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OBJECTIVE: Subjects exposed to laboratory animals are at a heightened risk of developing respiratory and allergic diseases. These diseases can be prevented by simple measures such as the use of personal protective equipment. We report here the primary findings of the Laboratory Animals and Respiratory Allergies Study regarding the prevalence of allergic diseases among laboratory animal workers, the routine use of preventive measures in laboratories and animal facilities, and the need for prevention programs. METHODS: Animal handlers and non-animal handlers from 2 Brazilian universities (University of São Paulo and State University of Campinas) answered specific questionnaires to assess work conditions and symptoms. These subjects also underwent spirometry, a bronchial challenge test with mannitol, and skin prick tests for 11 common allergens and 5 occupational allergens (rat, mouse, guinea pig, hamster, and rabbit). RESULTS: Four hundred fifty-five animal handlers (32±10 years old [mean±SD], 209 men) and 387 non-animal handlers (33±11 years old, 121 men) were evaluated. Sensitization to occupational allergens was higher among animal handlers (16%) than non-animal handlers (3%, p

Cockroaches are considered to be among the oldest insects on our planet, predating dinosaurs by more than 150 million years, and humans by more than 300 million (1). Some fossils attributed to cockroaches date back to the Carboniferous period, around 325 million B.C. However, an uncertain evolutionary link between cockroach-like fossils from the Carboniferous period (Paleozoic era), and “modern” cockroaches, originated in the Cretaceous period (Mesozoic era) as did most other insects, has also been suggested, due to morphological differences (2). Regardless of their origin, cockroaches have undergone very little changes over time, and the diversification of species and adaptation to multiple habitats are remarkable, reflecting their tremendous success at surviving throughout evolution (1). Over 4,000 cockroach species have been identified, with tropical forests as their preferred habitat (3). However, a few species, including the German cockroach (Blattella germanica) and the American cockroach (Periplaneta americana), brought from Africa to the Americas on slave ships, have adapted to live near people, turning into household pests.

O objetivo deste estudo foi analisar a expressão de alergias (dermatite, rinite e ou asma) e relacionar com a incidência de parasitoses. Foi feito um estudo clínico em crianças de 2 a 15 anos residentes na zona urbana ou rural. A avaliação se fez pelo questionário ISAAC adaptado e exames laboratoriais - dosagem de IgG4, IgE total e específico para Derp 1 2, Per a7, tropomiosina de ácaro e de Ascaris, contagem de eosinófilos e parasitológico de fezes. A prevalência de parasitoses foi reduzida na população estudada, mas elevada na população proveniente de zona rural. Observou-se correlação entre a concentração de IgE total e contagem relativa de eosinófilos. A ocorrência de alergias ou a procedência dos indivíduos não esteve relacionada com alterações laboratoriais, e nem com reatividade a antígenos parasitários e ambientais, por se tratar de uma interiorana, com poucas discrepâncias sociais, ambientais e climáticas entre zona rural e urbana.Descritores: Hipersensibilidade; Imunoglobulina E; Helmintos.

Methods Prospective study including 116 patients with positive history of hypersensitivity to NSAIDs, evaluated at the Allergy Clinic of the Clinical Hospital of Ribeirao Preto Medical School, between October 2010 and July 2014. Patients were evaluated using the European Network for Drug Allergy questionnaire, and underwent single blind, placebo controlled OCT, in a hospital-controlled environment. Patients were required to be clinically stable, and to withdraw antihistamines or corticosteroids seven days prior to the OCT. Patients were given 10%, 20%, 30% and 40% of the therapeutic NSAID dosage, selecting an NSAID different from the one implicated in the allergic reaction. Fifteen minutes after each dosage, patients were evaluated with Peak Flow and blood pressure measurements, heart and respiratory rates and general examination.

M O N D A Y 603 The Major Allergens of Birch Pollen and Cow Milk, Bet v 1 and Bos d 5, Are Structurally Related to Human Lipocalin 2, Enabling Them to Manipulate T-Helper Cells Depending on Their Load with Siderophore-Bound Iron Erika Jensen-Jarolim, MD, Cristina Gomez-Casado, PhD, Ass.Prof., Luis F. F. Pacios, PhD, Prof., Gerlinde Hofstetter, MSc, BSc, Nadine Mothes-Luksch, MD, Georg A. Roth, MD, Josef Singer, MD, PhD, Araceli Diaz-Perales, PhD, Prof, Franziska Roth-Walter, PhD, Ass.Prof.; Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Austria, Institute for Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria, Vienna, Austria, Biotechnology Department, Center for Plant Biotechnology and Genomics, Madrid, Spain, Biotechnology Department, Center for Plant Biotechnology and Genomics, Technical University of Madrid, Madrid, Spain, Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria, AllergyCare, Molecular Diagnostics and Study Center, Vienna, Austria, Department of Anesthesiology, General Intensive Care and Pain Medicine, Medical University of Vienna, Vienna, Austria. RATIONALE: Human lipocalin 2, LCN2, is highly expressed in encounter sites of allergens, as lung and gut. It has immuno-regulatory properties when carrying iron via siderophores (holo) or not (apo). We investigated whether major allergens of birch pollen and milk, Bet v 1 and Bos d 5, might structurally and biologically interfere with LCN2. METHODS: Structural comparison to LCN2 was performed using FATCATflex, CE algorithm and TM-Align methods. Ligand binding was analyzed with AutoDock Vina. Iron-binding was determined by Prussian blue staining. Activated human PBMCs were stimulated for 18h with apoor holo-allergens, or controls. Subsequently, cells and supernatants were analysed by flow cytometry and for their cytokine-content. RESULTS: Both tested allergens shared great structural homology to LCN2. Thus, besides Bos d 5, we could also classify Bet v 1 as a lipocalinlike protein. Both allergens were capable of binding iron via siderophores. When incubated with PBMCs, only the apo-forms of the allergens, but not the holo-forms, were able to promote CD4+ cells and the secretion of IL13. CONCLUSIONS: We conclude that Bet v 1 and Bos d 5 not only structurally mimic human LCN2, but also functionally by their ability to bind iron via siderophores. The apo-forms promote Th2 cells, whereas the holo-forms appear to be immunosuppressive. These results provide for the first time a functional understanding on the principle of allergenicity of major allergens from entirely independent sources, like birch and milk. Supported by Austrian Science Fund FWF F4606-B19,W1205-B09, BES2010-03462 (FPI-Programm, Spanish Government (MICINN/MINECO).

The aim of this article is to provide information on risk factors associated with the development of atopy and asthma in childhood.Several gene polymorphisms have been associated with susceptibility to asthma and allergy; complex gene-environmental interactions, however, appear to play a key role in the development of the disease. Early life sensitization to aeroallergens, presence of atopic dermatitis or allergic rhinitis, maternal smoking during pregnancy and children's environmental exposure to tobacco smoke, lower respiratory tract infections with respiratory syncytial virus and potentially with other viruses including rhinovirus and metapneumovirus, exposure to air pollutants, several perinatal factors other than maternal smoking, are among factors associated with an increased risk for development of chronic asthma.The prevalence of asthma and allergic diseases is increasing progressively. Those who are involved in the care of young children should be prepared to recognize risk factors for development of these diseases and to appreciate the role of gene-environment interactions. Preventive measures established at an early age may modify the natural history of asthma and other allergic diseases.

Epidemiologic studies have shown that the presence of IgE antibodies to house dust mite and other indoor allergens is an important risk factor for asthma.The aim of this study was to develop a reverse ELISA (rELISA) for measuring specific IgE to Der p 2, a major Dermatophagoides pteronyssinus (Dpt) allergen, as a potential tool for followup of allergen immunotherapy.Recombinant Der p 2 allergen or a monoclonal antibody to Der p 2 was used to coat plates in conventional ELISA (cELISA) and rELISA, respectively. Sera from 48 asthmatic patients with positive skin prick test (SPT+) to D. pteronyssinus extract were analyzed for total IgE and specific IgE to Der p 2, and the results were compared with a group of 41 SPT asthmatic and 30 SPT- control subjects.The sensitivity of the two assays for Der p 2-specific IgE was 3.9 EU/mL and their specificities were confirmed by inhibition tests, in a dose-dependent manner. There was a significant positive correlation between cELISA and rELISA (r = 0.74; P0.0001). However, rELISA was more sensitive than was cELISA, regarding both the positive sera percentage (70.8% vs 52.1%) and the Der p 2-specific IgE levels (28.4 vs 4.5 EU/mL) in SPT+ asthmatic patients.rELISA has shown to be a sensitive and alternative method for measuring Der p 2-specific IgE without using radioactive techniques. Detection of specific IgE to major allergens and relevant peptides, and identification of B cell epitopes in allergens will provide valuable information for the design of allergen analogs and peptides for immunotherapy.

Keywords: allergy; anaphylaxis; corn flour; dust mites; food allergy; storage mites

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (>108 copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections.

Invertebrates: Use of Recombinant Tropomyosin From Cockroach and Ascaris lumbricoides A. B. R. Santos1, V. S. F. Sales2, V. P. L. Ferriani3, M. D. Chapman4, L. K. Arruda1; 1Internal Medicine, School of Medicine of Ribeirao Preto University of Sao Paulo, Ribeirao Preto, BRAZIL, 2Pharmacology, Federal University of Rio Grande do Norte, Natal, BRAZIL, 3Pediatrics, School of Medicine of Ribeirao Preto University of Sao Paulo, Ribeirao Preto, BRAZIL, 4Indoor Biotechnologies Inc, Charlottesville, VA. RATIONALE: We have previously shown that infection with A. lumbricoides is an important risk factor for wheezing in children living in Brazil. We aimed to use recombinant tropomyosins to investigate IgE antibody responses to Ascaris and cockroach. METHODS: cDNA coding for A. lumbricoides tropomyosin was subcloned into pPIC-9 vector and expression of the recombinant protein was carried out in the Pichia pastoris system. Recombinant tropomyosins from A. lumbricoides and cockroach were used in chimeric ELISA to quantitate specific IgE antibodies in panels of sera from 137 children living in a parasite-endemic area, and 188 patients with asthma and/or rhinitis allergic to cockroach from our clinic. RESULTS: In children from a parasite-endemic area, IgE to tropomyosin was found in 99/137 (72.2 %) of sera; of those, 57 (41.6%) had IgE to both Ascaris and cockroach tropomyosin. Geometric mean (GM) levels of IgE to Ascaris and cockroach tropomyosin were 7.5 IU/mL and 8.1 IU/mL, respectively. There was a significant correlation of levels of IgE to A. lumbricoides and cockroach tropomyosin (r = 0.7, p < 0.0001). Among cockroach allergic patients, presence of IgE to tropomyosin was found in 79/188 (42%), and of those, 48/188 (25.5%) presented IgE to both Ascaris and cockroach tropomyosin, with GM levels of 2.56 UI/mL and 5.4 UI/mL , respectively. There was also a significant correlation of levels of IgE antibodies to both tropomyosins (r = 0.58, p < 0.0001). CONCLUSION: Tropomyosins from invertebrates are important IgEbinding proteins. Recombinant tropomyosins could be used to investigate IgE antibody responses to Ascaris and cockroach. Funding: FAPESP, CNPq

Rationale Phadiatop® is a respiratory allergy screening test for evaluation of serum IgE antibodies to inhalant allergens. Objectives To evaluate the positivity of Phadiatop® in children from several Brazilian pediatric allergology centers and to compare its results with the presence of serum specific IgE to inhalant and food allergens. Methods Phadiatop® and serum specific IgE levels (RAST) to inhalant and food allergens (UniCAP - Pharmacia®) were determined in 451 atopic children from several pediatric allergy centers and in a non-allergic control group (N=62), distributed in five age groups: Results Phadiatop was positive in 305 atopic children (67.6%) and in 25.8% of controls (p D. pteronyssinus, D. farinae and Blomia tropicalis ). Conclusions Phadiatop® is an useful tool for allergy diagnosis to domestic mites.

Rationale The aim of this study was to investigate the role of autoimmunity in children and adolescents with chronic urticaria. Methods We have studied 38 children aged 2 to 17 years-old (20 females), who were referred to the allergy clinic for evaluation of chronic urticaria in the preceding 4 years. Age at onset of symptoms varied from 6 months to 15 years (mean 5.3 years), duration of symptoms ranged from 6 weeks to 10 years (mean 2.4 years), and 63% patients also presented angioedema. Each patient underwent clinical evaluation, skin prick testing with inhalant and food allergens, and blood testing including detection of anti-nuclear antibodies, antithyroid antibodies, thyroxine and TSH levels and measurements of total IgE and specific IgE antibodies (Pharmacia CAP system). Autologous skin testing was carried out in 28/38 patients. Results Positive skin tests and/or serum IgE antibodies (CAP score ≥2) to inhalant and food allergens were detected in 65% and 76% of patients, respectively. Associated conditions were (number of patients): allergic rhinitis (14), asthma (5), precocious puberty (2), atopic dermatitis, JRA and anaphylaxis to insect sting (1 each). Evidence for autoimmunity was present in a subset of children: 15/28 (53%) had a positive autologous skin test and 3/30 had positive ANA (≥1:20). Two patients had elevated TSH (>4 mIU/L) with normal thyroxine levels, and no patient had detectable (≥35 IU/mL) antithyroid peroxidase antibodies. Conclusion An autoimmune mechanism may underlie the pathogenesis of chronic urticaria in a subset of children with this condition.

Rationale We have evaluated risk factors associated with persistence of wheezing beyond the first two years of life among children living in a subtropical area. Methods 80 children who had been seen at the Emergency Room for an episode of wheezing in the first 2 years of life were followed for 2 years in this prospective study. Children underwent clinical evaluation and skin prick testing to inhalant and food allergens two years following the index episode of wheezing. Detection of respiratory viruses and analysis of house dust samples for exposure to major allergens from mites, cockroach, cat and dog (by ELISA) were carried out at enrollment. Total IgE and specific IgE antibodies (Pharmacia CAP system) were measured at the beginning of the study and at the end of follow-up. Multivariate analysis was performed to identify factors associated with persistent wheezing. Results 73 children (44 boys) completed the study. After 2 years, 38 (52%) presented 3 or more wheezing episodes treated with beta-2 agonists in the past 12 months (persistent wheezers). Risk factors for persistence of wheezing were: family history of allergy (OR=48.6; p=0.03), exposure to levels of cockroach allergen Bla g 1 >2U/g in the kitchen (OR=7.6; p=0.03) and allergic sensitization at age 2-4 (OR=11.3; p=0.002). Breast-feeding for at least one month was a protective factor (OR=0.08; p=0.01). Conclusions Environmental exposure to high levels of cockroach allergen in infancy, sensitization to indoor allergens at age 2-4 and family history of allergy were strongly and independently associated with persistent wheezing in children.