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.

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.

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.

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.

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.

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.

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.

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.

Introduction of functional chimeric E/L-selectin by RNA electroporation to target dendritic cells from blood to lymph nodes.

BACKGROUND: Inefficient migration of dendritic cells (DC) to regional lymph nodes (LN) upon intracutaneous injection is a major obstacle for effective DC vaccination. Intravenous vaccination is unfavorable, because DC cannot migrate directly from the blood into LN. METHODS: To enable human monocyte-derived (mo)DC to enter LN directly from the blood, we manipulated them by RNA electroporation to express a human chimeric E/L-selectin (CD62E/CD62L) protein, which binds to peripheral node addressin expressed on high endothelial venules. RESULTS: Transfection efficiency exceeded 95%, and high E/L-selectin surface expression was detected for >48 h. E/L-selectin RNA-transfected DC displayed an identical mature DC phenotype as mock-transfected DC. Furthermore, E/L-selectin-transfected DC maintained their normal CCR7-mediated migration capacity, and their ability to prime and expand functional cytotoxic T cells recognizing MelanA. Most importantly, E/L-selectin-RNA-transfected DC gained the capability to attach to and roll on sialyl-Lewis(X) in vitro. OUTLOOK: The presented strategy can be readily translated into the clinic, as it involves no stable genetic manipulation or viral transformation, and allows targeting of a large number of LN.

Impaired induction of adhesion molecule expression in immortalized endothelial cells leads to functional defects in dynamic interactions with lymphocytes.

Immortalization should overcome the problem of short lifespan and difficult culture of endothelial cells that limited their use in functional studies. We used four different immortalized endothelial cell lines to study dynamic interactions with lymphocytes. Surprisingly, tumor necrosis factor (TNF)alpha-stimulated human umbilical vein endothelial cells (HUVECs) or human dermal microvascular endothelial cells (HDMECs) readily supported rolling and binding of lymphocytes, whereas none of the immortalized cell lines did. As rolling interactions are primarily mediated by selectins and vascular cell adhesion molecule (VCAM)-1, the endothelial cells were analyzed regarding expression of selectins and other adhesion molecules. Interestingly, cell surface expression of E-selectin could only be detected on HUVEC and HDMEC. Immunocytochemistry showed that some immortalized endothelial cells expressed E-selectin intracellularly following TNFalpha stimulation, suggesting translation but defective post-translational processing or transport of the molecule. In contrast, other immortalized cell lines did not have detectable levels of E-selectin mRNA, suggesting impaired transcription. VCAM-1 could only be induced on normal and human placental microvascular endothelial cell-A2 endothelial cells, whereas all cell lines expressed intercellular adhesion molecule-1 following TNF stimulation. The immortalized endothelial cells tested here have lost functions that are required for dynamic interactions with immune cells and that are common to primary endothelial cells.