The environmental pollutant BDE-47 modulates immune responses in invitro and in vivo murine models.

2,2',4,4'-tetra-bromodiphenyl ether (BDE-47) is widespread in the environment and biological samples. Its association with health risks is an increasing concern, yet information on BDE-47 immunotoxicity remains limited. This study investigated the impact of BDE-47 on innate and adaptive immune responses through in vitro and in vivo approaches. BDE-47's capacity to directly induce cell responses and modulate responses induced by known stimuli was studied in vitro using the RAW 264.7 murine macrophage cell line and spleen-derived lymphocytes, and in vivo using keyhole limpet hemocyanin (KLH)-immunized BALB/c mice orally administered (28 d) at dose levels (7.5, 15.0 and 30 mg/kg/bw/d) derived from relevant toxicokinetic data from rodent models. RAW 264.7 cells stimulated with lipopolysaccharide (LPS) and exposed to BDE-47 exhibited unchanged cell viability but decreased release of interleukin (IL)-6. Primary splenocytes from naïve mice stimulated with anti-CD3/anti-CD28 antibodies and exposed to BDE-47 showed a significant decrease of IL-17 A and IFNγ production. In vivo data showed that BDE-47 significantly reduced the KLH-specific antibody response. A generally decreasing trend of IFNγ, IL-10 and IL-5 production was observed after in vitro antigen-specific restimulation of spleen cells. Histopathological effects on liver, spleen, small intestine and thyroid were detected at the highest dose in the absence of general toxicity. In addition, the expression of Mm_mir155 and Mm_let7a was induced in livers of exposed mice. The data obtained in this study suggest that exposure to BDE-47 may perturb innate and adaptive immune responses, thus possibly decreasing resistance to bacterial and viral infections.

Effects of sub-chronic oral exposure to pyrogenic synthetic amorphous silica (NM-203) in male and female Sprague-Dawley rats: focus on reproductive systems.

Synthetic amorphous silica (SAS) consists of agglomerates and aggregates of primary particles in the nanorange (<100 nm) and it is the E551 authorized food additive. The potential risks for human health associated to dietary exposure to SAS are not completely assessed; in particular, data on male and female reproductive systems are lacking. A 90-day oral toxicity study with pyrogenic SAS nanomaterial NM-203 was carried out on the basis of the OECD test guideline 408 in the frame of the NANoREG project. Adult Sprague-Dawley rats of both sexes were orally treated for 90 days with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day. Dose levels were selected to be as close as possible to the expected human exposure to food additive E551. The present paper provides specific information on potential effects on male and female reproductive systems, through the evaluation of serum biomarkers, sperm count, histopathological analysis of testis, epididymis, ovary and uterus and real-time PCR on uterus; potential genotoxic alterations were evaluated by comet assay on testis, sperm and ovary. NM-203 did not induce histophatological and genotoxic effects in male reproductive system. In female rats, ovary is not target of NM-203 and only tissue-specific effects on uterus were recorded up to 10 mg/kg bw per day. To our best knowledge, this is the first study providing data on male and female reproductive systems after long-term, repeated oral exposure at dose levels close to dietary human exposure, which identifies a limited concern only for female reproductive health.

A harmonized and standardized in vitro approach produces reliable results on silver nanoparticles toxicity in different cell lines.

Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results.

Use of a common European approach for nanomaterials' testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

Hazard identification of pyrogenic synthetic amorphous silica (NM-203) after sub-chronic oral exposure in rat: A multitarget approach.

Food additive E551 consists of synthetic amorphous silica (SAS), comprising agglomerates and aggregates of primary particles in the nanorange (<100 nm), which potential nanospecific risks for humans associated to dietary exposure are not yet completely assessed. In NANoREG project, aim of the study was to identify potential hazards of pyrogenic SAS nanomaterial NM-203 by a 90-day oral toxicity study (OECD test guideline 408). Adult Sprague-Dawley rats of both sexes were orally treated with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day; dose levels were selected to be as close as possible to E551 dietary exposure. Several endpoints were investigated, the whole integrative study is presented here along with the results of dispersion characterization, tissue distribution, general toxicity, blood/serum biomarkers, histopathological and immunotoxicity endpoints. No mortality, general toxicity and limited deposition in target tissues were observed. NM-203 affected liver and spleen in both sexes. Proposed NOAEL 5 mg/kg bw per day in male rats for enlarged sinusoids in liver. In female rats, TSH and creatinine levels were affected, proposed LOAEL 2 mg/kg bw per day. Overall, these data provide new insight for a comprehensive risk assessment of SAS exposure by the oral route.

Allergenic activity of Pseudoterranova decipiens (Nematoda: Anisakidae) in BALB/c mice.

BACKGROUND: Anisakis simplex is the only fishery-product associated parasite causing clinical allergic responses in humans so far. However, other anisakids, due to the presence of shared or own allergens, could also lead to allergic reactions after sensitization. The aim of this study was to determine if Pseudoterranova decipiens belonging to the family Anisakidae has allergenic activity and is able to induce sensitization after oral administration in a murine (BALB/c mice) model. RESULTS: The ingestion of A. pegreffii proteins by BALB/c mice, which had been previously sensitized by intraperitoneal inoculation with the corresponding live L3 larvae, triggers signs of allergy within 60 min, whereas P. decipiens did to a lesser extent. Beside symptoms, allergic reactions were furtherly supported by the presence of histamine in sera of sensitized mice. Specific IgG1 and IgE responses were detected in sera of all sensitized mice from week four. Specific IgG2a response was detected in sera from mice sensitized to P. decipiens. After polyclonal or specific activation with anti-CD3/anti-CD28 or antigens, respectively, splenocytes from mice infected i.p. with A. pegreffii or P. decipiens larvae showed significantly higher production of IL-10 than naïve mice. After stimulation with specific antigens, significantly higher IL-5 and IL-13 amounts were produced by specific antigen stimulated splenocytes than by the naïve cells; only P. decipiens proteins induced IFN-É£. CONCLUSIONS: The overall results suggest that infection with P. decipiens can sensitize mice to react to subsequent oral challenge with anisakid proteins, as described for A. simplex (sensu stricto) and A. pegreffii infections. The results show that anisakid proteins induce a dominant Th2 response, although P. decipiens could also induce a mixed type 1/type 2 pattern.

Nanoparticles Adjuvants in Allergology: New Challenges and Pitfalls.

Allergen specific immunotherapy has been introduced in the clinic more than 100 years ago showing effectiveness and, so far, it represents the only curative approach to treat allergic disorders ameliorating the symptoms, reducing the medication costs and blocking the onset of new sensitizations. However, some questions are still open regarding to the safety of the treatment and the need to reduce the dose and time of administration to improve the compliance of the patients. All preparations that are currently available may trigger side effects. For these reasons, new formulations and route of administration have been exploited demonstrating that such products presented improved efficacy and safety. Nanotechnology for biomedical applications offers many advantages, such as improved stability and bioavailability, favourable biodistribution profiles and targeting to specific cell populations whose impact on the immune system has been evaluated in animal systems. Nanoparticles interact with the immune system, and the final outcome of this interaction depends on their physico-chemical characteristics. Concerns can be raised when immunotoxic effect are induced, resulting in inflammatory dangerous responses or in the reduction of the normal defensive activity of the immune system. In this paper, we will review the most relevant data about the synthesis of allergen/nanoparticles systems and will discuss their impact on the immune system in terms of immunomodulatory activity and immunotoxicity risk assessment.

Probiotic VSL#3-induced TGF-ß ameliorates food allergy inflammation in a mouse model of peanut sensitization through the induction of regulatory T cells in the gut mucosa.

SCOPE: Among food allergies, peanut allergy is frequently associated with severe anaphylactic reactions. In the need for safe and effective therapeutic strategies, probiotics may be considered on the basis of their immunomodulatory properties. The aim of the present study was to investigate the immunological mediators involved in the effects of probiotic VSL#3 oral supplementation on Th2 inflammation and anaphylaxis in a mouse model of peanut allergy. METHODS AND RESULTS: VSL#3 supplementation to peanut-sensitized mice was effective in ameliorating anaphylaxis and Th2-mediated inflammation, by promoting regulatory responses in the jejunum mucosa and in the mesenteric lymph node, as evaluated by ELISA, real-time PCR, histologic, and immunohistochemical analysis. Probiotic-induced TGF-ß mediates its protective effects through the induction of regulatory T cells expressing FOXP3 and/or latency-associated peptide, as proven by in vivo blockade of TGF-ß in VSL#3-treated mice with a neutralizing monoclonal antibody one day before challenge. CONCLUSION: TGF-ß, induced in the gut by VSL#3 supplementation, is capable of reducing the Th2 inflammation associated with food anaphylaxis in a mouse model of peanut sensitization. TGF-ß acts through the induction/maintenance of regulatory T cells expressing FOXP3 and/or latency-associated peptide. Probiotics supplementation may represent an effective and safe strategy for treating food allergies in adult population.

Structural and immunological characterization of recombinant Pan b 1, a major allergen of northern shrimp, Pandalus borealis.

BACKGROUND: Shellfish allergy is one of the major causes of life-threatening allergic reactions to food. The shrimp species Pandalus borealis is the commercially most important coldwater shrimp species, and its protein extract is commonly used in shrimp allergy diagnostics. However, the DNA sequence of its major allergen, tropomyosin, designated Pan b 1, was not previously described. Our aim was to identify the cDNA sequence of Pan b 1 and to generate a recombinant protein with similar structure and allergenicity as the natural protein. METHODS: P. borealis shrimps were caught in the Oslofjord (Norway). cDNA from Pan b 1 was generated, an N-terminal histidine tag was added, and the protein was expressed in Escherichia coli. The recombinant Pan b 1 was characterized by structural and IgE-binding studies and investigated further with basophil activation tests (BATs) and skin prick tests (SPTs) on Norwegian shrimp-allergic individuals. RESULTS: The open reading frame encoded 284 amino acids that shared 97-100% identity with other shrimp tropomyosins. Mass spectroscopy of natural Pan b 1 confirmed the protein's molecular mass and indicated the absence of posttranslational modifications. Circular dichroism spectroscopy revealed virtually identical spectra between recombinant and natural Pan b 1, which together with native PAGE and size exclusion chromatography results indicated a similar structure. Furthermore, immunoblot and ELISA studies as well as BATs and SPTs showed equivalent results of recombinant and natural Pan b 1. CONCLUSION: A recombinant tropomyosin from P. borealis was generated that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy.

Prevalence of self-perceived allergic diseases and risk factors in Italian adolescents.

The aim of the study was to assess the symptoms prevalence of allergic diseases in a population of 11-15 yr old schoolchildren, to evaluate the associations between asthma and other symptoms and identify risk factors for asthma, rhinitis and eczema syndromes. A sample of 481 students was studied using an International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. Prevalence of different kind of self-reported symptoms was calculated. Using a logistic regression approach, we tried to identify risk factors for three syndromes - rhinitis, eczema and asthma. The highest and the lowest prevalence rates of self-reported symptoms were recorded for rhinitis (43.6%) and for eczema (8.1%), respectively. The prevalence of asthma was 15.7%. Univariate analysis showed a mutual association between wheeze and rhinitis symptoms. Multivariate logistic regression model for eczema syndrome revealed female gender as a significant risk factor. The polytomic logistic multivariate regression revealed female gender and family history of allergy as significant risk factors for rhinitis syndrome only, and maternal smoking and familial allergy for rhinitis and asthma together. In particular, familial allergy yields a 400% higher chance of developing asthma and rhinitis together. The synergistic effect of familial allergy on rhinitis and asthma syndromes suggests the implementation of preventive measures in children with family history of these diseases.

Differences in the presence of allergens among several types of indoor environments.

Exposure to indoor allergens can occur both at home and in public places such as schools and workplaces. To investigate and compare the presence of indoor allergens in different kind of environments (schools, offices and homes), dust samples were collected from furniture, desks, mattresses and floors with a standardized procedure. Samples were analyzed for Der p 1, Der f 1, Mite group 2 (mites) and Fel d 1(cat) by monoclonal antibody ELISA assay. Mite allergens were detected with low frequencies in schools and workplaces and with high frequency in homes. Fel d 1 was found with high frequency in every examined environment. Homes rather than public places can represent the environment where people can easier incur in mite allergy. All environments could be at risk for cat allergen exposure.

Exposure to indoor allergens and association with allergy symptoms of employees in a work environment.

Exposure to indoor allergens is an important risk factor for sensitisation and respiratory allergy. The aim of this paper was to evaluate the levels of mite, cat and latex allergens in dust collected from an indoor workplace and to assess whether the exposure to these allergens was associated with the allergy symptoms reported by employees. Sixty dust samples were collected. Allergen concentrations were measured with antibody based ELISAs. All 144 participants compiled a questionnaire exploring possible symptoms of allergy. No association between latex allergen exposure and symptoms was found in spite of the high frequency of latex allergens. Mite allergens were detected in a minority of rooms. Cat allergen was the most important indoor allergen in the sampled workplace and exposure to this allergen could represent a risk for employees.

Use of probiotic bacteria for prevention and therapy of allergic diseases: studies in mouse model of allergic sensitization.

Probiotic bacteria as modulators of the immune response have been intensively studied in reducing the risk of immune-mediated diseases, including atopic diseases. Results from in vitro studies demonstrated that probiotics may modify the polarization of immune cells, supporting potential therapeutic effects in atopic diseases. Several clinical studies have been designed to explore the effective role of probiotics in the modulation of allergic diseases. The results of these studies, although promising, are not conclusive yet and are considered insufficient to recommend probiotics as a part of standard therapy in any allergic conditions. In vivo studies on animal models can provide useful information on the immunologic mechanisms responsible for the potential antiallergic effects of probiotic bacteria. The immunomodulatory activity of the probiotic mixture VSL#3 has been studied in the mouse models of allergic sensitization and anaphylaxis developed in our laboratory with inhalant and food allergens, according to a prophylactic setting by the intranasal route (inhalant allergy model) or a therapeutic setting by the oral route (food allergy model). Intranasally delivered probiotic bacteria prevented the development of Parietaria major allergen-specific response, by down-regulating T helper cell 2 responses at the local and systemic level. Oral therapeutic treatment was able to reduce both systemic and local anaphylactic symptoms induced by oral challenge with the sensitizing allergen Shrimp Tropomyosin. The induction of protective immune responses at the sites of allergen exposure linked to counterregulatory local and systemic immune responses by mucosal delivery of probiotic bacteria mixtures might become an effective strategy in the prevention and therapy of allergic diseases.

Oral sensitization with shrimp tropomyosin induces in mice allergen-specific IgE, T cell response and systemic anaphylactic reactions.

Appropriate murine models of shrimp tropomyosin (ST) allergy would be useful in investigating the mechanisms underlying food allergy in human subjects, as well as for the pre-clinical evaluation of efficacy and safety of novel therapeutic approaches. These models should mimic immune and clinical features of human disease, including anaphylactic response. We sensitized C3H/HeJ mice by the oral route with purified ST using cholera toxin (CT) as adjuvant. ST-specific IgE, IgG1, IgG2a and IgA responses were evaluated by ELISA. Spleen cell proliferation and cytokine production by allergen-specific activation were assessed. Jejunum and colon fragments were collected to evaluate the local expression of cytokine genes by PCR. Local and systemic anaphylactic reactions induced by oral ST challenge were scored according to symptoms observed. Faecal samples were collected to assess local IgA production and histamine levels. Oral sensitization with ST plus CT induced in mice significant levels of serum IgE and IgG1 and faecal IgA. ST-specific cell proliferation and IL-4, IL-13 and IFN-gamma cytokine production were induced in the spleen. After oral challenge, 100% of mice had anaphylactic symptoms while no symptoms were observed in challenged naive mice. Faecal histamine content after ST challenge appeared significantly increased in sensitized mice when compared with that observed in pre-immune mice. Jejunum mRNA expression of T(h)2 cytokines was up-regulated by ST sensitization. These results support the importance of the oral way of sensitization and of the in-depth characterization of the anaphylactic response for the development of a suitable in vivo model of food allergy.

Biochemical and molecular biological aspects of silverfish allergens.

Insects and insect-derived materials have been implicated as a risk factor for sensitization and subsequent elicitation of allergic rhinitis and allergic bronchial asthma. During the last decades, insects other than those known as allergenic, were investigated for their potential role in inducing and triggering an IgE immune response. Among these, the silverfish, an insect belonging to the Thysanura order, appeared to be of particular interest. Silverfish (Lepisma saccharina) is the most primitive living insect, and represents a descendent of the ancestral wingless insects. They are 3-12 mm long, have three tail feelers and are covered with shiny scales. They shun light and need a humid environment and their diet consists of carbohydrate materials such as paper and book-binding glue, crumbs of bread and flour. Because of these features, silverfish finds an optimal habitat both in dwellings and workplaces and in spite of its antiquity, silverfish has succeeded in exploiting the new opportunity created by man. Although its importance significantly increased when it has been demonstrated that house dust contains significant silverfish levels even in houses where the inhabitants were unaware of its presence, no silverfish extract for diagnosis of allergic diseases is commercially available yet. Identification of optimal extraction conditions and characterization of allergenic extracts are the first steps to obtain an effective allergen preparation suitable for diagnosis and therapy, and will be useful as a reference preparation for assessing silverfish exposure in different indoor environments. It has been cloned and characterized a silverfish tropomyosin, named Lep s 1, which represents the first allergen identified in silverfish extract and can be regarded as a molecule cross-reactive among inhalant and edible invertebrates allergenic sources. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.

Cloning and Expression of the Olea europaea allergen Ole e 5, the pollen Cu/Zn superoxide dismutase.

BACKGROUND: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. METHODS: cDNA of Ole e 5 was amplified by nested 3'-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients' IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). RESULTS: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. CONCLUSIONS: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts.

To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12-O-tetradecanoylphorbol-13-acetate induced, in 10-60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2-3x, while the enzyme activity increased 5x in total cell lysates, 2x in the plasma membrane, and 4x in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix. Pre-incubation of cells with the PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609), before PDGF-stimulation, not only reduced the enzyme activity in total cell lysates as well as in plasma membrane and nuclear fractions, but also blocked the mechanisms of PC-PLC subcellular redistribution. These effects were associated with a D609-induced long-lasting cell cycle block in Go.

Molecular, structural, and immunologic relationships between different families of recombinant calcium-binding pollen allergens.

BACKGROUND: Calcium-binding plant allergens can be grouped in different families according to the number of calcium-binding domains (EF hands). OBJECTIVE: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens. METHODS: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper). Purified recombinant Aln g 4 and Jun o 4 were used to determine the prevalence of IgE recognition in 210 patients sensitized to different pollens and to search, by means of ELISA competition, for the presence of cross-reactive epitopes in pollens from 16 unrelated plant species. IgE cross-reactivity among the allergen families was studied with purified rPhl p 7, rAln g 4, rBet v 3, and rJun o 4 and 2 synthetic peptides comprising the N-terminal and C-terminal EF hands of Phl p 7 by means of ELISA competition. RESULTS: Structural similarities were found by using molecular modeling among the allergens with 2, 3, and 4 EF hands. Pollens from 16 unrelated plants contained Aln g 4- and Jun o 4-related epitopes. Twenty-two percent of the patients with multiple pollen sensitization reacted to at least one of the calcium-binding allergens. A hierarchy of IgE cross-reactivity (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3) could be established that identified rPhl p 7 as the EF-hand allergen containing most IgE epitopes in the population studied. CONCLUSION: The demonstration that members of different families of calcium-binding plant allergens share similarities suggests that it may be possible to use representative molecules for the diagnosis and therapy of allergies to EF-hand allergens.

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.