Serum samples, skin prick test and studied groups
Sera containing αBt E IgE were obtained from blood samples collected from 32 randomly chosen individuals out of 80 individuals from a previous work, all positive for αBt E IgE, who lived in a poor area of Salvador, a major city in Northeastern Brazil. These individuals answered an ISAAC phase-I questionnaire, adapted to the Portuguese language, and had their skin response to seven regional allergens, including B. tropicalis, assessed. SPTs were performed by the introduction of the allergenic extracts (ALK-Abelló, São Paulo, Brazil) in the skin of the right forearm of children with a disposable lancet (ALK-lancet®; ALK-Abelló, São Paulo, Brazil). In negative- and positive-control reactions, saline and histamine at 10 mg/mL, respectively, substituted for the allergenic extracts. Readings were done 15 minutes after the puncture. The result of the test was considered positive if the mean diameter of the wheal was ≥ 3 mm after subtraction of the mean diameter of the negative-control wheal. A negative reaction to histamine was an exclusion criterion. These 32 individuals were further divided in two groups: a group of 12 individuals with negative Bt E SPT results (when tested with Bt E, one of them had a wheal with mean diameter of 2.5 mm, two had a wheal of 2.0 mm, one had a wheal of 1.0 mm and in the other eight no wheal was formed), with ages ranging from 6 to 48 years; and a group of 20 individuals with positive Bt E SPT results, with ages ranging from 5 to 46 years. Other 28 sera, that were used for investigating cross-reactivity of B. tropicalis and A. lumbricoides antigens with serum IgE antibodies, were selected from a serum bank of the Laboratório de Alergia e Acarologia of the Instituto de Ciências da Saúde, Federal University of Bahia, Brazil, so as to have similar α-Bt E IgE levels (all of them had α-Bt E IgE levels between 3.4 to 17.5 kIU/L, as determined by Pharmacia Immunocap System IgE FEIA (Pharmacia, Uppsala, Sweden). These 28 sera were divided into two groups, paired in accordance with α-Bt E IgE levels: 14 with positive Bt E SPT and 14 without any observable wheal formed when subjected to the Bt E SPT. Each serum from one group was paired with a serum from the other group that had similar α-Bt E IgE levels. Informed written consent was obtained from all individuals, and the study was approved by the Ethics Committee of the Maternity Hospital Climério de Oliveira, of the Federal University of Bahia, Brazil.
B. tropicalis and A. lumbricoides extracts
The B. tropicalis was collected from bed dust in Salvador, Brazil, cloned and cultured with a fish food medium, at 25°C and 75% humidity. The mites were purified from the medium by flotation on a 5 M sodium chloride solution, followed by several washings by filtration in a 100 μm-pore polystyrene sieve with endotoxin-free distilled water. The washings were carried out until no food residues were seen under microscopy. The mites were lysed in 0.15 M phosphate-buffered saline, pH 7.4 (PBS), in an electric blender (Waring Commercial, Torrington, CN, USA). Lipids from the lysate were extracted by five or six ether extractions and discarded. The protein content of the B. tropicalis aqueous extract was determined by the Folin reagent method [28], and was subsequently stored at -70°C until use. The amount of Bt E used in the experiments was standardized by measuring its Blo t 5 content, using a commercially available capture ELISA kit (INDOOR Biotechnologies, Charlottesville, VI, USA). All used batches contained 30-40 ng of this allergen per μg of protein. An aqueous extract of A. lumbricoides was prepared from adult worms obtained from albendazole-treated infected children. The worms were washed with saline and crushed in an electric grinder (Bead-Beartas; Biospec, NC, USA), in the presence of PBS containing protease inhibitors (1 mM phenylmethanesulfonylfluoride, 2 mM ethylenediamine tetra-acetic acid, 50 μm tosyl phenylalanyl chloromethyl ketone and 50 μm tosyl-L-lysine chloromethyl ketone). The suspension was centrifuged at 4000 g for 15 minutes. The supernatant was stored at -70°C, and its protein content was determined by the Folin reagent method [28].
Detection of serum anti-Bt E IgE antibodies
Microassay plate wells (Costar, Cambridge, ME, USA) were coated with Bt E by incubation with a Bt E solution containing 100 μg of protein.mL-1. After blocking the remaining free protein-binding sites with PBS containing 10% fetal bovine serum, serum samples, diluted 1:5 in PBS containing 0.05% Tween 20 and 5% fetal bovine serum, were applied to the wells in duplicates. Biotinylated anti-human IgE was added, followed by streptavidin-peroxidase (Pharmigem, San Diego, CA, USA). The reaction was developed using H2O2 and orthophenilenodiamine as substrate and chromogen, respectively (Sigma-Aldrich, St. Louis, ME, USA). The optical density to 480 nm light was measured in a plate reader spectrophotometer. Between all steps, the wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T) followed by three times with PBS alone. The cut-off of the assay corresponded to the mean plus two standard deviations of the results obtained using sera from 10 individuals without history of allergy and with negative SPT reaction to the tested allergens.
Detection of total serum IgE and calculation of anti-Bt E IgE/total IgE ratio
Total serum IgE levels were measured by capture ELISA. Briefly, microtiter plate wells (Costar 3590, flat bottom, polystyrene, high hinding; Cambrigde, MA, USA) were coated with 4 μg/mL of mouse anti-human IgE antibodies (Pharmigem, San Diego, CA, USA). The other assay steps were done as for the α-Bt E IgE assay described above. An IgE standard curve was obtained by using purified human IgE (Research Diagnostics Inc, Flanders, NJ, USA) and the results were expressed in international units (IU). A pool of sera from allergic patients who had positive SPT reactions to dust mite antigens was used as positive control. As negative control, an umbilical cord serum sample was used. An arbitrary α-Bt E IgE/total IgE ratio was calculated for each serum by dividing the value of α-Bt E IgE level that was detected in that serum (expressed in OD480 nm) by the value of total IgE level detected in the same serum (expressed as IU).
Prevention of the binding of α-Bt E IgE to Bt E carbohydrate epitopes
In the indirect ELISA described above, some Bt E-coated wells were treated with 10 mM sodium metaperiodate (VETEC, Rio de Janeiro, Brazil) in 50 mM acetate buffer, pH 4.5, for 1 hour at room temperature in the dark, so that carbohydrates were oxidized to aldehyde. In control wells, the solid-phase Bt E was incubated only with acetate buffer. The wells were then washed once with 200 μL of sodium acetate buffer, and the formed aldehyde groups were reduced to alcohol through incubation with 100 μL of 50 mM sodium borohydride (Sigma-Aldrich, St Louis, MO) during 30 minutes at room temperature. After the incubation, the wells were washed three times with PBS-T and two times with PBS and used in the ELISA. The percentage of carbohydrate determinant-reactive α-Bt E IgE, in relation to the total α-Bt E IgE, was calculated in accordance with the formula: % of carbohydrate-reactive α-Bt E IgE = [1.0 - (mean OD of the results obtained in the assay of serum duplicates in the metaperiodate/borohydride-treated Bt E-coated wells/mean OD of the results obtained from serum duplicates assayed in control wells)] × 100, where OD = optical density.
Blood cell cultivation and IL-10 detection
Venous blood samples were cultured at a dilution of 1:4 in RPMI (Gibco, Auckland, New Zealand) containing 10 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL of gentamicin (Sigma-Aldrich, St. Louis, MO, USA). The cultures were performed within 6 hours of the blood collection, in a humidified atmosphere, with 5% CO2, at 37°C, for 24 hours. The IL-10 concentrations in cultures with non-stimulated blood cells and with blood cells that were stimulated by Bt E (40 μg/mL), Al E (10 μg/mL), bacillus Calmette-Guérin (BCG) lysate (62.5 μg/mL; Fundação Ataulpho de Paiva, Rio de Janeiro, Brazil) or pokeweed mitogen (3.125 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), were measured using commercially available anti - IL-10 antibodies and a standard curve constructed with recombinant IL-10, by sandwich ELISA, following manufacturer's instructions (BD Pharmingen San Diego, CA, USA). The detection range was 31.25 to 500.00 pg/mL.
Investigation of cross-reactivity of B. tropicalis and A. lumbricoides antigens with serum IgE
The presence of IgE antibodies that cross-reacted with B. tropicalis and A. lumbricoides antigens was determined by a competitive ELISA. Briefly, different concentrations (0.3 to 300 μg/mL) of Al E, or the same volume of PBS, were incubated with the sera diluted at 1:2.5 overnight at 4°C. α-Bt E IgE were semi-quantified in these treated sera in a final dilution of 1:5, as described above. As a control for the specificity of the reduction in α-Bt E IgE levels, the concentration of total IgE was determined in the same sera. The proportions of α-Bt E IgE and total IgE levels that were reduced by incubation with Al E were calculated comparing the absorbance obtained in control sera (sera not pre-incubated with Al E) and in sera pre-incubated with Al E. Results were expressed in percentage of reduction, calculated as follows: percentage of reduction in α-Bt E IgE or total IgE level = [1 - (mean OD detected in the assay of duplicates of serum that had been pre-incubated with Al E/mean OD detected in the assay of untreated serum duplicates)] × 100.
Statistical analysis
All statistical analyses were performed using the SPSS Version 13.0 program (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk test was used for verification of the data normality. The statistical significance of differences between the groups was assessed by the Mann-Whitney's U test. The Spearman's correlation between serum α-Bt E IgE level and the average diameter of SPT reactivity was calculated. A p value equal to or less than 0.05 was considered statistically significant.