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.

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.

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.

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.

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.

Shape matters: effects of silver nanospheres and wires on human alveolar epithelial cells.

BACKGROUND: In nanotoxicology, the exact role of particle shape, in relation to the composition, on the capacity to induce toxicity is largely unknown. We investigated the toxic and immunotoxic effects of silver wires (length: 1.5 - 25 µm; diameter 100 - 160 nm), spherical silver nanoparticles (30 nm) and silver microparticles (<45 µm) on alveolar epithelial cells (A549). METHODS: Wires and nanoparticles were synthesized by wet-chemistry methods and extensively characterized. Cell viability and cytotoxicity were assessed and potential immunotoxic effects were investigated. To compare the effects on an activated and a resting immune system, cells were stimulated with rhTNF-α or left untreated. Changes in intracellular free calcium levels were determined using calcium imaging. Finally, ion release from the particles was assessed by ICP-MS and the effects of released ions on cell viability and cytotoxicity were tested. RESULTS: No effects were observed for the spherical particles, whereas the silver wires significantly reduced cell viability and increased LDH release from A549 cells. Cytokine promoter induction and NF-κB activation decreased in a concentration dependent manner similar to the decrease seen in cell viability. In addition, a strong increase of intracellular calcium levels within minutes after addition of wires was observed. This toxicity was not due to free silver ions, since the samples with the highest ion release did not induce toxicity and ion release control experiments with cells treated with pre-incubated medium did not show any effects either. CONCLUSIONS: These data showed that silver wires strongly affect the alveolar epithelial cells, whereas spherical silver particles had no effect. This supports the hypothesis that shape is one of the important factors that determine particle toxicity.

Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects.

BACKGROUND: With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated. RESULTS: In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin. CONCLUSION: The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

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.

Passive dosing for producing defined and constant exposure of hydrophobic organic compounds during in vitro toxicity tests.

Toxicity testing of hydrophobic organic compounds (HOCs) in plastic cell culture plates is problematic due to compound losses through volatilization and sorption to the wells and culture medium constituents. This leads to poorly defined exposure and reduced test sensitivity. Passive dosing can overcome these problems by the continual partitioning of HOCs from a dominating reservoir loaded in a biologically inert polymer such as silicone, providing defined and constant freely dissolved concentrations and also eliminating spiking with cosolvents. This study aimed to select a suitable passive dosing format for in vitro tests in multiwell plates and characterize its performance at 37 degrees C. Silicone O-rings were the most suitable format; they were both practical and demonstrated excellent passive dosing performance. (1) The rings were loaded by partitioning from a methanol solution containing polycyclic aromatic hydrocarbons (PAHs) (log K(OW), 3.33-6.43) that served as model compounds, followed by removal of the methanol with water. This resulted in highly reproducible HOC concentrations in the silicone O-rings. (2) The release of PAHs into aqueous solutions was rapid and reproducible, with equilibrium partitioning being reached within hours. (3) The buffering capacity of the O-rings was sufficient to maintain stable concentrations over more than 72 h. The O-rings were then applied to test a range of PAHs at their aqueous solubility in an array of established in vitro cell culture assays with human cells and cell lines. These included the formation of reactive oxygen species, induction of the IL-8 cytokine promoter, and secretion of MCP-1 by the cells. The biological responses depended on the melting point of the individual PAHs and their maximum chemical activities (a(max)). Only those PAHs with the highest a(max) stimulated the formation of reactive oxygen species and MCP-1 secretion, while they inhibited the induction of the IL-8 cytokine promoter.

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.

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.].

Computer-aided analysis of cell interactions under dynamic flow conditions.

Experimentally, initial steps of leucocyte extravasation, including tethering and rolling, are analysed in endothelial cell flow chambers. Given the complexity and speed of endothelial-immune cell interaction, computer-aided advances of this analysis are highly desirable. Herein, we compared two established methods, hand counting and tracking software, with novel analysis software using defined movies recorded at standard conditions of endothelial-leucocyte interactions. As a first validation, cell counts and velocity parameters determined by seven experienced experts revealed no statistic differences to both semi-automated tracking and fully computerized analyses. Nevertheless, interindividual variations were substantial for hand counting. In additional experiments, velocity distributions between 1 and 800 microm/s picked up by the fully computerized analysis matched well with the tracking software as indicated by speed vector histograms. With respect to the time consumed for a defined set of movies, hand counting took 3.6 +/- 1.6 h, tracking software 4.5 +/- 1.2 h, whereas fully automated analysis consumed less than 15 min, reaching real-time mode. Thus, a validated and fully computerized method yielded functional flow chamber data unbiased, independent from an examiner, and reaching high-throughput level, which in turn will allow a substantial progress in understanding this process central for skin inflammation.

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.

PECAM-1 polymorphism affects monocyte adhesion to endothelial cells.

BACKGROUND: Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. METHODS: We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. RESULTS: There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. CONCLUSIONS: This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

Diminished thrombus formation and alleviation of myocardial infarction and reperfusion injury through antibody- or small-molecule-mediated inhibition of selectin-dependent platelet functions.

BACKGROUND AND OBJECTIVES: P-selectin ctin has been implicated in important platelet functions. However, neither its role in thrombus formation and cardiovascular disorders nor its suitability as a therapeutic target structure is entirely clear. DESIGN AND METHODS: Platelet aggregation was assessed in complementary in vitro settings by measurements of static aggregation, standardized aggregometry and dynamic flow chamber assays. Degradation of aggregates was also analyzed under flow conditions using video microscopy. In vivo, platelet rolling in cutaneous venules was assessed by intravital microscopy in wild-type mice treated with selectin-blocking compounds as well as in P-selectin-deficient mice. FeCl3-induced arterial thrombosis was studied by intravital microscopy in untreated mice or mice treated with an inhibitor of selectin functions. Finally, inhibition of selectin functions was studied in an ischemia/reperfusion injury model in rats. RESULTS: Antibody- or small-molecule-mediated inhibition of P-selectin functions significantly diminished platelet aggregation (p<0.03) and platelet-neutrophil adhesion in vitro (p<0.01) as well as platelet aggregate sizes under flow (p<0.03). Established aggregates were degraded, either via detachment of single platelets following addition of efomycine M, or via detachment of multicellular clumps when P-selectin-directed Fab-fragments were used. In vivo, selectin inhibition resulted in a greater than 50% reduction of platelet rolling in cutaneous venules (p<0.01), producing rolling fractions similar to those observed in P-selectin-deficient mice (p<0.05). Moreover, inhibition of selectin functions significantly decreased the thrombus size in FeCl3-induced arterial thrombosis in mice (p<0.05). In an ischemia/reperfusion injury model in rats, small-molecule-mediated selectin inhibition significantly reduced myocardial infarct size from 18.9% to 9.42% (p<0.001) and reperfusion injury (p<0.001). INTERPRETATION AND CONCLUSIONS: Inhibition of P-selectin functions reduces platelet aggregation and can alleviate platelet-related disorders in disease-relevant preclinical settings.

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.

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.

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.

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.