Importance of albumin in cross-reactivity among cat, dog and horse allergens.

Different allergenic proteins have been involved in cross-reactivity among animals. Albumins seem to be cross-sensitizing allergenic components. The aim of this study was to assess the importance of albumin as a cross-reactive allergen in patients sensitized to cat, dog and horse. One hundred and seventeen patients sensitized to cat were tested for IgE reactivity using skin prick tests and RAST assays with cat, dog and horse hair/dander extracts and their purified albumin extracts. RAST-inhibition studies were carried out to assess cross-reactivity among cat, dog and horse and among their purified albumins. It was found that 22% of patients exhibited specific IgE to cat albumin; 41% of patients sensitized to cat were also sensitized to dog and horse. Out of these patients, 21% had IgE to three albumins and 17% to two. Reciprocal inhibitions were observed among cat, dog and horse albumins and also among cat, dog and horse hair/dander extracts, using in the latter experiment sera from patients not sensitized to albumins. IgE binding to horse extract was inhibited 30% by its homologous albumin and IgE binding to cat and dog extracts in almost 15% by their respective albumins. It was concluded that albumins from these three animals share some epitopes that account for the cross-reactivity observed in around one-third of patients sensitized to cat, dog and horse. Nevertheless, more than 50% of specific IgE that cross-reacts among these three animals is directed to allergens other than albumin.

An immunoblotting analysis of cross-reactivity between melon, and plantago and grass pollens.

It is known that most patients with type I allergy to pollens also suffer intolerance to fruits. Recently, an epidemiological and CAP-inhibition study has shown a new clustering of allergy between melon and Plantago and grass pollens. The aim of the present study was to confirm these results by immunoblotting analysis and inhibition of immunoblotting. Sera from 3 patients with confirmed allergy to melon, and Dactylis glomerata and Plantago lanceolata pollens were used for the in vitro studies. SDS-PAGE and immunoblotting analysis with a pool of sera revealed that several distinct protein bands were shared by the three extracts at 14, 31, and a spectrum between 40 and 70 kDa, approximately. Immunoblotting inhibition experiments, performed with extracts of melon, Plantago and Dactylis, showed that all allergens of melon blotting were almost completely inhibited by grass and Plantago pollen extracts. Inversely, the melon extract was capable of inhibiting IgE-binding to various allergens of Dactylis at high mol mass and partially to the band at 14 kDa. Moreover, the melon almost totally inhibited the IgE-binding capacity to the proteins of Plantago extract. Taken together, the results support the presence of structurally similar allergens in melon, Plantago and grass pollens, and that all allergenic epitopes of the melon are present in these pollens.

Indoor mite allergens in patients with respiratory allergy living in Porto, Portugal.

We investigated the levels of mite allergens (Der p 1, Der f 1, Der 2, and Lep d 1) in dust samples from the homes of 59 patients with asthma, 36 sensitized to house-dust mites (HDM) and 23 to grass pollen (controls), living in Porto, northern Portugal. The relationship between exposure and sensitization to HDM and the influence of housing conditions on mite-allergen levels were also evaluated. Der p 1 (median 9.2 micrograms/g) and Der 2 (4.6 micrograms/g) were the main allergens, while Der f 1 and Lep d 1 levels were always < 1 microgram/g dust and undetectable in 11% and 47% of samples, respectively. All HDM-sensitized asthmatics were exposed to Der p 1 levels > 2 micrograms/g and their homes contained significantly higher levels of Der p 1 (median 12.5 vs 6.4 micrograms/g; P = 0.008) and Der 2 (6.2 vs 3.0 micrograms/g; P = 0.004) when compared to the control group. A significant correlation was observed between the exposure to Der p 1 and the wheal area at skin testing with the Dermatophagoides pteronyssinus (Dp) extract (P = 0.01) as well as with serum specific IgE levels to Dp (P = 0.03). Patients with higher levels of serum specific IgE (> or = 17.5 HRU/ml) were also more frequently exposed to Der p 1 levels > or = 10 micrograms/g (P = 0.002). Old homes, presence of carpets, and signs of dampness were conditions associated with significantly higher levels of mite allergens. In conclusion, we found high levels of Der p 1 and Der 2 particularly in the homes of HDM-sensitized patients and we confirm the relationship between exposure and sensitization to HDM, assessed by both in vivo and in vitro methods. In additional to a favorable outdoor climate, we found in our region housing conditions propitious to mite growth, suggesting that specific geographic characteristics must also be taken into account for the correct planning of mite-avoidance measures.

Lepidoglyphus destructor acarus in the urban house environment.

To assess the presence of Lepidoglyphus destructor in the household environment of sensitized children living in an urban environment, samples of house dust were collected at the homes where two groups of patients were living, as well as in two bakeries in the city of Valencia, which were taken as a reference. Patients were divided into two groups. Group A included atopic children suffering from rhinitis and/or asthma, who were sensitized to L. destructor, as proven by prick test and specific IgE (CAP). Group B included children with the same features as those included in Group A, who were sensitized to Dermatophagoides pteronyssinus, with prick and CAP tests showing no significant sensitization to L. destructor. The samples of dust were analyzed, and the amounts of Der p I, Der f I, Der II and Lep d I per gram of dust were assessed through a solid-phase ELISA with monoclonal antibodies. In Group A, all patients but two showed a sensitization to D. pteronyssinus by prick test and serum IgE. At the homes of the patients from both groups, significant levels of Dermatophagoides were found. In Group A, only three houses showed levels of L. destructor which were comparable to those found in bakeries. Lep d I was not found in the houses of Group B patients. This means that a sensitization to L. destructor, as assessed with full extracts, is not always an indicator of its presence at the patient's house environment; it may rather refer to cross-reactivity to Dermatophagoides. Thus, availability of the main antigen Lep d I seems necessary to increase the specificity of the allergologic study.

Immunotherapy with the storage mite lepidoglyphus destructor.

We carried out a double-blind clinical trial of immunotherapy on 35 patients sensitized to the storage mite Lepidoglyphus destructor (Ld). Before and after 12 months of specific hyposensitization (Abelló Lab., Spain) we performed in vivo (skin tests with Ld, methacholine and challenge tests), and in vitro tests (specific IgE, IgG, IgG1 and IgG4 to Ld and specific IgE, IgG, IgG1 and IgG4 to their major allergen Lep dI). We also monitored the efficacy and safety of the immunotherapy with clinical and analytical controls (symptoms and medication score, detection of immune complexes). After therapy we found a significant decrease in specific skin reactivity, dose of positive challenge tests, and hyperresponsiveness to methacholine. Sputum eosinophilia decreased. Specific IgE to Ld was increased and we also observed an increase in specific IgG1 and IgG4 to Ld and Lep DI. The placebo group showed no changes in these variables. There were no severe secondary reactions after treatment with the extract. Patients-self-evaluation was favourable and their labour absence decreased. No development of circulating immune complexes was associated with this immunotherapy.

Quantitation of major allergens in dust samples from urban populations collected in different seasons in two climatic areas of the Basque region (Spain).

We present the results of allergen content evaluation in 80 dust samples from 31 homes of atopic patients from two climatic areas (humid and subhumid), collected in two seasons of the year (autumn and winter). Monoclonal antibody-based immunoassays were used to quantify Der p 1, Der f 1, Der 2, Lep d 1, and Fel d 1. The results were compared according to climate, season, and the type of sensitization (Pyroglyphidae mites, storage mites, or grass pollens). We underline the predominance of Dermatophagoides pteronyssinus (89% of samples) over D. farinae (16% of samples) in our environment. Der p 1 rates were higher in the humid area (Mann-Whitney P < 0.001), especially in the autumn (Wilcoxon P < 0.05). Lep d 1 was detected in 23% of samples and Lep d 1 levels were higher in the homes of patients sensitized to storage mites (Mann-Whitney P < 0.05), whereas this allergen was not detected in the homes of pollen-allergic patients. Fel d 1 was detected in nine of the 31 homes (16% of samples) although there was a cat in only one home.

Primary structure of Lep d I, the main Lepidoglyphus destructor allergen.

The most relevant allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been characterized. Lep d I is a monomer protein of 13273 Da. The primary structure of Lep d I was determined by N-terminal Edman degradation and partially confirmed by cDNA sequencing. Sequence polymorphism was observed at six positions, with non-conservative substitutions in three of them. No potential N-glycosylation site was revealed by peptide sequencing. The 125-residue sequence of Lep d I shows approximately 40% identity (including the six cysteines) with the overlapping regions of group II allergens from the genus Dermatophagoides, which, however, do not share common allergenic epitopes with Lep d I.

Seasonal versus perennial immunotherapy: evaluation after three years of treatment.

We have performed a comparative study to evaluate seasonal and perennial schedules after 3 years of immunotherapy. Sixty patients suffering from rhinitis and/or asthma due to grass pollen sensitization were randomly allocated to receive a semi-depot extract of Phleum pratense according to a perennial or seasonal schedule. The last year of the study, 14 patients were recruited as a control group without immunotherapy. The cumulative dose was 602 BU in the perennial group and 372 BU in the seasonal group. The frequency and severity of side-effects were similar and very low in both treated groups. The IgE level was significantly lower after perennial immunotherapy at the end of the first 2 years. A seasonal decrease in specific IgG levels was observed in patients who interrupted immunotherapy, while this was not observed in patients under the perennial schedule. Symptoms and medication scores did not show differences between groups. Nevertheless, we found a significant difference between treated patients and the control group.

Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor.

A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.

Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.

The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.

[Evaluation of the total biological activity and allergenic composition of allergenic extracts]. / Evaluación de la actividad biológica total y composición alergénica de extractos alergénicos.

In the present study, a complete procedure is presented in order to standardize allergenic extracts, the meaning of which is the measurement of the total allergenic activity and the determination of the allergenic composition. The measurement of the biological activity comprises 2 steps: Preparation of Reference Extracts and determination of their "in vivo" activity. Evaluation of the total allergenic activity of extracts for clinical use. Reference extracts were prepared from the main allergens and their "in vivo" biological activity was determined by a quantitative skin prick test in a sample of at least 30 allergic patients. By definition, the protein concentration of Reference Extract that produces, in the allergic population, a geometric mean wheal of 75 mm.2 has an activity of 100 biological units (BUs). The determination of the biological activity of a problem extract is made by RAST inhibition. The sample is compared with the corresponding Reference Extract by this technique and, from this comparison, it is possible to quantify the activity of the problem extract in biologic units (BUs) with clinical significance. Likewise, different techniques have been used to determine the allergenic composition of extracts. These techniques comprise 2 steps: Separation of the components of the extract. Identification of the components that bind specific human IgE. The separation of the components of the extract has been carried out by isoelectric focusing (IEF) and electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). In order to identify the allergenic components, an immunoblotting technique has been employed. The separated components in the IEF gel or SDS-PAGE gel are transferred to a nitrocellulose sheet and later on, this membrane is overlaid with a serum pool from allergic patients and a mouse monoclonal anti-human IgE, labelled with 125I. Finally, the autoradiography of the nitrocellulose membrane is obtained. In this way it is possible to compare the allergenic composition of an extract with the corresponding Reference Extract and so to employ for clinical use only those extracts with the right allergenic composition.