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Notes: Six monoclonal antibodies against Bet v I, the major cross-reactive allergen of birch pollen (Betula verrucosa), were obtained. Four did not react with fruits, but two monoclonal antibodies (mAbs) (5H8 and 9C11) were reactive with apple and other fruits. These two cross-reactive antibodies reacted with identical or overlapping sites, but differed in their relative degree of cross-reactivity toward various fruits and hazelnut. Cross-reactive human IgE antibodies reacted with a nonoverlapping epitope, as indicated by results of a two-site radioimmunoassay (RIA) with the fruit-reactive mAb 9C11. By isoelectric focusing (IEF) in conjunction with immunoblotting, a maximum of seven isoforms could be distinguished. Depletion of birch-pollen extract for Bet v I with the most reactive mAb (7F7) removed approximately 95% of the IgE cross-reactivity between birch pollen and apple extract. The remaining 5% cross-reactive material was still capable of inhibiting the binding of IgE to apple allergen completely, and was reactive with mAbs 5H8 and 3C4. By means of IEF/immunoblot, it was shown that these mAbs recognize an isoform of Bet v I that is poorly, if at all, recognized by mAb 7F7. These results illustrate the heterogeneity of Bet v I, both with respect to the cross-reactive sites as well as to the backbone structure. This type of heterogeneity has possible implications for the use of monoclonal antibodies in allergen standardization.

Notes: The cross-reactivity of IgE antibodies is of interest for various reasons, three of which are discussed. Firstly, from the clinical view, it is important to know the patterns of cross-reactivity, because they often (but not always) reflect the pattern of clinical sensitivities. We discuss the cross-reactivities associated with sensitization to pollen and vegetable foods: PR-10 (Bet v 1-related), profilin, the cross-reactive carbohydrate determinant (CCD), the recently described isoflavone reductase, and the (still elusive) mugwort allergen that is associated with celery anaphylaxis; cross-reactivities between allergens from invertebrates, particularly tropomyosin, paramyosin, and glutathione S-transferase (GST); and latex-associated cross-reactivities. Clustering cross-reactive allergens may simplify diagnostic procedures and therapeutic regimens. Secondly, IgE cross-reactivity is of interest for its immunologic basis, particularly in relation to the regulation of allergic sensitization: are IgE antibodies to allergens more often cross-reactive than IgG antibodies to “normal” antigens? If so, why? For this discussion, it is relevant to compare not only the structural relation between the two allergens in question, but also the relatedness to the human equivalent (if any) and how the latter influences the immune repertoire. Thirdly, prediction of IgE cross-reactivity is of interest in relation to allergic reactivity to novel foods. Cross-reactivity is a property defined by individual antibodies to individual allergens. Quantitative information (including relative affinity) is required on cross-reactivity in the allergic population and with specific allergens (rather than with whole extracts). Such information is still scarce, but with the increasing availability of purified (usually recombinant) allergens, such quantitative information will soon start to accumulate. It is expected that similarity in short stretches of the linear amino-acid sequence is unlikely to result in relevant cross-reactivity between two proteins unless there is similarity in the protein fold.

Notes: Background High levels of allergen-specific IgG have been associated with clinical efficacy in immunotherapy studies, but whether this antibody isotype is associated with clinical tolerance in the setting of environmental exposure remains unclear.Objective To determine if mouse allergen-specific IgG (mIgG) and IgG4 (mIgG4) levels are associated with mouse-related symptoms among IgE-sensitized laboratory workers.Methods Fifty-eight workers with either skin test or serologic evidence of IgE-mediated mouse sensitization were studied. Symptom data were obtained by a questionnaire. Serum levels of mouse-specific IgG, IgG4, and IgE were quantified by a solid-phase antigen-binding assay (IgG) and RAST (IgG4 and IgE), and the relationships between mouse-specific serologic responses and mouse-related symptoms were analysed.Results Twenty-three (39.7%) participants reported mouse-related symptoms. Mouse-specific IgG and IgG4 levels were not associated with mouse-related symptoms among the study population as a whole. Among the 29 (50%) participants with detectable mouse-specific IgE (mIgE), higher mouse-specific IgG and IgG4 levels were associated with a decreased risk of symptoms, after adjusting for mIgE level (odds ratio (OR) 0.3, 95% confidence interval (CI): 0.1–1.4, and OR 0.3, 95% CI: 0.04–2.6, respectively). Higher levels of mIgG and mIgG4 remained associated with a decreased risk of symptoms after additional adjustment for sex and handling of mice (OR 0.1, 95% CI: 0.02–0.7, and OR 0.2, 95% CI: 0.02–2.1, respectively). Higher mIgG : IgE and mIgG4 : IgE ratios were also associated with a decreased risk of symptoms after adjusting for these confounders (OR 0.1, 95% CI: 0.02–0.7, and OR 0.2, 95% CI: 0.02–0.92, respectively).Conclusion Among workers with detectable mIgE, higher mIgG and mIgG4 levels are associated with a decreased risk of mouse-related symptoms. High serum levels of mIgG or mIgG4 may be markers for clinical tolerance among laboratory mouse workers with detectable mIgE, but these findings need to be confirmed in larger, prospective studies.

Notes: Background It has been suggested that the period immediately after birth is a sensitive period for the development of atopic disease.Objective We investigated whether birth characteristics and environmental factors are associated with the development of atopic dermatitis in the first year of life.Methods Seventy-six children with and 228 without atopic dermatitis, all children of mothers with respiratory allergy or asthma (PIAMA birth cohort study) were included in the study. Atopic dermatitis was defined as a positive history of an itchy skin condition with at least two of the following characteristics: visible dermatitis, history of outer arms/leg involvement, or general dry skin. Multiple logistic regression analysis was performed to study the independent effects of various risk factors.Results A birth weight 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:09547894:CEA1751:ges" location="ges.gif"/〉4000 g compared to 3000–4000 g was a significant risk factor for atopic dermatitis (odds ratio (OR)=2.4; 95% CI: 1.1–5.1) as was day care attendance (OR=2.9; 95% CI: 1.5–5.9). Exclusive breastfeeding in the first 3 months was negatively associated with atopic dermatitis (OR=0.6; 95% CI: 0.3–1.2), especially with visible dermatitis (OR=0.4; 95% CI: 0.2–1.0). Gender, gestational age, the presence of siblings or pets, and parental smoking were not significantly associated with atopic dermatitis.Conclusion This study shows that a high birth weight and day care attendance increase the risk of atopic dermatitis in the first year of life, while exclusive breastfeeding is a protective factor when dermatitis is found on inspection.

Notes: Background It is presently unknown which factors determine the occurrence and persistence of asthma in house dust mite-allergic individuals. The level of allergen-specific IgE antibodies does not seem to be decisive for asthmatic symptoms. Moreover, levels of exposure to mite allergens do not seem to differ significantly between asthmatic and non-asthmatics individuals.Aim It was hypothesized that the presence or absence of asthmatic symptoms in house dust mite-allergic patients is associated with quantitative or qualitative differences in the cellular bronchial inflammatory response during the late phase of the allergic reaction. This hypothesis was tested in the bronchial allergen challenge model.Material and methods Whole lung challenges with house dust mite extract were performed in 52 house dust mite-allergic subjects, of whom 26 had asthma and 26 had perennial rhinitis without asthmatic symptoms. Primary outcomes were parameters for bronchial inflammation in serial samples of induced sputum (cell differentials, eosinophil cationic protein (ECP), interleukin-8 (IL-8), myeloperoxydase (MPO)). In addition, lung function, non-specific bronchial hyper- responsiveness and serial blood samples (eosinophils and IL-5) were analysed.Results At baseline sputum eosinophils and ECP were similar in both groups but neutrophils and IL-8 were higher in asthmatics. The early bronchoconstriction after allergen challenge was similar in asthma and non-asthmatic rhinitis (median decrease in FEV1: asthma − 31.7% vs. non-asthmatics − 29.1%, P 〉 0.1). The late phase bronchoconstriction was significantly greater in asthma (median decrease in FEV1: asthma − 27.6% vs. non-asthmatics − 18.9%, P = 0.02). Induction of bronchial hyper-responsiveness was similar in both groups. Bronchial allergen challenge elicited significant increases in sputum eosinophils and ECP, which were indistinguishable for both groups (P 〉 0.1 and P = 0.07, respectively). In contrast, higher numbers of neutrophils persisted in asthma 24 h after challenge and were accompanied by significant increases in IL-8 and MPO, which were absent in non-asthmatics (difference between groups P = 0.007 and P = 0.05, respectively).Conclusion Allergen challenge induced very similar increases in eosinophils and ECP in induced sputum in allergic asthmatics and in allergic non-asthmatic patients. The difference in bronchial inflammation between asthma and non-asthmatic rhinitis appeared to be more closely related to indices for neutrophilic inflammation.

Notes: Background The models for exposure to house dust in research and clinical practice are selected with respect to their role in IgE-mediated immediate hypersensitivity. The use of isolated major allergens instead of complex allergen extracts is becoming increasingly popular as it offers some important advantages for quantitative measures in diagnosis and research.Objective To compare house dust mite extract and isolated mite major allergens with respect to their ability to induce early and late asthmatic responses and bronchial hyperreactivity.Methods Bronchial responses to house dust mite (HDM, Dermatophagoides pteronyssinus) extract and isolated major allergens from HDM (Der p 1 and Der p 2) were compared in a double-blind, randomized, cross-over study in 20 patients with mild to moderate asthma who were allergic to HDM. Allergen was titrated to a standardized early asthmatic response. Bronchial hyper-responsiveness to histamine (PC20histamine) was determined before and after allergen inhalation to assess allergen-induced bronchial hyper-responsiveness and IL-5 was measured in serum. In addition, the allergens were applied in intracutaneous skin tests and activation of basophil leucocytes and proliferation of peripheral blood mononuclear cells was tested in vitro.Results After a similar early asthmatic response (mean Δforced expiratory volume in 1 s (FEV1),max−29.4 (SD 7.2) vs. −33.1 (8.6) %; mean difference 3.6 (95% CI −0.9 to 8.2) %), the late asthmatic response (mean ΔFEV1,max−45.9 (21.9) vs. −32.7 (22.3) %; mean difference 13.2 (3.8–22.3) %), the degree of allergen-induced bronchial hyper-responsiveness (mean ΔPC20histamine, 1.8 (1.0) vs. 1.2 (0.9) doubling dose; mean difference 0.6 (0.2–1.1) doubling dose) and serum IL-5 at 6 h were found to be significantly higher after bronchial challenge with HDM extract than after challenge with an isolated HDM major allergen. Likewise, there was an increased late skin reaction with HDM compared with isolated major allergen after a similar early skin reaction.Conclusion Constituents of HDM extract, other than Der p 1 or Der p 2, with no significant influence on the IgE-mediated early asthmatic response contribute significantly to the allergen-induced late asthmatic response and bronchial hyper-reactivity.

Notes: Recombinant Der p 2, expressed in yeast, lacked reactivity with 5 monoclonal antibodies against natural Der p 2.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveThe aim of this study was to investigate whether the lack of reactivity with recombinant Der p 2 can be explained by the existence of isoforms.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsBy site-directed mutagenesis three recombinant isoforms of Der p 2 were produced. Reactivity with monoclonal antibodies and human IgE was analysed by means of RAST and RAST-inhibition.〈section xml:id="abs1-4"〉〈title type="main"〉ResultsAll five monoclonals that lacked reactivity with the originally selected isoform, showed reactivity upon replacement of aspartic acid by asparagine at position 114. The other two substitutions (at position 26 and 47) had no effect. Binding of human IgE (n = 10) was not significantly influenced by the isogenetic variation at position 114.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionsMonoclonal antibodies raised against natural Der p 2 can sometimes discriminate between different isoforms, allowing the study of the natural occurrence of isoforms. For application in allergen-measurement assays, non-discriminating monoclonal antibodies should be selected.

Notes: Two assays have been developed to measure arthropod levels in house dust. The first assay measures silverfish antigens. The second assay measures invertebrate tropo-myosin and gives a global assessment of the level of atthropod-derived material. These assays and a Der p 1 and Der p 2 assay were used to analyse 53 dust samples. In most dust samples the ratio of tropomyosin/Der p 2 was higher than in mite body extract, indicating that the assay measures other arthropods besides mites. Silverfish antigen was detectable in most of the dust samples. In many homes in which the inhabitants were unaware of the presence of silverfish, silverfish antigen was detectable. Therefore for information on exposure an immunochemical analysis is superior to a questionnaire.

Notes: IgE- and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fei d) I. Recombinanl chain 1 and chain 2 were coupled to sepharose and tested in IgE- and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE- and IgG4 binding was found. The fraction of Pel d I-specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for lgG4 (P= 0.05). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P= 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Pel d I, compared with IgG4.

Notes: Leucocytes from normal non-allergic donors were passively sensitized with sixty-five atopic sera that contained IgE directed against house dust mite (HDM) or guinea-pig dander (GPD). In no serum was there any IgE with an abnormal basophil anaphylactic-sensitizing activity.In sera from patients hypersensitive to GPD, both GPD-specific IgE and IgG4 were measured. A significant correlation was found between the GPD-IgE RAST and the basophil-sensitizing capacity of GPD-positive sera, but no correlation was found between this sensitizing capacity and the GPD-lgG4 RAST.Leucocytes were passively sensitized with serum mixtures that contained the same amounts of total and HDM-specific IgE; these mixtures differed as to sensitizing activity. It is unclear whether this variation is due to differences in IgE or to influences of other factors in serum.