Multiplex Assays in Allergy Diagnosis: Allergy Explorer 2 versus ImmunoCAP ISAC E112i.

ImmunoCAP ISAC E112i (ISAC) and Allergy Explorer 2 (ALEX2) detect specific immunoglobulin E (IgE) reactivity. Both multiplex assays contain molecular allergens and ALEX2 additionally includes allergen extracts and inhibitors that block the binding of IgE to cross-reacting carbohydrate determinants (CCDs). This study aimed to compare the performance of ISAC and ALEX2 by determining the IgE reactivity against allergen extracts and/or allergen components and by using qualitative, semiquantitative, and quantitative analyses of all comparable allergen components in sera from 216 participants recruited in South Tyrol/Italy. For extract sensitization in ALEX2, the analysis revealed negative corresponding allergen components in 18.4% and at least one positive corresponding allergen component in 81.6% of all cases. For ISAC, the corresponding results were 23.5% and 76.5% of cases, respectively. The ALEX2 CCD inhibitor eliminated CCD-positive signals detected by ISAC in 88.5% of cases. Based on sensitization values of 0.3-14.9 ISU or kUA/L, there was good agreement between ALEX2 and ISAC, although ALEX2 showed higher values than ISAC. The addition of allergen-extract tests in ALEX2 resulted in the detection of more sensitizations than with corresponding allergen components alone. In the range of <15 ISU or kUA/L, ALEX2 may be more effective in detecting sensitizations.

Quantification of anomalies in rats' spinal cords using autoencoders.

Computed tomography (CT) scans and magnetic resonance imaging (MRI) of spines are state-of-the-art for the evaluation of spinal cord lesions. This paper analyses micro-CT scans of rat spinal cords with the aim of generating lesion progression through the aggregation of anomaly-based scores. Since reliable labelling in spinal cords is only reasonable for the healthy class in the form of untreated spines, semi-supervised deviation-based anomaly detection algorithms are identified as powerful approaches. The main contribution of this paper is a large evaluation of different autoencoders and variational autoencoders for aggregated lesion quantification and a resulting spinal cord lesion quantification method that generates highly correlating quantifications. The conducted experiments showed that several models were able to generate 3D lesion quantifications of the data. These quantifications correlated with the weakly labelled true data with one model, reaching an average correlation of 0.83. We also introduced an area-based model, which correlated with a mean of 0.84. The possibility of the complementary use of the autoencoder-based method and the area feature were also discussed. Additionally to improving medical diagnostics, we anticipate features built on these quantifications to be useful for further applications like clustering into different lesions.

Erratum: Generative Adversarial Networks in Digital Pathology: A Survey on Trends and Future Potential.

[This corrects the article DOI: 10.1016/j.patter.2020.100089.].

Generative Adversarial Networks in Digital Pathology: A Survey on Trends and Future Potential.

Image analysis in the field of digital pathology has recently gained increased popularity. The use of high-quality whole-slide scanners enables the fast acquisition of large amounts of image data, showing extensive context and microscopic detail at the same time. Simultaneously, novel machine-learning algorithms have boosted the performance of image analysis approaches. In this paper, we focus on a particularly powerful class of architectures, the so-called generative adversarial networks (GANs) applied to histological image data. Besides improving performance, GANs also enable previously intractable application scenarios in this field. However, GANs could exhibit a potential for introducing bias. Hereby, we summarize the recent state-of-the-art developments in a generalizing notation, present the main applications of GANs, and give an outlook of some chosen promising approaches and their possible future applications. In addition, we identify currently unavailable methods with potential for future applications.

Immune Effects of the Nitrated Food Allergen Beta-Lactoglobulin in an Experimental Food Allergy Model.

Food proteins may get nitrated by various exogenous or endogenous mechanisms. As individuals might get recurrently exposed to nitrated proteins via daily diet, we aimed to investigate the effect of repeatedly ingested nitrated food proteins on the subsequent immune response in non-allergic and allergic mice using the milk allergen beta-lactoglobulin (BLG) as model food protein in a mouse model. Evaluating the presence of nitrated proteins in food, we could detect 3-nitrotyrosine (3-NT) in extracts of different foods and in stomach content extracts of non-allergic mice under physiological conditions. Chemically nitrated BLG (BLGn) exhibited enhanced susceptibility to degradation in simulated gastric fluid experiments compared to untreated BLG (BLGu). Gavage of BLGn to non-allergic animals increased interferon-γ and interleukin-10 release of stimulated spleen cells and led to the formation of BLG-specific serum IgA. Allergic mice receiving three oral gavages of BLGn had higher levels of mouse mast cell protease-1 (mMCP-1) compared to allergic mice receiving BLGu. Regardless of the preceding immune status, non-allergic or allergic, repeatedly ingested nitrated food proteins seem to considerably influence the subsequent immune response.

Evaluation of reference intervals of haematological and biochemical markers in an Austrian adolescent study cohort.

Background Reference intervals are a prerequisite for the interpretation of laboratory data related to diagnostic issues and treatment strategies. In adolescents, biomarker concentrations change with age, necessitating a continuous age-related definition of the reference intervals. The purpose of this pilot study was to evaluate the reference intervals for a healthy population of adolescents in Salzburg and compare these, when possible, with age- and gender-matched published data. Methods Anthropometrical parameters and blood samples were collected from adolescents (male and female; 14-17 years) in a school setting. Haematological samples were measured using Sysmex XS-1000i, lipid and carbohydrate metabolism markers as well as enzymes and hormones were determined by Cobas c311, Vitros ECiQ® or ELISA. The reference intervals were calculated according to the CLSI guidelines C28-A3c. Results Samples of 102 participants were included. Compared to age- and gender-matched reference intervals, the BMI levels were in the lower normal rage. Most haematological parameters and biomedical makers reveal similar ranges to values published in other studies. Conclusions This data analysis allowed for a partial comparison of reference values with published data and enabled a new determination of paediatric reference intervals for an Austrian cohort.

Influence of Intrinsic and Lifestyle Factors on the Development of IgE Sensitization.

BACKGROUND: IgE sensitization is a prerequisite for the development of allergic symptoms. The investigation of factors influencing the development of IgE is therefore crucial for understanding the onset of allergic diseases. METHODS: This epidemiological study investigated personal, intrinsic, and lifestyle factors in a nonselected cohort of 501 Austrian adolescents (aged 12-21 years). IgE levels to 112 allergen molecules were analyzed in the serum of participants using the ImmunoCAP ISAC®. Allergic sensitization, IgE levels to single allergens, and ISAC score sums were correlated with results obtained from a questionnaire. RESULTS: In this adolescent cohort, male participants showed a higher sensitization frequency (56.8%) compared to females (50.9%) and significantly increased IgE levels to profilins. Underweight subjects demonstrated a stronger IgE sensitization. Family size inversely correlated with IgE levels to PR-10 allergens, and predominately paternal allergies were a predictive factor for IgE sensitization in the children. Vaccination, breastfeeding, and delivery mode showed no influence, while a highly protective effect was observed for growing up on a farm. Of all of the investigated lifestyle factors, only smoking significantly influenced the risk for IgE development. Participants with moderate frequencies of colds showed increased sensitization levels. CONCLUSION: A hereditary predisposition and lifestyle factors such as a farming environment, smoking, family size, body weight, or frequency of colds significantly influenced the development of allergen-specific IgE in this cohort of adolescents.

Exposure to Indoor Allergens in Different Residential Settings and Its Influence on IgE Sensitization in a Geographically Confined Austrian Cohort.

BACKGROUND: Exposure to indoor allergens is crucial for IgE sensitization and development of allergic symptoms. Residential settings influence the allergen amount in house dust and hence allergic sensitization. Within this study, we investigated allergen exposure and molecule-based IgE levels in a geographically confined region and evaluated the impact of housing, pets and cleaning. METHODS: 501 adolescents from Salzburg, Austria participated in this cross-sectional study. House dust samples were examined regarding major mite, cat, dog, and mold allergens using a multiplex assay. Serum samples of participants were analyzed for specific IgE to Der p 1, Der p 2, Fel d 1, Can f 1 and Alt a 1 using the multiplex array ImmunoCAP ISAC. Information on allergies, living areas, dwelling form (house, flat, farm), pets, and household cleanliness were obtained by a questionnaire. RESULTS: In investigated house dust samples, the concentration of cat allergen was highest while the prevalence of mold allergens was very low. Participants showed IgE sensitization to Der p 1 (13.2%), Der p 2 (18.2%), Fel d 1 (14.4%), Can f 1 (2.4%) and Alt a 1 (2.0%). In alpine regions, lower mite allergen concentrations were detected which correlated with reduced IgE levels. A trend for increased sensitization prevalence from rural to alpine to urban regions was noted. Living on farms resulted in lower sensitization prevalence to mite and cat allergens, even though exposure to mites was significantly elevated. The presence of cats was associated with a lower sensitization rate and IgE levels to cat and mite allergens, and less frequent allergic diseases. Cleaning did not impact allergen concentrations, while IgE reactivity to mites and allergic diseases were more pronounced when living in cleaner homes. CONCLUSION: Allergen exposure to indoor allergens was influenced by setting of homes. Living in a farm environment and having a cat at home showed a protective effect for IgE sensitization and allergies. This cross-sectional study in combination with hereditary and lifestyle factors enables development of risk schemes for a more efficient management and potential prevention of allergic diseases.

Nitration of ß-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice.

BACKGROUND: We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins. OBJECTIVE: The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy. DESIGN: BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen ß-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood. RESULTS: A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation. CONCLUSION: Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.

Training of carbohydrate estimation for people with diabetes using mobile augmented reality.

BACKGROUND: Imprecise carbohydrate counting as a measure to guide the treatment of diabetes may be a source of errors resulting in problems in glycemic control. Exact measurements can be tedious, leading most patients to estimate their carbohydrate intake. In the presented pilot study a smartphone application (BE(AR)), that guides the estimation of the amounts of carbohydrates, was used by a group of diabetic patients. METHODS: Eight adult patients with diabetes mellitus type 1 were recruited for the study. At the beginning of the study patients were introduced to BE(AR) in sessions lasting 45 minutes per patient. Patients redraw the real food in 3D on the smartphone screen. Based on a selected food type and the 3D form created using BE(AR) an estimation of carbohydrate content is calculated. Patients were supplied with the application on their personal smartphone or a loaner device and were instructed to use the application in real-world context during the study period. For evaluation purpose a test measuring carbohydrate estimation quality was designed and performed at the beginning and the end of the study. RESULTS: In 44% of the estimations performed at the end of the study the error reduced by at least 6 grams of carbohydrate. This improvement occurred albeit several problems with the usage of BE(AR) were reported. CONCLUSIONS: Despite user interaction problems in this group of patients the provided intervention resulted in a reduction in the absolute error of carbohydrate estimation. Intervention with smartphone applications to assist carbohydrate counting apparently results in more accurate estimations.

Differential immunomodulatory responses to nine polycyclic aromatic hydrocarbons applied by passive dosing.

Studying the effects of hydrophobic chemicals using in vitro cell based methods is hindered by the difficulty in bringing and keeping these chemicals in solution. Their effective concentrations are often lower than their nominal concentrations. Passive dosing is one approach that provides defined and stable dissolved concentrations during in vitro testing, and was applied to control and maintain freely dissolved concentrations of polycyclic aromatic hydrocarbons (PAHs) at levels up to their aqueous solubility limit. The immunomodulatory effects of 9 different PAHs at aqueous solubility on human bronchial epithelial cells were determined by analysing the cytokine promoter expression of 4 different inflammatory cytokines using stably transfected recombinant A549 cell lines. Diverse immunomodulatory responses were found with the highest induction observed for the most hydrophobic PAHs chrysene, benzo(a)antracene and benzo(a)pyrene. Cytokine promoter expression was then studied in dose response experiments with acenaphthene, phenanthrene and benzo(a)anthracene. The strongest induction was observed for benzo(a)anthracene. Cell viability analysis was performed and showed that none of the PAHs induced cytotoxicity at any of the concentrations tested. Overall, this study shows that (1) immunomodulatory effects of PAHs can be studied in vitro at controlled freely dissolved concentrations, (2) the most hydrophobic PAHs were the strongest inducers and (3) induction was often higher at lower exposure levels and decreased then with concentration despite the apparent absence of cytotoxicity.

The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101.

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Can smartphone-based logging support diabetologists in solving glycemic control problems?

Treatment of diabetic patients strongly relies on the continuous logging of parameters relevant to glycemic control. Keeping diabetes diaries can be tedious which can affect the data quality and completeness. Mobile technologies could provide means to overcome these limitations. However, studies analyzing the direct effect on the treatment of patients are rare. In the presented study diabetic patients were supplied with a smartphone application to record various parameters relevant for glycemic control. Questions regarding the completeness of diabetes diaries were answered by the patients before and after the study. The attending diabetologist analyzed the data obtained from the smartphone-based diaries to determine whether these provided solutions for problems in glycemic control. The analysis of the available smartphone data provided the basis for therapeutic recommendations that can improve the daily glycemic control for almost all participants. Importantly, especially the newly developed implicit-activity logging, registering the participants' movements, provided important means to generate these recommendations.

Nitration of the birch pollen allergen Bet v 1.0101: efficiency and site-selectivity of liquid and gaseous nitrating agents.

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO(-)), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO(-), and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.

Determination of nitration degrees for the birch pollen allergen Bet v 1.

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration.

Development of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular effects.

Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful than the exposure to a larger number of micrometer-sized particles. An in vitro exposure system was established to expose human alveolar epithelial cells to freshly generated exhaust. Computer simulations were used to determine the optimal flow characteristics and ensure equal exposure conditions for each well of a 6-well plate. A selective particle size sampler was used to continuously deliver diesel soot particles with different particle size distributions to cells in culture. To determine, whether the system could be used for cellular assays, alterations in cytokine production and cell viability of human alveolar A549 cells were determined after 3h on-line exposure followed by a 21-h conventional incubation period. Data indicated that complete diesel engine emission slightly affected pre-stimulated cells, but naive cells were not affected. The fractions containing large or small particles never affected the cells. The experimental set-up allowed a reliable exposure of the cells to the complete exhaust fraction or to the fractions containing either large or small diesel engine emission particles.

Nitration of the pollen allergen bet v 1.0101 enhances the presentation of bet v 1-derived peptides by HLA-DR on human dendritic cells.

Nitration of pollen derived allergens can occur by NO(2) and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.

Hardening of the nanoparticle-protein corona in metal (Au, Ag) and oxide (Fe3O4, CoO, and CeO2) nanoparticles.

The surface modifications of metal and metal oxide nanoparticles with sizes ranging from 7 to 20 nm dispersed in commonly used cell culture medium supplemented with serum are investigated. All the tested nanoparticles adsorb proteins onto their surface, thereby forming a protein corona through a dynamic process evolving towards an irreversible coating (hard protein corona). Despite the fact that the studied nanomaterials have similar characteristics of hydrophobicity and surface charge, different temporal patterns of the protein corona formation are observed that can be considered a fingerprint for nanoparticle identification. Some of the biological and toxicological implications of the formation of the nanoparticle-protein corona are studied using the human monocytic cell line THP-1 exposed to cobalt oxide nanoparticles. Results show that production of reactive oxygen species is decreased if the nanoparticles are preincubated for 48 h with serum.

IL-37: a new anti-inflammatory cytokine of the IL-1 family.

The IL-1 family of cytokines encompasses eleven proteins that each share a similar ß-barrel structure and bind to Ig-like receptors. Some of the IL-1-like cytokines have been well characterised, and play key roles in the development and regulation of inflammation. Indeed, IL-1α (IL-1F1), IL-1ß (IL-1F2), and IL-18 (IL-1F4) are well-known inflammatory cytokines active in the initiation of the inflammatory reaction and in driving Th1 and Th17 inflammatory responses. In contrast, IL-1 receptor antagonist (IL-1Ra, IL-1F3) and the receptor antagonist binding to IL-1Rrp2 (IL-36Ra, IL-1F5) reduce inflammation by blocking the binding of the agonist receptor ligands. In the case of IL-37 (IL-1F7), of which five different splice variants have been described, less is known of its function, and identification of the components of a heterodimeric receptor complex remains unclear. Some studies suggest that IL-37 binds to the α chain of the IL-18 receptor in a non-competitive fashion, and this may explain some of the disparate biological effects that have been reported for mice deficient in the IL-18R. The biological properties of IL-37 are mainly those of down-regulating inflammation, as assessed in models where human IL-37 is expressed in mice. In this review, an overview of the role of IL-37 in the regulation of inflammation is presented. The finding that IL-37 also locates to the nucleus, as do IL-1α and IL-33, for receptor-independent organ/tissue-specific regulation of inflammation is also reviewed.

Biocompatible micro-sized cell culture chamber for the detection of nanoparticle-induced IL8 promoter activity on a small cell population.

In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds.PACS: 85.35.Be, 81.16.Nd, 87.18.Mp.