Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice.

BACKGROUND: In genetically modified (GM) crops there is a risk that the inserted genes may introduce new allergens and/or adjuvants into the food and feed chain. The MON810 maize, expressing the insecticidal Cry1Ab toxin, is grown in many countries worldwide. In animal models, intranasal and intraperitoneal immunisations with the purified Cry1Ab proteins have induced immune responses, and feeding trials with Cry1Ab-containing feed have revealed some altered immune responses. Previous investigations have primarily measured antibody responses to the protein, while investigations of clinical food allergy symptoms, or allergy promotion (adjuvant effect) associated with the Cry1Ab protein are largely missing. We aimed to investigate immunogenic, allergenic and adjuvant properties of purified Cry1Ab toxin (trypCry1Ab, i.e., trypsin activated Cry1Ab) in a mouse model of food allergy. METHOD: Female C3H/HeJ mice were immunized by intragastric gavage of 10 µg purified, trypsin activated Cry1Ab toxin (trypCry1Ab) alone or together with the food allergen lupin. Cholera toxin was added as a positive control for adjuvant effect to break oral tolerance. Clinical symptoms (anaphylaxis) as well as humoral and cellular responses were assessed. RESULTS: In contrast to results from previous airway investigations, we observed no indication of immunogenic properties of trypCry1Ab protein after repeated intragastric exposures to one dose, with or without CT as adjuvant. Moreover, the results indicated that trypCry1Ab given by the intragastric route was not able to promote allergic responses or anaphylactic reactions against the co-administered allergen lupin at the given dose. CONCLUSION: The study suggests no immunogenic, allergenic or adjuvant capacity of the given dose of trypCry1Ab protein after intragastric exposure of prime aged mice.

In utero exposure to compounds with dioxin-like activity and birth outcomes.

BACKGROUND: Maternal exposure to dioxins and dioxin-like compounds may affect fetal growth and development. We evaluated the association between in utero dioxin-like activity and birth outcomes in a prospective European mother-child study. METHODS: We measured dioxin-like activity in maternal and cord blood plasma samples collected at delivery using the Dioxin-Responsive Chemically Activated LUciferase eXpression (DR CALUX) bioassay in 967 mother-child pairs, in Denmark, Greece, Norway, Spain, and England. Multiple linear regression models were used to investigate the associations with birth weight, gestational age, and head circumference. RESULTS: Plasma dioxin-like activity was higher in maternal sample than in cord samples. Birth weight was lower with medium (-58 g [95% confidence interval (CI) = -176 to 62]) and high (-82 g [-216 to 53]) tertiles of exposure (cord blood) compared with the lowest tertile. Gestational age was shorter by approximately half a week in the highest compared with the lowest (-0.4 weeks [95% CI = -0.8 to -0.1]). This association was stronger in boys than in girls, although the statistical evidence for interaction was weak (P = 0.22). Analysis based on CALUX-toxic equivalents expressed per milliliter of plasma showed similar trends. We found no association between dioxin-like activity in maternal plasma and birth outcomes. CONCLUSIONS: Results from this international general population study suggest an association between low-level prenatal dioxin-like activity and shorter gestational age, particularly in boys, with weaker associations for birth weight.

Particles influence allergic responses in mice--role of gender and particle size.

Epidemiological evidence suggesting that exposure to traffic air pollution may enhance sensitization to common allergens in children is increasing, and animal studies support biological plausibility and causality. The effect of air pollution on respiratory symptoms was suggested to be gender dependent. Previous studies showed that allergy-promoting activity of polystyrene particles (PSP) increased with decreasing particle size after footpad injection of mice. The primary aim of this study was to confirm the influence of particle size on the immunoglobulin E (IgE)-promoting capacity of particles in an airway allergy model. A second aim was to examine whether the allergy-promoting capacity of particles was influenced by gender. Female and male mice were intranasally exposed to the allergen ovalbumin (OVA) with or without ultrafine, fine, or coarse PSP modeling the core of ambient air particles. After intranasal booster immunizations with OVA, serum levels of OVA-specific IgE antibodies, and also markers of airway inflammation and cellular responses in the lung-draining mediastinal lymph nodes (MLN), were determined. PSP of all sizes promoted allergic responses, measured as increased serum concentrations of OVA-specific IgE antibodies. Further, PSP produced eosinophilic airway inflammation and elevated MLN cell numbers as well as numerically reducing the percentage of regulatory T cells. Ultrafine PSP produced stronger allergic responses to OVA than fine and coarse PSP. Although PSP enhanced sensitization in both female and male mice, significantly higher IgE levels and numbers of eosinophils were observed in females than males. However, the allergy-promoting effect of PSP was apparently independent of gender. Thus, our data support the notion that ambient air particle pollution may affect development of allergy in both female and male individuals.

Long-term bisphenol A exposure accelerates insulitis development in diabetes-prone NOD mice.

Exposure to the endocrine disruptor (ED) bisphenol A (BPA) used in polycarbonate plastic and epoxy resins appears ubiquitous since BPA can be found in over 90% of analyzed urine samples from all age groups. There is a parallel occurrence of increased prevalence in type 1 diabetes mellitus (T1DM) and an increased exposure to EDs the last decades. T1DM is caused by insulin deficiency due to autoimmune destruction of insulin producing pancreatic beta cells and has been suggested to be induced by various environmental factors acting together with a genetic predisposition. The objective of the present study was to investigate the effect of BPA (0, 1 and 100 mg/l BPA in the drinking water) on T1DM development in nonobese diabetic (NOD) mice, spontaneously developing T1DM. Histological evaluation of pancreas from 12-weeks-old female mice revealed significantly increased insulitis in mice exposed to 1 mg/l BPA, while the insulitis was less severe at the higher BPA exposure. Serum glucose levels in the 1 mg/ml BPA group tended to be hyperglycaemic, also indicating an accelerated onset of T1DM. The high BPA exposure seemed to counteract the diabetes development in females and also in male NOD mice for both BPA concentrations. Prior to insulitis, both BPA concentrations resulted in increased apoptosis and reduced numbers of tissue resident macrophages in pancreatic islets. In conclusion, long-term BPA exposure at a dose three times higher than the tolerable daily intake of 50 µg/kg, appeared to accelerate spontaneous insulitis and diabetes development in NOD mice.

Early life interventions to prevent allergy in the offspring: the role of maternal immunization and postnatal mucosal allergen exposure.

BACKGROUND: Childhood allergy is influenced by maternal factors and allergen exposure in early life, but the factors that determine the development of allergy and tolerance are unknown. Therefore, we compared the effects of two early life interventions, i.e. maternal allergen immunization and postnatal intranasal allergen exposure, as well as a combination of both treatments on allergic responses in the offspring. METHODS: Female mice were immunized with ovalbumin (OVA) or vehicle during pregnancy. After birth, half the offspring from each group were exposed intranasally to low doses of OVA or vehicle weekly for 5 weeks before intraperitoneal immunization with OVA. RESULTS: Maternal immunization reduced OVA-specific IgE and IgG1, but increased IgG2a and T(H)2 cytokine responses in the offspring after immunization. Postnatal intranasal OVA exposure similarly reduced both IgE and IgG1, but also spleen cell numbers and cytokine secretion. Following airway challenges of the offspring, IgE and airway inflammation were suppressed only by intranasal exposure, but not by maternal immunization, the effect of which also waned with age. Differential gene expression in the spleen of offspring supported that IgE suppression by the two interventions was caused by different mechanisms. Despite this, tolerance development after mucosal exposure was obtained in the offspring of immunized dams. CONCLUSIONS: The study suggests that prevention of allergy is possible if initiated in early life, and early mucosal allergen exposure may play a protective role independent of the maternal immune responses.

Maternal allergen immunisation to prevent sensitisation in offspring: Th2-polarising adjuvants are more efficient than a Th1-polarising adjuvant in mice.

BACKGROUND: Allergy has been an increasing problem in several parts of the world. Prenatal exposure to allergen and microbial components may affect the development of allergies in childhood, as indicated by epidemiological and experimental studies. We investigated the capacity for allergic sensitisation in offspring after induction of a Th1- or a Th2-polarised immune response to the same allergen in mothers during pregnancy. RESULTS: During pregnancy, mice were immunised with ovalbumin (OVA) given with either one of the Th2-adjuvants pertussis toxin (PT) or Al(OH)3 (aluminium hydroxide), or with the Th1 adjuvant CpG. Offspring were immunised with OVA in Al(OH)3 as young adults. Serum and supernatants from ex vivo stimulated or non-stimulated spleen cells from mothers and offspring were analysed for OVA-specific antibodies and cytokines, respectively. Mothers immunised with OVA together with either Al(OH)3 or PT had increased levels of OVA-specific IgE and IgG1 compared to naive mothers, whereas mothers immunised with OVA together with CpG had increased levels of OVA-specific IgG2a compared to naive mothers. In general the highest levels of IL-5, IL-10, and IFNgamma were observed in spleen cells from mothers immunised with PT and OVA. Upon immunisation, offspring from mothers immunised with OVA and either PT or Al(OH)3 showed reduced levels of OVA-specific IgE and IgG1 and increased levels of OVA-specific IgG2a antibodies compared to offspring from naive mothers. Maternal immunisation with CpG and OVA did not affect antibody responses in offspring. CONCLUSION: Allergic sensitisation in the offspring was affected by the type of adjuvant used for immunisation of the mothers with the same allergen. Th2 polarisation of the immune response in the mothers was found to give reduced IgE levels upon sensitisation of the offspring, whereas no reduction was achieved with Th1 polarisation in the mothers.

Fine ambient particles from various sites in europe exerted a greater IgE adjuvant effect than coarse ambient particles in a mouse model.

In the European Union (EU)-funded project Respiratory Allergy and Inflammation due to Ambient Particles (RAIAP), coarse and fine ambient particulate matter (PM) was collected at traffic dominated locations in Oslo, Rome, Lodz, and Amsterdam, in the spring, summer, and winter 2001/2002. PM was also collected in de Zilk, a rural seaside background location in the Netherlands. The aim of this study was to screen the ambient PM fractions for allergy adjuvant activity measured as the production of allergen- (ovalbumin-) specific immunoglobulin (Ig) E following subcutaneous (sc) injection into the footpad of mice. A second aim was to determine whether the 6-d popliteal lymph node (PLN) assay can be used to detect an allergy adjuvant activity. Allergy screening for IgE adjuvant activity showed that in the presence of ovalbumin (Ova) 12 out of 13 of the fine ambient PM fractions exerted a significant IgE adjuvant activity. In contrast, only 3 out of 13 of the coarse PM fractions had significant adjuvant activity. Overall, fine ambient PM exerted significantly greater IgE adjuvant activity per unit mass than coarse PM. No significant differences were observed between locations or seasons. Substantial higher levels of specific components of PM such as vanadium (V), nickel (Ni), zinc (Zn), ammonium (NH(4)), and sulfate (SO(4)) were present in the fine compared to coarse PM fractions. However, differences in the content of these components among fine PM fractions did not reflect the variation in the levels of IgE anti-Ova. Still, when comparing all seasons overall, positive correlations were observed between V, Ni, and SO(4) and the allergen specific IgE levels. The PLN responses (weight and cell number) to Ova and ambient PM in combination were significantly higher than to Ova or PM alone. Still, the PLN assay appears not to be useful as a quantitative assay for screening of allergy adjuvant activity since no correlation was observed between PLN responses and allergen specific IgE levels. In conclusion, fine ambient PM fractions consistently were found to increase the allergen-specific IgE responses more than the coarse ones. Our finding is in agreement with the notion that traffic-related air pollution contributes to the disease burden in asthma and allergy, and points to fine and ultrafine ambient PM as the most important fractions in relation to allergic diseases.

Particles from wood smoke and road traffic differently affect the innate immune system of the lung.

The effect of particles from road traffic and wood smoke on the innate immune response in the lung was studied in a lung challenge model with the intracellular bacterium Listeria monocytogenes. Female Balb/cA mice were instilled intratracheally with wood smoke particles, particles from road traffic collected during winter (studded tires used; St+), and during autumn (no studded tires; St-), or diesel exhaust particles (DEP). Simultaneously with, and 1 or 7 days after particle instillation, 10(5) bacteria were inoculated intratracheally. Bacterial numbers in the lungs and spleen 1 day after Listeria challenge were determined, as an indicator of cellular activation. In separate experiments, bronchoalveolar lavage (BAL) fluid was collected 4 h and 24 h after particle instillation. All particles tested reduced the numbers of bacteria in the lung 24 h after bacterial inoculation. When particles were given simultaneously with Listeria, the reduction was greatest for DEP, followed by St+ and St-, and least for wood smoke particles. Particle effects were no longer apparent after 7 days. Neutrophil numbers in BAL fluid were increased for all particle exposed groups. St+ and St- induced the highest levels of IL-1beta, MIP-2, MCP-1, and TNF-alpha, followed by DEP, which induced no TNF-alpha. In contrast, wood smoke particles only increased lactate dehydrogenase (LDH) activity, indicating a cytotoxic effect of these particles. In conclusion, all particles tested activated the innate immune system as determined with Listeria. However, differences in kinetics of anti-Listeria activity and levels of proinflammatory mediators point to cellular activation by different mechanisms.

Allergy adjuvant effect of particles from wood smoke and road traffic.

There is growing evidence that in addition to augmenting the severity of asthma and allergic diseases, particulate air pollution also increases the incidence of allergy and asthma. We studied the adjuvant effect of particles from wood smoke and road traffic on the immune response to the allergen ovalbumin (OVA). OVA with and without particles was injected into one hind footpad of Balb/cA mice. All particles together with OVA significantly increased the level of OVA-specific immunoglobulin E (IgE) in serum, compared to groups given OVA or particles alone. Reference diesel exhaust particles (DEP) with OVA induced the highest levels of IgE, whereas no clear difference was observed between particles from road traffic and wood smoke. Road traffic particles collected in the autumn induced higher IgE values with OVA than corresponding particles collected during the winter season when studded tires are used, suggesting that studded tire-generated road pavement particles have less allergy adjuvant activity than exhaust particles. Compared to OVA or particles alone, all particles with OVA increased popliteal lymph node cell numbers, cell proliferation, ex vivo secretion of IL-4 and IL-10 after ConA stimulation, and the expression of several cell surface molecules (CD19, MHC class II, CD86 and CD23). Wood smoke particles with OVA induced somewhat higher cellular responses than road traffic particles, but less than DEP with OVA which seemed to be the most potent particle in inducing cellular as well as antibody responses. Thus, wood smoke particles had about the same capacity to enhance allergic sensitization as road traffic particles, but less than diesel exhaust particles.

Guidance on allergenicity assessment of genetically modified plants.

This document provides supplementary guidance on specific topics for the allergenicity risk assessment of genetically modified plants. In particular, it supplements general recommendations outlined in previous EFSA GMO Panel guidelines and Implementing Regulation (EU) No 503/2013. The topics addressed are non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity. New scientific and regulatory developments regarding these three topics are described in this document. Considerations on the practical implementation of those developments in the risk assessment of genetically modified plants are discussed and recommended, where appropriate.

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.

Relation between sources of particulate air pollution and biological effect parameters in samples from four European cities: an exploratory study.

Given that there are widely different prevalence rates of respiratory allergies and asthma between the countries of Europe and that exposure to ambient particulate matter (PM) is substantial in urban environments throughout Europe, an EU project entitled "Respiratory Allergy and Inflammation Due to Ambient Particles" (RAIAP) was set up. The project focused on the role of physical and chemical composition of PM on release of cytokines of cells in vitro, on respiratory inflammation in vivo, and on adjuvant potency in allergy animal models. Coarse (2.5-10 microm) and fine (0.15-2.5 microm) particles were collected during the spring, summer and winter in Rome (I), Oslo (N), Lodz (PL), and Amsterdam (NL). Markers within the same model were often well correlated. Markers of inflammation in the in vitro and in vivo models also showed a high degree of correlation. In contrast, correlation between parameters in the different allergy models and between allergy and inflammation markers was generally poor. This suggests that various bioassays are needed to assess the potential hazard of PM. The present study also showed that by clustering chemical constituents of PM based on the overall response pattern in the bioassays, five distinct groups could be identified. The clusters of traffic, industrial combustion and/or incinerators (TICI), and combustion of black and brown coal/wood smoke (BBCW) were associated primarily with adjuvant activity for respiratory allergy, whereas clusters of crustal of material (CM) and sea spray (SS) are predominantly associated with measures for inflammation and acute toxicity. The cluster of secondary inorganic aerosol and long-range transport aerosol (SIALT) was exclusive associated with systemic allergy. The present study has shown that biological effect of PM can be linked to one or more PM emission sources and that this linkage requires a wide range of bioassays.

The allergy adjuvant effect of particles - genetic factors influence antibody and cytokine responses.

BACKGROUND: There is increasing epidemiological and experimental evidence for an aggravating effect of particulate air pollution on asthma and allergic symptoms and, to a lesser extent, on allergic sensitization. Genetic factors appear to influence not only the magnitude, but also the quality of the adjuvant effect of particles with respect to allergen-specific IgE (Th2-associated) and IgG2a (Th1-associated) responses. In the present study, we aimed to investigate how the genetic background influences the responses to the allergen and particles alone and in combination. We examined how polystyrene particles (PSP) affected the IgE and IgG2a responses against the model allergen ovalbumin (OVA), after subcutaneous injection into the footpad of BALB/cA, BALB/cJ, NIH and C3H/HeN mice, Further, ex vivo IL-4, IFN-gamma and IL-10 cytokine secretion by Con A-stimulated cells from the draining popliteal lymph node (PLN) five days after injection of OVA and PSP separately or in combination was determined. RESULTS: PSP injected with OVA increased the levels of OVA-specific IgE antibodies in all strains examined. In contrast, the IgG2a levels were significantly increased only in NIH and C3H/HeN mice. PSP in the presence of OVA increased cell numbers and IL-4, IL-10 and IFN-gamma levels in BALB/cA, NIH and C3H/HeN mice, with the exception of IFN-gamma in NIH mice. However, each mouse strain had their unique pattern of response to OVA+PSP, OVA and PSP, and also their unique background cytokine response (i.e. the cytokine response in cells from mice injected with buffer only). CONCLUSION: Genetic factors (i.e. the strain of mice) influenced the susceptibility to the adjuvant effect of PSP on both secondary antibody responses and primary cellular responses in the lymph node, as well as the cellular responses to both OVA and PSP given separately. Interestingly, PSP alone induced cytokine responses in the lymph node in some of the mouse strains. Furthermore, we found that the ex vivo cytokine patterns did not predict the in vivo Th2- and Th1-associated antibody response patterns in the different mouse strains. The results indicate that insoluble particles act by increasing the inherent response to the allergen, and that the genetic background may determine whether an additional Th1-associated component is added to the response.

The IgE adjuvant effect of particles: characterisation of the primary cellular response in the draining lymph node.

Diesel exhaust particles, and polystyrene particles (PSP) as a model for the insoluble particle core, have an adjuvant effect on allergen-specific IgE production in mice. We therefore examined the primary immune response in the draining popliteal lymph node (PLN) to the allergen ovalbumin (OVA) injected together with polystyrene particles into the footpad of BALB/cA mice. Similar numbers of particle-containing cells were observed in the draining lymph node on day 1 after injection of PSP alone or OVA + PSP, the numbers increasing continuously until day 21. The total lymph node cell numbers increased three to four times in the OVA + PSP group compared to both OVA and PSP groups, peaking on day 5. The increase in B cell numbers was twice the increase in T cell numbers. On day 5, OVA + PSP increased the expression of most surface markers measured (MHC class II, CD86, CD23, CD69) compared to OVA and PSP. Further, the ex vivo production of IL-4 and IL-10 by PLN cells from OVA + PSP-injected animals was increased. In conclusion, whereas PSP alone did not influence any of the immunologic markers studied, the adjuvant effect of PSP on the IgE antibody response to OVA was associated with an early increased primary cellular response in the draining lymph node.

Ambient air particles from four European cities increase the primary cellular response to allergen in the draining lymph node.

In the RAIAP (respiratory allergy and inflammation due to ambient particles) project, qualitative properties of ambient air particles from Amsterdam, Oslo, Lodz and Rome were investigated in relation to inflammation and allergy. Most collected particle fractions were found to increase the allergen-specific IgE and IgG2a responses after subcutaneous injection of particles with allergen in mice. However, some fractions appeared to skew the antibody response towards more Th1- or Th2-associated antibody isotypes, and the fine fractions were found to be more potent than the coarse fractions with regard to IgE adjuvant activity. In the present study we investigated the cellular response in the draining lymph node 5 days after a subcutaneous injection of selected RAIAP particle fractions. The particles (100 microg) were injected into both hind footpads of BALB/cA mice, in the presence or absence of the allergen ovalbumin (OVA, 50 microg). We also studied if the coarse and fine RAIAP particle fractions affected the cellular responses to OVA differently. The number of lymph node cells, as well as the relative number of B and T lymphocytes and T helper cells were determined. Expression of cell surface molecules (MHC class II, CD86 and CD23) and ex vivo cytokine production (IL-4, IL-10 and IFN-gamma) by the lymph node cells were measured. Overall, particles in the presence of allergen enhanced the levels of the various cellular parameters compared to allergen alone or particles alone. In the absence of allergen, ambient air particles, in contrast to diesel exhaust particles, marginally affected some cellular parameters. By histological examination of the lymph node, the particles appeared to be scattered between the lymphocytes, often localised within macrophage-like (acid phosphatase positive) cells. The cell parameters measured could, for the individual sample, neither predict the degree of a Th2- or Th1-skewed antibody response, nor the stronger antibody adjuvant capacity of the fine than the coarse particle fractions. In conclusion, we have shown that coarse and fine ambient air particles from different European cities enhance the cellular response in the draining lymph node after injection with an allergen. In the absence of allergen, ambient particles only marginally affected the cellular parameters.

IgE-mediated sensitisation, rhinitis and asthma from occupational exposures. Smoking as a model for airborne adjuvants?

OBJECTIVE: Airborne pollutants with adjuvant effect, called airborne adjuvants, may promote IgE-sensitisation and development of allergic airway diseases. Smoking and occupational allergen exposures were reviewed to establish a general and verified framework for hazard identification and risk assessment of adjuvant effects of airborne pollutions. METHODS: The relative risks and the attributable risks of adjuvant effect of smoking were determined for co-exposures with green coffee and castor beans, ispaghula, senna, psyllium, flour and grain dust, latex, laboratory animals, seafood, enzymes, platinum salts, organic anhydrides, or reactive dyes. RESULTS: Adjuvant effects of smoking depended on the types of allergen, but not on whether sensitisation or allergy was promoted by atopy-the hereditarily increased ability to increase IgE formation. CONCLUSION: Promotion of IgE sensitisation in humans and in animals may serve for hazard identification of adjuvant effects. Risk assessment has been based mainly on epidemiological studies, which are sensitive to confounding factors. This highlights the need to develop appropriate animal models for risk assessment.

Detection of immunotoxicity using T-cell based cytokine reporter cell lines ("Cell Chip").

Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype "Cell Chip" to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included.

Development of the "Cell Chip": a new in vitro alternative technique for immunotoxicity testing.

Predictive testing of immunotoxicity associated with chemical compounds is complicated and cannot be accomplished with a single test. As most of the existing tests for immunotoxicity employ experimental animals, there is an increasing need for alternative tests in vitro. We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. This system named "fluorescent cell chip" (FCC) is based on a number of genetically modified cell lines that regulate the expression of a transgene coding for fluorescent protein enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-1beta, IL-2, IL-4, IFN-gamma, IL-10, TNF-alpha, and beta-actin. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promotors were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells. Two chemicals with known immunotoxic activities, cyclosporine A and potassium tetrachloro-platinate(II), mediated compound-specific pattern of inhibition and activation of reporter gene expression. Thus, the "fluorescent cell chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well defined biological activity.

[Mustard--an important food allergen?]. / Sennep--eit viktig matallergen?

BACKGROUND: The European Parliament and the Council of the European Union have issued a new directive on information on the label about the ingredients present in foodstuffs. Twelve foods causing hypersensitivity are now subject to particularly strict labelling rules. Among these foods is mustard, which until now has been little recognised as a food allergen in Norway. MATERIALS AND METHODS: Literature searches in Medline were performed and supplemented by manual searches of reference lists. RESULTS: Since the first published case of mustard allergy in 1980, a number of case reports and patient series have been published. Most reports come from certain regions of France, and a majority of the studies have been performed by a limited number of investigators. Symptoms triggered by mustard cover the whole range from full-blown systemic anaphylactic reactions to pollen-food allergy syndrome-like local reactions. Frequently, reactions appear to be serious. Mustard allergy has on many instances been found in small children. Cross-reactivity apart from that among different types of mustard does not appear to be a clinical problem. Mustard allergens are resistant to heat and food processing. INTERPRETATION: With more attention being given to mustard as a food allergen because of the new labelling rules, it will most likely soon become apparent whether mustard is a food allergen of importance in Norway. The Norwegian national reporting system and register of severe allergic reactions to food will be an important tool in this context.