Bengt Härfast

Identification of allergen components of the opportunistic yeast Pityrosporum orbiculare by monoclonal antibodies

The yeast Pityrosporum orbiculare (P. orbiculare) is a member of the normal human cutaneous flora, but it is also associated with several clinical manifestations of the skin. We have previously observed IgE‐binding components in P. orbiculare extracts, using sera from patients with atopic dermatitis. In the present study, we raised several monoclonal antibodies (MoAbs) against P. orbiculare to characterize some of its antigens, and used Candida albicans (C. albicans) as a control. We obtained several IgGI MoAbs which specifically recognized P. orbiculare in ELISA. Two of these were selected for immunoblotting studies on P. orbiculare and two patterns of reactivity emerged. Firstly, one MoAb showed a distinct band at a molecular mass of 67 kDa. In the second pattern, a sharp band at about 37 kDa appeared. In contrast, the IgM antibodies raised reacted with a 14‐kDa component; but they reacted with C. albicans in addition to P. orbiculare The IgGI antibodies seemed to react with proteins, as their ability to react in ELISA with extract pretreated with protease was greatly reduced. In contrast, IgM MoAbs were much less affected, suggesting that they recognized nonprotein components. To determine whether these MoAbs‐binding components were also recognized by human IgE, we adopted a radioimmunoassay (RIA) using the MoAbs as catcher antibodies. Both the 67‐kDa and the 37‐kDa components were IgE‐binding proteins. P. orbicular RAST positive sera were scored as positive in the RIA, whereas the control serum was not.

Specific induction of interleukin-4-producing cells in response to in vitro allergen stimulation in atopic individuals

Background and Objective CD4+ T cells can be divided into two major subsets. T helper (TH)1 and TH2 cells. Interleukin‐4 (IL‐4) is produced by TH2 cells and induces switching of immunoglobulin (Ig) M/lgG to IgE. Interferon‐γ (IFNγ) produced by TH1 cells counteracts the IgE‐promoting effects of IL‐4. In this study we wanted to investigate whether the number of IL4‐producing cells could be a direct measurement of allergen exposure in vitro, and whether this was correlated to the elevated serum IgE‐levels seen in atopic persons.

Dispersion of horse allergen in the ambient air, detected with sandwich ELISA

Background: The objective was to establish an ELISA to detect horse allergen in ambient air and settled dust.

Increased allergen‐specific Th2 responses in vitro in atopic subjects receiving subclinical allergen challenge

The study aimed to determine whether inhalation of subclinical allergen doses leads to a shift in the balance between T helper (Th) 1 and Th2 cells in asthmatic patients. Elevated IgE requires allergen‐specific T cells producing cytokines such as interleukin (IL)‐4 or 1L‐13. Interferon‐gamma (IFN‐y) produced by Till cells counteracts the effects of IL‐4. In nature, allergic persons are often exposed to low levels of allergen, leading to hyperreactivity, but not to acute allergic reactions. In this study, nine allergic persons inhaled low doses of allergen or placebo in a double‐blind manner over seven consecutive weekdays. During the study, the bronchial responsiveness to histamine challenge increased, but no subject exhibited asthmatic symptoms. Blood was drawn on days 0,1, 4, and 9, and the number of IL‐4– and IFN‐γ‐producing cells was measured by enzyme‐linked immunospot (ELISPOT) assay after in vitro stimulation with a low‐dose phytohemagglutinin (PHA) mixed with the relevant allergen or with PHA alone. In three of the four subjects receiving allergen, the IL‐4/IFN‐y ratio increased during the time of the study. No increase was seen in the placebo group. No increase was seen in serum IgE levels in any of the groups. We conclude that a shift in the balance between Thl and Th2 cells can be detected in subjects exposed to subclinical allergen doses.

Characterization of the mite fauna (Acari) in Swedish barn dust

Abstract The mite fauna in barn dust from 30 farms on the Swedish island of Gotland was analysed. All samples contained mites, but densities varied from 660 to 200,500 specimens/g dust. Twelve species of astigmatic mites were identified and among them was Lepidoglyphus destructor from all the farms and abundant in most samples. Tyrophagus spp., Acarus siro -complex and Glycyphagus domesticus were also present in high numbers. Of the prostigmatic mites, the families Cheyletidae, Tarsonemidae and Tydeidae were represented in almost all samples. Significant positive correlations were obtained between the acarids and the predatory cheyletid mites and between the glycyphagids and the cheyletids (p <0.05).

No signs of activity markers in peripheral blood despite increased bronchial reactivity after repeated low-dose allergen exposure.

The allergen inhalation test can be used as an experimental model to study pathophysiological events in allergic asthma. Repeated low‐dose inhalations of allergen induce increased bronchial hyperresponsiveness (BHR) and resemble natural allergen exposure. The objective of the present study was to investigate whether eosinophil recruitment and activation in peripheral blood, differences in expression of lymphocyte surface antigens and increased bronchial responsiveness to histamine occur during and after repeated low‐dose bronchial allergen challenge. Fourteen atopic asthmatic patients were challenged in a randomized double‐blind manner for 7 days with either allergen in very low doses or placebo. We measured the concentration of eosinophils, eosinophil cationic protein (ECP) and the expression of the EG2‐epitope on intracellular ECP in eosinophils and the expression of lymphocyte surface antigen markers in peripheral blood. The challenge period started and ended with a histamine provocation. The repeated low‐dose allergen exposure resulted in a significant increase in BHR. No changes were seen in the placebo group. Concerning the inflammatory parameters in peripheral blood, no significant changes were seen during or after the week of low‐dose allergen inhalations. Our results show that very low, repeated doses of allergen induce increased airway reactivity despite lack of evident clinical symptoms or signs of activation of inflammatory cells in peripheral blood.

Localization of major allergens in the dust mite Lepidoglyphus destructor with confocal laser scanning microscopy

The dust mite Lepidoglyphus destructor is the dominating source of allergens giving rise to asthma and rhinitis among farmers. In a previous study of the localization of allergens in L. destructor we demonstrated that the 39 kDa allergen is associated with digestion. Here we describe the localization of the principal 15 kDa allergen and the high molecular weight allergen complex (79 and 93 kDa) in L. destructor with confocal laser scanning microscopy (CLSM). Cryostat‐cut sections of mite bodies and faecal pellets were probed with mouse monoclonal antibodies (MoAbs) raised against the allergens. The 15 kDa allergen disclosed labelling of the mite body and most of the faecal pellets but left the exoskeleton unlabelled. The binding was widespread, and most intense in the mouth region. However, some staining was also observed around the gastrointestinal tract. In contrast, the 79 and 93 kDa allergen complex stained the exoskeleton and the front part of the mile. Interestingly, we detected no labelling of the faecal pellets with the MOAb against the 79/93 kDa allergen. The study indicates that the 15 kDa allergen is associated with the digestive tract whereas the function of the 79 and 93 kDa allergen complex remains to be elucidated.

Identification of a new major allergen of 39 kilodaltons of the storage mite Lepidoglyphus destructor.

The allergen composition of a non-denatured extract of the storage mite Lepidoglyphus destructor was studied by a combination of hybridoma technology, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a sandwich radio-allergosorbent test (four-step RAST). Using hybridoma technology, monoclonal antibodies (mAbs) were generated against the non denatured extract of the L. destructor. After screening by ELISA, mAb 42B6 was selected for further studies. This mAb specifically recognized an antigen of L. destructor of 39 kDa in SDS-PAGE. By a sandwich RAST, sera from 8 patients with known allergy to L. destructor were compared with control sera from 8 allergic patients. The results showed the presence of specific IgE against the 39-kDa protein in the sera of the test group, but not in the control group. There was also a good correlation observed between these data and the results obtained with the classical RAST. These results indicate that the newly identified antigen of 39 kDa is a major allergen of the storage mite Lepidoglyphus destructor.

T‐cell subsets in adenoids and peripheral blood related to age, otitis media with effusion and allergy

Adenoids and peripheral blood samples from 29 children (20–120 months of age) undergoing adenoidectomy for long‐standing otitis media with effusion (OME) (n=16) or obstructive adenoid hyperplasia (n=13) were investigated by flow cytometry for their T‐lymphocyte profile. Eleven of the enrolled children were allergic to inhalant and/or food allergens. For the whole group, the percentage of helper T cells belonging to the memory phenotype (CD4+/CD45RO+ cells) was significantly higher in adenoids than in blood (p<0.0001), while the same cell category increased with age in peripheral blood (p<0.01). A highly significant negative regression (p<0.001) was found between age and the percentage ratio of CD4+ cells that were CD45RO+ in adenoids and blood. Allergic children had a higher CD4+/CD8+ ratio for cells expressing CD45RO+ (p<0.05) in adenoids. The results of this study indicate that adenoids participate in the development of an immunological memory. Our findings support a relationship between allergy and memory cells in adenoids.

ELISA method for detection of mite allergens in barn dust: comparison with mite counts

ELISA (enzyme‐linked immunosorbent assay) inhibition with a monoclonal antibody (mAb) (42B6) to Lepidoglyphus destructor was used to detect and quantify the storage‐mite allergens in 30 dust samples collected from barns. Regarding the mite fauna, microscopic inspection of the barn dust and mite counts showed that L. destructor infested all 30 barns investigated (range 430–195400 mites/g dust). In 29/30 barns, L. destructor constituted more than 70% of the Astigmata species. Acarus siro was found in 26 samples, the highest value being 16 155 nites/g. No Dermatophagoides species were found. As to mites of the suborder of Prostigmata, species belonging to seven different families were detected. Besides the predominant L. destructor, allergens derived from other storage mites such as Glycyphagus domesticus, A. siro, and Tyrophagus putrescentiue have previously been assessed by this ELISA method. The correlation between number of mites and concentrations of mite antigen as measured by ELISA was assessed by linear regression (r2= 0.83). Thus, inhibition of mAb 42B6 in ELISA would seem to offer a simple and reliable method to detect levels of dust‐mite species belonging to the Acaridae and Glycyphagidae families.