Leonardo Puerta

Mite fauna, Der p I, Der f I and Blomia tropicalis allergen levels in a tropical environment

Fifty dust samples were collected from the mattresses and bedroom floors of 25 subjects with allergic asthma in Cartagena, Colombia, in order to identify house dust mites and quantitate Der p I, Derf I and Blomia tropicalis allergens. The geometric mean of the total mite density per gram of dust was 418 (range, 40–2280). Twenty‐two samples (44%) had more than 500 mites and four, less than 100. B. tropicalis and Dermatopha‐goides pteronyssinus were found in 96% and 90% of the samples, accounting for 40.1% and 35.7% of the total mites, respectively. Cheyletus malaccensis, Chortoglyphus arcuatus, Pyroglyphus africanus, Orihatids, Grallacheles bakeri. Tarsonemus spp., Suidasia spp., Dermatophagoides farinae and unidentified mites accounted for the rest. The geometric mean of the total mites/gram of dust in mattresses (563.9) was significantly higher than in floor dust (309. 1), P < 0.01. Allergen concentrations and mite numbers were analysed by Spearman rank correlations: B. tropicalis mites vs B. tropicalis allergen, r= 0.54, P<0.001; D. pteronyssinus mites vs Der p I, r= 0.52, P< 0.001. A negative correlation was obtained between B. tropicalis mites and Der p I. Allergens derived from B. tropicalis and other domestic mite species may play an important role in sensitization and allergic symptoms in Cartagena, Colombia.

IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1

Background:  Analysis of cross‐reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross‐reactivity between Ascaris and two domestic mites in the tropics.

Cloning and IgE Binding of a Recombinant Allergen from the Mite Blomia tropicalis, Homologous with Fatty Acid-Binding Proteins

To characterize the allergens of Blomia tropicalis, a cDNA library was constructed and screened with allergic sera from asthmatic patients. One clone, Bt6, was subcloned and sequenced. The nucleotide sequence of 934 bp length shows a 390-bp reading frame which encodes a 130-amino acid protein with a MW 14.8 kD. No potential glycosylation site was found in the predicted protein. The inferred amino acid sequence has no homology to known allergens. It has a cytosolic fatty acid-binding protein (FABP) signature at 5-22 amino acid residues, 42.3% identity with the Sm14-FABP of Schistosoma mansoni and 36% identity with FABPs from rat, mouse, bovine and human. The protein was expressed as a GST fusion protein and the purified GST-Bt6 used for dot blot, RAST and RAST inhibition assays. The frequency of IgE binding of allergic sera to Bt6 was low (11%) and usually weak. One positive serum did, however, show strong reactivity by RAST and dot blot and Bt6 could inhibit 60% of the IgE binding of this serum to the B. tropicalis extract. These data show that Bt6 encodes a mite FABP with allergenic properties, which are pronounced in some atopic subjects.

Identification of allergens from the mite Blomia tropicalis

In some tropical areas the mite Blomia tropicalis is a clinically important allergenic component of house dust, inducing specific IgE immune response in patients with allergic respiratory diseases such as asthma and rhinitis. The identification of allergens of this mite is necessary to obtain appropriate reagents for diagnostic and treatment procedures. We carried out this study using immunoblotting to detect the allergens of B. tropicalis. Our results demonstrate that this mite has one major allergen (11–13kDa) and three other important allergens with about 50% binding (64, 36 and 33 kDa). Therefore, B. tropicalis should be regarded as an important source of allergens in the house dust in tropical areas, besides those derived from other mites.

Association between total immunoglobulin E and antibody responses to naturally acquired Ascaris lumbricoides infection and polymorphisms of immune system-related LIG4, TNFSF13B and IRS2 genes

The 13q33–34 region harbours a susceptibility locus to Ascaris lumbricoides, although the underlying genes are unknown. Immunoglobulin (Ig)E and IgG confer protective immunity and here we sought to investigate in an endemic population whether LIG4, TNFSF13B and IRS2 genes influence IgE and IgG levels against Ascaris and the ABA‐1 allergen as a putative resistance marker. Mite‐allergic asthmatic patients were analysed for potential relationships between Ascaris predisposition and allergy. One thousand and sixty‐four subjects from Cartagena, Colombia, were included. Single nucleotide polymorphisms (SNPs) were genotyped using TaqMan assays. Antibody levels were measured by enzyme‐linked immunosorbent assay. Linear and logistic regressions were used to model effects of genotypes on antibody levels. The GG genotype of LIG4 (rs1805388) was associated with higher IgE levels to Ascaris compared with other genotypes. TNFSF13B (rs10508198) was associated positively with IgG levels against Ascaris extract and IgE levels against ABA‐1. In asthmatics, IRS2 (rs2289046) was associated with high total IgE levels. Associations held up after correction by population stratification using a set of 52 ancestry markers, age, sex and disease status. There was no association with asthma or mite sensitization. In a tropical population, LIG4 and TNFSF13B polymorphisms are associated with specific IgE and IgG to Ascaris, supporting previous linkage studies implicating the 13q33 region. Our results suggest that genes protecting against parasite infections can be different to those predisposing to asthma and atopy.

Sensitization to Chortoglyphus arcuatus and Aleuroglyphus ovatus in Dermatophagoides spp. allergic individuals

The prevalence of specific IgE to the storage mites Aleuroglyphus ovatus (Ao) and Chortoglyphus arcuatus (Ca) was studied in 77 individuals with allergic asthma and/or chronic allergic rhinitis. All these individuals had a positive skin test (weal ≥ 3 mm) to extracts of Dermatophagoides pteronyssimis (Dp) and/or Dermatophagoides farinac (Df). Sera from 29 non‐atopic individuals were used as controls. A RAST was considered positive when a serum bound ≥1% of the total counts added. The prevalence of a positive RAST to Dp was 75.3%, and to at least one of the two storage mites (Ao and Ca), 76.6%. Among patients with a positive RAST to Dp. 79.3% and 75.8% were RAST positive to Ao and Ca, respectively. RAST inhibition studies with a pool of sera from 13 subjects with high RAST binding to all three mites showed significant crossreactivity between Ao and Ca and minimal to moderate crossreactivity between Dp and Ao and Co, This study demonstrates that Sensitization to Ao and Ca is common in individuals with respiratory allergies in Cartagena, Colombia and suggests that Ao, Ca and Dp have unique and common allergenic determinants.

Sequential determinations of Blomia tropicalis allergens in mattress and floor dust samples in a tropical city

BACKGROUND The mite species Blomia tropicalis is commonly found in tropical and subtropical regions, and it is an important source of allergen in the city of Cartagena, Columbia. AIM The study was designed to determine seasonal allergen levels of B. tropicalis in homes of patients with asthma and mite allergy. METHODS Dust samples from mattresses and floors in 20 homes were collected on a monthly basis for 1 year. Outdoor temperature, relative humidity, and rainfall were recorded. RAST inhibition was performed on extracts of dust samples. Allergen levels were compared with variations in climate. RESULTS B. tropicalis allergens were detected in all mattress samples. More than 50% RAST inhibition was detected in 30% of mattress samples and in 4.3% of floor samples, reflecting a high concentration of allergen. Significant correlations were only found between allergen levels and absolute humidity. CONCLUSION Levels of B. tropicalis allergen fluctuated minimally in Cartagena, Colombia.

Cloning and expression of complementary DNA coding for an allergen with common antibody-binding specificities with three allergens of the house dust mite Blomia tropicalis

The mite Blomia tropicalis is a potent source of allergens in tropical and subtropical regions. So far, most of these allergens have only been studied by immunoblotting. To characterize them at the molecular level, a lambda gt11 complementary DNA library was constructed from messenger RNA isolated from whole B. tropicalis mites. This library was screened by using pooled sera from patients allergic to B. tropicalis in a plaque IgE radioimmunoassay. A B. tropicalis IgE-positive clone (Bt-M) was selected for immunologic studies. After subcloning into pBluescript (Stratagene, La Jolla, Calif.), it produced a sequence of 310 bp, with a probable amino acid sequence of 72 residues for the expressed peptide. The recombinant protein was transferred to nitrocellulose filters and probed with 100 sera from patients allergic to B. tropicalis. Forty-seven percent of sera reacted with the recombinant allergen. Immunoblottings performed with allergic serum and B. tropicalis-affinity-purified IgE demonstrated that the recombinant protein shares allergenic epitopes with the 11-13, 14, and 16 kd native allergens of B. tropicalis, which are known to be important allergens of this mite.

Structural and Ligand Binding Analysis of Recombinant Blo t 13 Allergen from Blomia tropicalis Mite, a Fatty Acid Binding Protein

Background: We have previously described a cDNA (clone Bt6) encoding a novel allergen from Blomia tropicalis, which showed sequence similarities to the FABP/P2/cellular retinoic acid binding protein/cellular retinol binding protein, a family of cytosolic lipid transport proteins (cLTPs). This work was planned to better characterize this allergen to which the official name Blo t 13 had been assigned. Methods: Fluorescence–based lipid ligand binding assays and secondary structure analysis by circular dicroism were carry out using recombinant Blo t 13 (rBlo t 13) protein. Structural predictions and molecular modelling were performed based on the amino acid sequence inferred from the open reading frame of Bt6 cDNA sequence. Results: rBlot t 13 binds the natural fluorescent fatty acid cis–parinaric acid and oleic acid by competition, but not retinol, retinoic acid, cholesterol, dansylated or anthroxylated fatty acids such as dansyl–DL–aminocaprylic acid and 12–(9–anthroyloxy)–stereate. Circular dichroism analysis indicated that rBlo t 13 comprises 45% β–sheet and 13% α–helix. The amino acid sequence of Blo t 13 modelled well to known crystal structures of cLTPs providing a tertiary structural model comprising ten β–strands organized into two β–sheets, and two short α–helices. Conclusion: Blo t 13 is a fatty acid–specific member of the β–rich cLTP family of proteins.

Analysis of the Cross–Reactivity between BtM and Der p 5, Two Group 5 Recombinant Allergens from Blomia tropicalis and Dermatophagoides pteronyssinus

Background: In tropical climates, sensitization to Bloma tropicalis and Dermatophagoides pteronyssinus is high and mainly directed to species–specific allergens. There is some cross–reactivity between extracts of these mites, probably due to the group 5 allergens that have high sequence homology. Objective and Methods: We used the radioallergosorbent test (RAST), RAST inhibition and immunoblotting inhibition experiments to investigate the cross–reactivity between the recombinant allergens BtM and Der p 5, expressed as glutathione S–transferase fusion proteins, to detect the epitopes involved and to analyze the importance of this cross–reactivity. Results: Seventy–nine percent of 48 patients sera were RAST positive to both recombinants, with a strong correlation (r = 0.8, p<0.0001). BtM inhibited 25 and 21.1% of IgE–binding to B. tropicalis and D. pteronyssinus extracts respectively and Der p 5 inhibited 22 and 24% of IgE–binding to D. pteronyssinus and B. tropicalis extracts. Furthermore, BtM inhibited 74.5% of IgE binding to Der p 5 and Der p 5 inhibited 72.4% of IgE–binding to BtM. RAST inhibition with BtM–derived synthetic peptides showed that peptide 4 (residues 35–50) and peptide 5 (residues 46–61) inhibited 37 and 16% of IgE–binding to BtM while peptides 5 and 2 (residues 14–30) were able to inhibit the IgE binding (32 and 28%, respectively) to Der p 5. Conclusion: There is cross–reactivity between BtM and Der p 5, which explains almost all the cross–reactivity between the two mite extracts. This cross–reactivity seems to be related to epitope(s) at the C–terminal segment of these allergens.