Suspected gut barrier disruptors and development of food allergy: Adjuvant effects and early immune responses.

Food allergy is an increasing public health challenge worldwide. It has recently been hypothesized that the increase in exposure to intestinal epithelial barrier-damaging biological and chemical agents contribute to this development. In animal models, exposure to adjuvants with a food allergen has been shown to promote sensitization and development of food allergy, and barrier disrupting capacities have been suggested to be one mechanism of adjuvant action. Here, we investigated how gut barrier disrupting compounds affected food allergy development in a mouse model of peanut allergy. Sensitization and clinical peanut allergy in C3H/HEOuJ mice were assessed after repeated oral exposure to peanut extract together with cholera toxin (CT; positive control), the mycotoxin deoxynivalenol (DON), house dust mite (HDM) or the pesticide glyphosate (GLY). In addition, we investigated early effects 4 to 48 h after a single exposure to the compounds by assessing markers of intestinal barrier permeability, alarmin production, intestinal epithelial responses, and local immune responses. CT and DON exerted adjuvant effects on peanut allergy development assessed as clinical anaphylaxis in mice. Early markers were affected only by DON, observed as increased IL-33 (interleukin 33) and thymic stromal lymphopoietin (TSLP) alarmin production in intestines and IL-33 receptor ST2 in serum. DON also induced an inflammatory immune response in lymph node cells stimulated with lipopolysaccharide (LPS). HDM and GLY did not clearly promote clinical food allergy and affected few of the early markers at the doses tested. In conclusion, oral exposure to CT and DON promoted development of clinical anaphylaxis in the peanut allergy mouse model. DON, but not CT, affected the early markers measured in this study, indicating that DON and CT have different modes of action at the early stages of peanut sensitization.

Reduced polyfunctional T cells and increased cellular activation markers in adult allergy patients reporting adverse reactions to food.

BACKGROUND: The underlying cellular mechanisms causing adverse reactions to food are complex and still not fully understood. Therefore, in this study we aimed to identify functional and/or phenotypical immune cell signatures characteristic for adult patients reporting adverse reactions to food. By mass cytometry, we performed high-dimensional profiling of peripheral blood mononuclear cells (PBMC) from adult patients reporting adverse reactions to food and healthy controls. The patients were grouped according to sIgE-positive or sIgE-negative serology to common food and inhalant allergens. Two broad antibody panels were used, allowing determination of major immune cell populations in PBMC, as well as activation status, proliferation status, and cytokine expression patterns after PMA/ionomycin-stimulation on a single cell level. RESULTS: By use of data-driven algorithms, several cell populations were identified showing significantly different marker expression between the groups. Most striking was an impaired frequency and function of polyfunctional CD4+ and CD8+ T cells in patients reporting adverse reactions to food compared to the controls. Further, subpopulations of monocytes, T cells, and B cells had increased expression of functional markers such as CD371, CD69, CD25, CD28, and/or HLA-DR as well as decreased expression of CD23 in the patients. Most of the differing cell subpopulations were similarly altered in the two subgroups of patients. CONCLUSION: Our results suggest common immune cell features for both patient subgroups reporting adverse reactions to food, and provide a basis for further studies on mechanistic and diagnostic biomarker studies in food allergy.

Feasibility of desensitizing children highly allergic to peanut by high-dose oral immunotherapy.

BACKGROUND: There are limited data on the feasibility, efficacy and safety of high-dose oral immunotherapy (OIT) in children highly allergic to peanuts. OBJECTIVE: In children highly allergic to peanut, we primarily aimed to determine the feasibility of reaching the maximum maintenance dose (MMD) of 5000 mg peanut protein or, alternatively, a lower individual maintenance dose (IMD), by OIT up-dosing. Secondarily, we aimed to identify adverse events (AEs) and determine factors associated with reaching a maintenance dose. METHODS: The TAKE-AWAY peanut OIT trial enrolled 77 children 5-15 years old, with a positive oral peanut challenge. Fifty-seven were randomized to OIT with biweekly dose step-up until reaching MMD or IMD and 20 to observation only. Demographic and biological characteristics, AEs, medication and protocol deviations were explored for associations with reaching maintenance dose. RESULTS: All children had anaphylaxis defined by objective symptoms in minimum two organ systems during baseline challenge. The MMD was reached by 21.1%, while 54.4% reached an IMD of median (minimum, maximum) 2700 (250, 4000) mg peanut protein, whereas 24.5% discontinued OIT. During up-dosing, 19.4% experienced anaphylaxis. Not reaching the MMD was caused by distaste for peanuts (66.7%), unacceptable AEs (26.7%) and social reasons (6.7%). Increased peanut s-IgG4 /s-IgE ratio (OR [95% CI]: 1.02 [1.00, 1.04]) was associated with reaching MMD. CONCLUSION: Although 75.5% of children with peanut anaphylaxis reached a maintenance dose of 0.25-5 g, only 21.1% reached the MMD. Distaste for peanuts and AEs, including high risk of anaphylaxis, limited the feasibility of reaching MMD.

Exposure of Norwegian toddlers to perfluoroalkyl substances (PFAS): The association with breastfeeding and maternal PFAS concentrations.

High exposure to perfluoroalkyl substances (PFASs) has been associated with adverse health effects in children. PFASs exposure pathways of toddlers might differ from those of infants and adults, and the investigations on determinants of PFASs exposure in early childhood are scarce. Our aims were to examine the PFAS blood concentrations in Norwegian toddlers and to assess their relationship with maternal PFAS concentrations in pregnancy and breastfeeding duration. We determined PFAS concentrations in 112 plasma samples of 3-year-old children collected at 2010-2011 and 99 maternal serum samples collected around delivery at 2007-2008. PFAS concentrations in children were regressed on duration of breastfeeding, and the effect modification by maternal prenatal PFAS concentrations was examined in 55 mother-child pairs. Six PFASs were quantifiable in >50% of both maternal and children samples. Positive and significant correlations ranging between 0.50 and 0.66 were found between maternal and child concentrations of the same PFAS congeners. Nevertheless, toddlers had higher total PFAS blood concentrations than their mothers, due to higher concentrations of PFOA, PFNA and PFHxS. Every month of breastfeeding was associated with an increase of 3.3% (95% Confidence Intervals (CI): 0.8-5.8) for PFOS, 4.7% (95%CI: 2.8-6.6) for PFOA and 6.1% (95% CI: 2.6-9.7) for PFHpS in toddlers' plasma and a dose-response association was found, after adjustment for confounders. However, PFNA and PFUnDA concentrations in children were not associated with either maternal concentrations or breastfeeding duration. Our findings suggest that transplacental transfer, prenatally, and breastfeeding, postanatally, are among the main determinants of PFOS, PFOA, PFHxS and PFHpS concentrations in toddlers, while that was not the case for PFNA and PFUnDA. Nevertheless, due to the small number of mother child-pairs in our study, our results should be interpreted with caution.

Cord blood gene expression supports that prenatal exposure to perfluoroalkyl substances causes depressed immune functionality in early childhood.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds that have widespread use in consumer and industrial applications. PFAS are considered environmental pollutants that have various toxic properties, including effects on the immune system. Recent human studies indicate that prenatal exposure to PFAS leads to suppressed immune responses in early childhood. In this study, data from the Norwegian BraMat cohort was used to investigate transcriptomics profiles in neonatal cord blood and their association with maternal PFAS exposure, anti-rubella antibody levels at 3 years of age and the number of common cold episodes until 3 years. Genes associated with PFAS exposure showed enrichment for immunological and developmental functions. The analyses identified a toxicogenomics profile of 52 PFAS exposure-associated genes that were in common with genes associated with rubella titers and/or common cold episodes. This gene set contains several immunomodulatory genes (CYTL1, IL27) as well as other immune-associated genes (e.g. EMR4P, SHC4, ADORA2A). In addition, this study identified PPARD as a PFAS toxicogenomics marker. These markers can serve as the basis for further mechanistic or epidemiological studies. This study provides a transcriptomics connection between prenatal PFAS exposure and impaired immune function in early childhood and supports current views on PPAR- and NF-κB-mediated modes of action. The findings add to the available evidence that PFAS exposure is immunotoxic in humans and support regulatory policies to phase out these substances.

Peanut sensitization pattern in Norwegian children and adults with specific IgE to peanut show age related differences.

BACKGROUND: Peanuts contain potent food allergens and the prevalence of allergy is reported to increase, especially in children. Since peanut sensitization may differ between different geographical regions, we wanted to investigate the sensitization pattern to the individual peanut allergens in a Norwegian population. METHODS: Cases reported to the Norwegian Food Allergy Register with sera positive to peanut extract were analyzed for specific IgE (sIgE) to the recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 8 and Ara h 9 and to birch pollen extract. Serum samples negative to the above allergens were analyzed for sIgE to Ara h 6, and sIgE to Pru p 3 in peach were analyzed in sera positive to the cross-reactive allergen Ara h 9. RESULTS: Highest frequency of sIgE to Ara h 2, often co-sensitized to Ara h 1 and 3, were found in the small children up to 6 years of age. From the age of 6 years, sensitization to Ara h 8 was predominant. The sIgE levels to the storage proteins Ara h 1, 2 and 3 were strongly correlated, as was the sIgE levels to Ara h 8 and birch pollen extract. A low sensitization rate of sIgE to Ara h 9 in young adults was observed, which sIgE levels were very strongly correlated to Pru p 3. CONCLUSION: The sensitization to peanut allergens in a Norwegian population shows a clear age dependent pattern. The results add to the previously published research on the sensitization patterns of peanut sensitized patients in different geographical areas.

The effect of dietary estimates calculated using food frequency questionnaires on micronuclei formation in European pregnant women: a NewGeneris study.

The use of biomarkers of early genetic effects, predictive for cancer, such as micronuclei (MN) in lymphocytes, may help to investigate the association between diet and cancer. We hypothesised that the presence of mutagens in the diet may increase MN formation. A 'pooled' standardised analysis was performed by applying the same experimental protocol for the cytokinesis block micronucleus assay in 625 young healthy women after delivery from five European study populations (Greece, Denmark, UK, Spain and Norway). We assessed MN frequencies in mono- and binucleated T-lymphocytes (MNMONO and MNBN) and the cytokinesis blocked proliferation index using a semi-automated image analysis system. Food frequency questionnaires (FFQs) were used to estimate intake of fatty acids and a broad range of immunotoxic and genotoxic/carcinogenic compounds through the diet. Pooled difference based on delivery type revealed higher MNMONO frequencies in caesarean than in vaginal delivery (P = 0.002). Statistical analysis showed a decrease in MNMONO frequencies with increasing calculated omega-6 PUFA concentrations and a decrease in MNBN frequencies with increasing calculated omega-3 PUFA concentrations. The expected toxic compounds estimated by FFQs were not associated with MN formation in mothers after delivery. In pregnant women, an omega-3 and -6 rich diet estimated by FFQ is associated with lower MN formation during pregnancy and delivery.

Pre-natal exposure to perfluoroalkyl substances may be associated with altered vaccine antibody levels and immune-related health outcomes in early childhood.

Perfluoroalkyl substances (PFAS) are suggested to have immunosuppressive effects; exposure in utero and in the first years of life is of special concern as fetuses and small children are highly vulnerable to toxicant exposure. The objective of this study was to investigate the effect of pre-natal exposure to PFAS on responses to pediatric vaccines and immune-related health outcomes in children up to 3 years of age. In the prospective birth-cohort BraMat, a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa), pregnant women from Oslo and Akershus, Norway, were recruited during 2007-2008. Three annual questionnaire-based follow-ups were performed. Blood samples were collected from the mothers at the time of delivery and from the children at the age of 3 years. As a measure of pre-natal exposure to PFAS, the concentrations of perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) were determined in maternal blood from 99 BraMat participants. Main outcome measures were anti-vaccine antibody levels, common infectious diseases and allergy- and asthma-related health outcomes in the children up to the age of 3 years. There was an inverse association between the level of anti-rubella antibodies in the children's serum at age 3 years and the concentrations of the four PFAS. Furthermore, there was a positive association between the maternal concentrations of PFOA and PFNA and the number of episodes of common cold for the children, and between PFOA and PFHxS and the number of episodes of gastroenteritis. No associations were found between maternal PFAS concentrations and the allergy- and asthma-related health outcomes investigated. The results indicate that pre-natal exposure to PFAS may be associated with immunosuppression in early childhood.

Prenatal exposure to polychlorinated biphenyls and dioxins from the maternal diet may be associated with immunosuppressive effects that persist into early childhood.

We investigated whether prenatal exposure from the maternal diet to the toxicants polychlorinated biphenyls (PCBs) and dioxins is associated with the development of immune-related diseases in childhood. Children participating in BraMat, a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa), were followed in the three first years of life using annual questionnaires (0-3years; n=162, 2-3years; n=180), and blood parameters were examined at three years of age (n=114). The maternal intake of the toxicants was calculated using a validated food frequency questionnaire from MoBa. Maternal exposure to PCBs and dioxins was found to be associated with an increased risk of wheeze and more frequent upper respiratory tract infections. Furthermore, maternal exposure to PCBs and dioxins was found to be associated with reduced antibody response to a measles vaccine. No associations were found between prenatal exposure and immunophenotype data, allergic sensitization and vaccine-induced antibody responses other than measles. Our results suggest that prenatal dietary exposure to PCBs and dioxins may increase the risk of wheeze and the susceptibility to infectious diseases in early childhood.

Global gene expression analysis in cord blood reveals gender-specific differences in response to carcinogenic exposure in utero.

BACKGROUND: It has been suggested that fetal carcinogenic exposure might lead to predisposition to develop cancer during childhood or in later life possibly through modulation of the fetal transcriptome. Because gender effects in the incidence of childhood cancers have been described, we hypothesized differences at the transcriptomic level in cord blood between male and female newborns as a consequence of fetal carcinogenic exposure. The objective was to investigate whether transcriptomic responses to dietary genotoxic and nongenotoxic carcinogens show gender-specific mechanisms-of-action relevant for chemical carcinogenesis. METHODS: Global gene expression was applied in umbilical cord blood samples, the CALUX-assay was used for measuring dioxin(-like), androgen(-like), and estrogen(-like) internal exposure, and acrylamide-hemoglobin adduct levels were determined by mass spectrometry adduct-FIRE-procedure(TM). To link gene expression to an established phenotypic biomarker of cancer risk, micronuclei frequencies were investigated. RESULTS: While exposure levels did not differ between sexes at birth, important gender-specific differences were observed in gene expressions associated with these exposures linked with cell cycle, the immune system and more general cellular processes such as posttranslation. Moreover, oppositely correlating leukemia/lymphoma genes between male and female newborns were identified in relation to the different biomarkers of exposure that might be relevant to male-specific predisposition to develop these cancers in childhood. CONCLUSIONS/IMPACT: This study reveals different transcriptomic responses to environmental carcinogens between the sexes. In particular, male-specific TNF-alpha-NF-kB signaling upon dioxin exposure and activation of the Wnt-pathway in boys upon acrylamide exposure might represent possible mechanistic explanations for gender specificity in the incidence of childhood leukemia.

Severe allergic reactions to food in Norway: a ten year survey of cases reported to the food allergy register.

The Norwegian Food Allergy Register was established at the Norwegian Institute of Public Health in 2000. The purpose of the register is to gain information about severe allergic reactions to food in Norway and to survey food products in relation to allergen labelling and contamination. Cases are reported on a voluntary basis by first line doctors, and submitted together with a serum sample for specific IgE analysis. The register has received a total of 877 reports from 1 July, 2000 to 31 December, 2010. Two age groups, small children and young adults are over-represented, and the overall gender distribution is 40:60 males-females. The legumes lupine and fenugreek have been identified as two "new" allergens in processed foods and cases of contamination and faults in production of processed foods have been revealed. The highest frequency of food specific IgE is to hazelnuts and peanuts, with a marked increase in reactions to hazelnuts during the last three years. The Food Allergy Register has improved our knowledge about causes and severity of food allergic reactions in Norway. The results show the usefulness of population based national food allergy registers in providing information for health authorities and to secure safe food for individuals with food allergies.

Prenatal exposure to polychlorinated biphenyls and dioxins is associated with increased risk of wheeze and infections in infants.

The birth cohort BraMat (n = 205; a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health) was established to study whether prenatal exposure to toxicants from the maternal diet affects immunological health outcomes in children. We here report on the environmental pollutants polychlorinated biphenyls (PCBs) and dioxins, as well as acrylamide generated in food during heat treatment. The frequency of common infections, eczema or itchiness, and periods of more than 10 days of dry cough, chest tightness or wheeze (called wheeze) in the children during the first year of life was assessed by questionnaire data (n = 195). Prenatal dietary exposure to the toxicants was estimated using a validated food frequency questionnaire from MoBa. Prenatal exposure to PCBs and dioxins was found to be associated with increased risk of wheeze and exanthema subitum, and also with increased frequency of upper respiratory tract infections. We found no associations between prenatal exposure to acrylamide and the health outcomes investigated. Our results suggest that prenatal dietary exposure to dioxins and PCBs may increase the risk of wheeze and infectious diseases during the first year of life.

Transcriptomic profile indicative of immunotoxic exposure: in vitro studies in peripheral blood mononuclear cells.

Investigating the immunotoxic effects of exposure to chemicals usually comprises evaluation of weight and histopathology of lymphoid tissues, various lymphocyte parameters in the circulation, and immune function. Immunotoxicity assessment is time consuming in humans or requires a high number of animals, making it expensive. Furthermore, reducing the use of animals in research is an important ethical and political issue. Immunotoxicogenomics represents a novel approach to investigate immunotoxicity able of overcoming these limitations. The current research, embedded in the European Union project NewGeneris, aimed to retrieve gene expression profiles that are indicative of exposure to immunotoxicants. To this end, whole-genome gene expression was investigated in human peripheral blood mononuclear cells in response to in vitro exposure to a range of immunotoxic chemicals (4-hydroxy-2-nonenal, aflatoxin B1, benzo[a]pyrene, deoxynivalenol, ethanol, malondialdehyde, polychlorinated biphenyl 153, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and nonimmunotoxic chemicals (acrylamide, dimethylnitrosamine, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine). Using Agilent oligonucleotide microarrays, whole-genome gene expression profiles were generated, which were analyzed using Genedata's Expressionist software. Using Recursive Feature Elimination and Support Vector Machine, a set of 48 genes was identified that distinguishes the immunotoxic from the nonimmunotoxic compounds. Analysis for enrichment of biological processes showed the gene set to be highly biologically and immunologically relevant. We conclude that we have identified a promising transcriptomic profile indicative of immunotoxic exposure.

Evaluation of the genotoxicity of 10 selected dietary/environmental compounds with the in vitro micronucleus cytokinesis-block assay in an interlaboratory comparison.

Complex exposure to xenobiotics is one of the reasons for the reported increase of respiratory diseases, cancer and immunological disturbances. Among such xenobiotics there are food mutagens whose effects on human health in the low level and/or chronic exposure still remains unknown. In the present manuscript, the compounds ethanol (EtOH), 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl (PCB 153), benzo[a]pyrene (BaP), 2-amino-3-methylimidazol[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP), N-Nitrosodimethylamine (NDMA) and acrylamide (AA) were evaluated in an interlaboratory comparison in the in vitro cytokinesis-block micronucleus assay (CBMN) with objective of assessing the induction of micronuclei, buds and nucleoplasmic bridges in dose responses. Statistically significant increase in MNBN frequency in binucleated cells was recorded by both laboratories for the compound PhIP (2.5µM). The compounds PCB (250 microM) and AA (500 microM) induced statistically significant increase of MNBN although it was recorded by one of the two laboratories. Induction of buds and nucleoplasmic bridges was only observed for BaP (100 microM) and AA (500 microM) by one of the laboratories. Data generated in this study may assist in the interpretation of the mother/newborn biomonitoring study being carried out within project NewGeneris and will contribute to overall knowledge on the genotoxic potential of dietary/environmental toxicants.

Allergenicity and antigenicity of fenugreek (Trigonella foenum-graecum) proteins in foods.

BACKGROUND: Fenugreek is an ingredient in Indian-style spiced foods. Reports of adverse reactions reflect a trend toward a more international cuisine. Fenugreek allergy has not been systematically investigated so far. OBJECTIVE: Study the allergenicity and antigenicity of fenugreek proteins using patient sera and a newly developed polyclonal antifenugreek antibody. METHODS: Allergenic fenugreek proteins were identified by immunoblotting, using sera from 29 patients with specific IgE to peanut and other legumes. In addition, 2 patients were evaluated by skin prick test and open food challenge with native fenugreek powder. Spiced and flavored food products were analyzed for fenugreek by semiquantitative IgE and IgG immunoblotting. RESULTS: High levels of specific IgE to both peanut and fenugreek were seen in most sera. Fenugreek sensitization is believed to be a consequence of cross-reactivity in patients with peanut allergy. Primary fenugreek allergy was suspected in only 1 case. The fenugreek dose eliciting objective symptoms was about 2 mg in the open food challenge. Major fenugreek allergens were identified at 50, 52, and 74 kd and peanut proteins at 22, 36, and 40 kd. A specific polyclonal antifenugreek antibody was found suitable for food analysis. In a food survey, about 1/3 of the fenugreek-containing products were labeled correctly. CONCLUSION: Fenugreek seed powder, an ingredient in spiced foods, contains several potential allergens. There is evidence for a high rate of cross-reactivity to peanut.

Pro-inflammatory potential of wood smoke and traffic-derived particles in a monocytic cell line.

Lung inflammation is an important process in host defence to inhaled particulate matter. To what extent physicochemical properties of particles from different sources influence their inflammatory potential has not been fully clarified. The aim of this study was to investigate the potential of particles from wood smoke and traffic to induce a release of pro-inflammatory cytokines in the monocytic cell line THP-1. The influence of endotoxin on cytokine release was investigated using the inhibitor polymyxin B sulphate, whereas the responses to native particles, washed particles and their organic extracts were compared to determine the role of the organic fraction. Particles from the two sources showed a similar inflammatory potential, but the response was mediated by different particle characteristics. The organic fraction of wood smoke accounted for the majority of the cytokine release, whereas the response to the traffic-derived particles was in addition influenced by endotoxin and the particle core. The sum of the cytokine release induced by the organic extract and washed particles was lower than that induced by native particles, suggesting that the organic fraction must be adsorbed to the particles to exert biological activity. The results also indicated that different particle characteristics may activate different signalling pathways, since inhibition of endotoxin reduced release of TNF-alpha, IL-1beta and IL-8, whereas organic extraction only affected release of TNF-alpha and IL-8. Together, these data illustrate that a similar inflammatory response may be mediated by different particle characteristics and possibly through different signalling pathways.

Quantification and characterisation of IgG binding to mould spores by flow cytometry and scanning electron microscopy.

The concentration of mould-specific IgG antibodies in serum may objectively indicate mould exposure and can help identifying exposed individuals. Although inhaled spores probably are the most important source of mould exposure, the commonly used methods for detecting mould-specific IgG antibodies are based on extracts from all mould components, with only low contribution from spores. We have developed a flow cytometric method using surface antigens on mould spores for quantifying mould-specific IgG antibodies in serum. Flow cytometric results were evaluated by comparison with ImmunoCap and ELISA measurements. The flow cytometric assay showed a broad linear dose-dependency and correlated moderately to strongly (r=0.41-0.97) with ImmunoCap and ELISA measurements. The IgG antibody binding was studied in detail by immunolabelling in scanning electron microscopy (SEM), revealing that morphology and IgG antibody binding differed among spores, both within and between mould strains. Germination studies by flow cytometry and SEM showed that IgG antibody binding to mould spores was altered during germination due to loss of coat. The present spore based antibody assay are simple and suitable for quantification of mould-specific IgG antibodies in serum, and includes specificity to other and possibly more relevant antigens than existing methods.

Mechanisms of silica-induced IL-8 release from A549 cells: initial kinase-activation does not require EGFR activation or particle uptake.

Understanding how mineral particles trigger cellular responses is crucial in order to elucidate what characteristics determine their harmful effects. It is not clear whether cellular effects are triggered through the cell membrane or require particle uptake. However, studies with asbestos suggest that activation of the epidermal growth factor receptor (EGFR) may be important. We have previously reported that crystalline silica-induced interleukin (IL)-8 release from human lung epithelial cells (A549) was regulated through Src family kinases (SFKs) and the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK)-1 and -2. The present study shows that SFK and p38 phosphorylation increased almost immediately upon crystalline silica exposure, whereas ERK1/2 phosphorylation increased after 10 min of exposure. The p38 inhibitor SB202190 increased the silica-induced ERK1/2 phosphorylation suggesting that p38 activity may attenuate activation of ERK1/2. Scanning electron microscopy showed that some silica particles were phagocytosed between 1 and 4h of exposure, but that the majority remained bound by microvilli on the cell surface. The EGFR inhibitor AG1478 attenuated both silica-induced IL-8 release and phosphorylation of SFKs and ERK1/2. However, AG1478 also inhibited the respective background levels, and the EGFR was not phosphorylated at the onset of silica exposure. The results suggest that crystalline silica triggers p38 and SFK-ERK1/2 signaling through interactions with membrane components as both pathways were rapidly activated prior to particle internalization. However, the silica-induced up-regulation of IL-8 release through the SFK-ERK1/2 pathway does not appear to be initiated through activation of the EGFR, although basal EGFR activity may affect the magnitude of the responses.

Detection of allergens adsorbed to ambient air particles collected in four European cities.

Air pollution has been implicated as one of the factors responsible for the increased incidence of allergic diseases observed in recent years. High concentrations of air pollutants may promote airway sensitization by acting as adjuvants. Ambient particles as carriers of adsorbed allergens are, therefore, of special interest since they may act as mediators of inflammatory as well as allergic responses. Ambient air particles from four cities in Europe were collected, in three different seasons, to examine the variation of allergens and their possible binding to the pollution particles. The particle fraction, PM10, was collected on polycarbonate filters using a low-volume sampling regime. The presence of pollen allergens, latex and beta-glucans was investigated using an immunogold labelling method directly on the collection filters. Scanning electron microscopy revealed mainly the classical carbon particles and aggregates determined to originate from vehicle exhaust. The immunogold labelling visualised in the backscatter electron imaging mode, showed that allergens from pollens, latex and also beta-glucans were bound to and, hence, transported by the combustion particles in ambient air. Thus, combustion particles in ambient air are carriers of allergens and act as depots of allergens inhaled into the airways.

Physicochemical characterisation of combustion particles from vehicle exhaust and residential wood smoke.

BACKGROUND: Exposure to ambient particulate matter has been associated with a number of adverse health effects. Particle characteristics such as size, surface area and chemistry seem to influence the negative effects of particles. In this study, combustion particles from vehicle exhaust and wood smoke, currently used in biological experiments, were analysed with respect to microstructure and chemistry. METHODS: Vehicle exhaust particles were collected in a road tunnel during two seasons, with and without use of studded tires, whereas wood smoke was collected from a stove with single-stage combustion. Additionally, a reference diesel sample (SRM 2975) was analysed. The samples were characterised using transmission electron microscopy techniques (TEM/HRTEM, EELS and SAED). Furthermore, the elemental and organic carbon fractions were quantified using thermal optical transmission analysis and the content of selected PAHs was determined by gas chromatography-mass spectrometry. RESULTS: Carbon aggregates, consisting of tens to thousands of spherical primary particles, were the only combustion particles identified in all samples using TEM. The tunnel samples also contained mineral particles originating from road abrasion. The geometric diameters of primary carbon particles from vehicle exhaust were found to be significantly smaller (24 +/- 6 nm) than for wood smoke (31 +/- 7 nm). Furthermore, HRTEM showed that primary particles from both sources exhibited a turbostratic microstructure, consisting of concentric carbon layers surrounding several nuclei in vehicle exhaust or a single nucleus in wood smoke. However, no differences were detected in the graphitic character of primary particles from the two sources using SAED and EELS. The total PAH content was higher for combustion particles from wood smoke as compared to vehicle exhaust, whereas no source difference was found for the ratio of organic to total carbon. CONCLUSION: Combustion particles from vehicle exhaust and residential wood smoke differ in primary particle diameter, microstructure, and PAH content. Furthermore, the analysed samples seem suitable for assessing the influence of physicochemical characteristics of particles on biological responses.