of chemiresistive immunosensors based on single polypyrrole (Ppy) nanowire for highly sensitive, specific, label free, and direct detection of viruses. Bacteriophages T7 and MS2 were used as safe models for viruses for demonstration. Ppy nanowires were electrochemically polymerized into alumina template, and single nanowire based devices were assembled on a pair of gold electrodes by ac dielectrophoretic alignment and anchored using maskless electrodeposition. Anti-T7 or anti-MS2 antibodies were immobilized on single Ppy nanowire using EDC-NHS chemistry to fabricate nanobiosensor for the detection of corresponding bacteriophage. The biosensors showed excellent sensitivity with a lower detection limit of 10(-3) plaque forming unit (PFU) in 10 mM phosphate buffer, wide dynamic range and excellent selectivity. The immunosensors were successfully applied for the detection of phages in spiked untreated urban runoff water samples. The results show the potential of these sensors in health care, environmental monitoring, food safety and homeland security for sensitive, specific, rapid, and affordable detection of bioagents/pathogens.

Virtual-core flow cytometry.

Traditional flow cytometers use a sheath fluid to position particles or cells for cytometric measurements, but the need for sheath fluid greatly complicates flow cytometric instrumentation. A cytometric detector that is free of the requirements of sheath fluid can simplify the design of flow cytometers and can extend their use into a number of areas. We designed a flow cytometer that uses a combination of three photodetectors to sense the position of a particle in sample stream. The position-sensitive detectors create a virtual core in the sample stream that eliminates the need for sheath fluid. In this article, we demonstrate the efficacy of a virtual-core flow cytometer (VCFC) using test particles, immunofluorescently labeled thymocytes, and raw seawater. The VCFC performs accurate measurements that can be used for a number of uses including environmental monitoring or simple immunology tests.

Concentration of Listeria monocytogenes in skim milk and soft cheese through microplate immunocapture.

Microplate immunocapture is an inexpensive method for the concentration of foodborne pathogens using an antibody-coated microplate. The objective of this study was to determine the efficacy of microplate immunocapture as an alternative to traditional enrichment for concentrating Listeria monocytogenes to levels detectable with selective plating or real-time PCR. L. monocytogenes isolates serologically characterized as Type 1 (1/2a) and Type 4 (untypeable) were grown overnight and diluted to 100 to 106 colony-forming units (CFU)/mL. The isolates were used to optimize microplate immunocapture in tryptic soy broth with 0.6% yeast extract (TSBYE), skim milk, and queso fresco samples. Following microplate immunocapture, the bacteria were streaked onto polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) agar, followed by incubation at 37 °C for 24 ±â€¯2 h. The bacteria also underwent real-time polymerase chain reaction (PCR). The optimized microplate immunocapture method was tested in triplicate for its ability to capture L. monocytogenes in broth and food samples. Overall recovery rates for L. monocytogenes in food samples at cell populations of 100, 102, and 104 CFU/25 g using microplate immunocapture with real-time PCR were 88.9%, 94.4%, and 100%, respectively. Recovery in these matrices using microplate immunocapture with selective plating was comparatively lower, at 0%, 44.4%, and 100%, respectively. Conventional culture method showed 100% detection at each inoculation level. Microplate immunocapture combined with real-time PCR shows high potential to reduce the time required for detection, with concentration of L. monocytogenes to detectable levels within 1-4 h. The incorporation of a short enrichment step may improve recovery rates at low cell levels.

Design and evaluation of cryodevice, an easy to use apparatus for maintenance of optimum temperature during cryoglobulin assay.

INTRODUCTION: Maintenance of temperature during collection and transport of blood is an important pre-requisite for cryoglobulin assays. In this manuscript, we describe 'cryodevice', a low-cost device for transportation and/or incubation of vials of whole blood at 37°C. Such a device would reduce false negatives in cryoglobulin assays. METHOD: The 'cryodevice' takes the embodiment of a portable, light, insulated water bath, which can be used as an incubator in a plugged-in state, or as a transport container after it is set up and disconnected from the power supply. The design of the cryodevice is described here, with focus on its construction and electronic control circuit. Computer simulations and in vitro trials were performed to study the temperature drop in the blood samples placed in the device. Subsequently, the cryodevice was also used with actual patient blood samples. RESULTS: Thermal simulations and in vitro testing of the cryodevice predicted that the design would meet the temperature maintenance goals. When the cryodevice was put in to use for screening 45 patient blood samples, it helped identify positive cryoglobulinemia in three of the samples. CONCLUSION: The description of the cryodevice envisions enabling the construction of a low-cost device in resource-limited healthcare settings in India created with locally available resources. On testing, the device was found to be satisfactory in performance and is expected to bring down incidences of false negatives in cryoglobulin tests.

Biocompatibility of nanoporous alumina membranes for immunoisolation.

Cellular immunoisolation using semi-permeable barriers has been investigated over the past several decades as a promising treatment approach for diseases such as Parkinson's, Alzheimer's, and Type 1 diabetes. Typically, polymeric membranes are used for immunoisolation applications; however, recent advances in technology have led to the development of more robust membranes that are able to more completely meet the requirements for a successful immunoisolation device, including well controlled pore size, chemical and mechanical stability, nonbiodegradability, and biocompatibility with both the graft tissue as well as the host. It has been shown previously that nanoporous alumina biocapsules can act effectively as immunoisolation devices, and support the viability and functionality of encapsulated beta cells. The aim of this investigation was to assess the biocompatibility of the material with host tissue. The cytotoxicity of the capsule, as well as its ability to activate complement and inflammation was studied. Further, the effects of poly(ethylene glycol) (PEG) modification on the tissue response to implanted capsules were studied. Our results have shown that the device is nontoxic and does not induce significant complement activation. Further, in vivo work has demonstrated that implantation of these capsules into the peritoneal cavity of rats induces a transient inflammatory response, and that PEG is useful in minimizing the host response to the material.

Environmental analysis by on-line immunoextraction and reversed-phase liquid chromatography: optimization of the immunoextraction/RPLC interface.

The use of antibodies in HPLC columns for on-line immunoextraction combined with reversed-phase liquid chromatography (RPLC) is of growing interest in environmental and agricultural analysis. This technique is typically performed by using a small RPLC precolumn to capture and concentrate analytes as they elute from the immunoextraction column; however, there is little information on the conditions required for optimizing this interface. This study examined the behavior of this interface by using 2,4-dichlorophenoxyacetic acid (2,4-D) and related herbicides as model analytes. It was found that analyte dissociation from immunoextraction columns followed first-order decay and that the elution of these analytes through the immunoextraction/RPLC interface gave an exponentially modified Gaussian profile. Computer simulations were used to see how analyte elution through the interface changed with different dissociation and retention conditions. Several guidelines were developed from this work that could be used for developing and optimizing on-line immunoextraction/RPLC systems for other chemicals of environmental or agricultural interest.

Immunoisolation of TCR signaling complexes from Jurkat T leukemic cells.

The formation of multimolecular assemblies of signaling molecules at the plasma membrane is key to triggering signaling cascades following activation of transmembrane receptors at the plasma membrane. We have developed a method to immunoisolate activated T cell receptors (TCRs) and associated signaling molecules in plasma membrane subdomains from Jurkat T leukemic cells. The immunoisolation procedure for the signaling complexes uses magnetic beads, which are coupled to TCR-activating antibodies. Following mechanical cellular disruption using nitrogen cavitation, conjugates of these beads with Jurkat cells are formed and isolated. TCR-signaling complexes within plasma membrane fragments associate with the antibody-coupled beads and are retrieved along with the beads using a magnet. We found that the immunoisolated plasma membrane fragments are highly enriched for activated TCRs and associated signaling proteins. This isolation procedure allows a detailed and precise biochemical analysis of the assembly of signaling proteins in plasma membrane subdomains.

Medical devices; immunology and microbiology devices; classification of cystic fibrosis transmembrane conductance regulator gene mutation detection system. Final rule.

The Food and Drug Administration (FDA) is classifying the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection systems into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: CFTR Gene Mutation Detection Systems." The agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance document that will serve as the special control for the device.

Medical devices; immunology and microbiology devices; classification of AFP-L3% immunological test systems. Final rule.

The Food and Drug Administration (FDA) is classifying AFP-L3% (alpha-fetoprotein L3 subfraction) immunological test systems into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: AFP-L3% Immunological Test Systems." The agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of a guidance document that will serve as the special control for the device.

A convenient multipurpose mouse restrainer.

A convenient, inexpensive, easily constructed mouse restrainer is described and illustrated. The restrainer has the advantage over other models that while the animal is effectively immobilized, time-consuming and potentially injurious binding of extremities is avoided, and selected areas such as the back, extremities and tail remain accessible for manipulations.