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Notes: For determination of allergen-specific IgE in cell culture supernatants and other highly diluted IgE preparations a radioallergosorbent test (RAST) based on high adsorption polystyrene test tubes has been developed (“Maxisorp RAST”). Cladosporium herbarum extract was used as a model allergen but timothy grass pollen, house dust mite and dog dander showed similar results. The test showed specificities of both allergen and immunoglobulin isotype and significant correlations (r= 0.67–0.88) with established RAST procedures were found. Based on immunosorbent-purified allergen-specific IgE the estimated sensitivity was within the order of 150-300 pg allergen-specific IgE per ml. The within-assay variation was 4-9% and the inter-assay-variation 17–29%. The Maxisorp RAST is useful as an inhibition assay for quantitating allergenic activity down to 0.1 biological units/ml of allergen extracts.

Notes: The interrelation of in vitro IgE-mediated parameters, i.e. serum-specific IgE (RAST), basophil cell-bound specific IgE, and histamine release from basophil leucocytes was investigated in a 1-year placebo-controlled, double-blind Cladosporium immunotherapy study involving 22 adult asthmatics. The intense and early burst (within 6 weeks of immunotherapy) of serum-specific IgE did not result in a corresponding increased binding of specific IgE molecules to basophils. Cell-bound IgE increased in the Cladosporium season in both groups at the same time as serum levels of specific IgE declined in the Cladosporium group. In the placebo group histamine release from circulating basophils paralleled changes in basophil-bound IgE. In Cladosporium-treated patients, histamine release cell sensitivity after a lag phase (during immunotherapy dose-increase) declined two log steps, i.e. the cells became less responding in spite of a significant increase in cell-bound IgE. To further evaluate the sensitizing capacity of circulating specific IgE, passive sensitization studies were performed using basophils from a single donor. Although sera taken at the maximal IgE-response showed an enhanced capacity of passive sensitization, the ratio between RAST and passive sensitization capacity increased significantly in Cladosporium-treated patients, implying a less than expected sensitization capacity of immunotherapy-induced specific IgE. The lack of active binding of IgE to basophils might be explained by a reduced Fc-affinity of immunotherapy-induced IgE in contrast to the Cladosporium seasonally induced IgE. Regarding the decrease in histamine release in Cladosporium-treated patients in spite of an increased amount of cell-bound specific IgE, immunotherapy may initiate a decrease in mediator releasibility which is not caused by a reduction in the number of Fc-receptors but rather some yet unknown subcellular mechanisms regulating the histamine release. The described changes in IgE-mediated parameters do not seem to be caused by interference with either specific IgG, or IgG4. Changes in histamine release in the Cladosporium, season were the only IgE-mediated parameter significantly related to the graded clinical efficacy of immunotherapy.

Notes: The IgG subclass response was evaulated by a sensitive allergen- and subclass-specific solid phase immunoradiometric assay during a 1-year placebo-controlled, double-blind study of immunotherapy with Cladosporium herbamm in 22 adult asthmatics. The IgG response was mainly restricted to subclasses 1 and 4 but a few patients were IgG2 and IgG3 responders. An intense and early IgG1 response was observed during the first clusters of injection followed by a levelling down of the titer. The IgG4 response had a later onset and showed slowly increasing levels during the 12 months of immunotherapy. The graded clinical efficacy estimated by symptom-medication score was significantly correlated to the preseasonal IgG1 value, with high values indicating a deleterious response of immunotherapy (deterioration of disease activity). Likewise, the fold increase of IgG1 and IgG4 after two clusters of immunotherapy (i.e. after 4 weeks) was significantly related to the clinical outcome, Little or no increase of IgG1 and IgG4 was associated with improvement, i.e. decrease in symptom-medication score. The magnitude of the IgG1 response during the dose-increase phase was directly correlated to the number of systemic side effects. No relation of IgG1, IgG4 or IgG4/IgG1 ratio to changes in the IgE-mediated parameters (skin prick test, bronchial challenge and circulating specific IgE) was observed. Our data, which are based on few patients and only one allergen system, do not support the hypothesis of IgG acting as blocking antibody being the immunologic mechanism of immunotherapy. The association between high IgG4 values and a deleterious efficacy of immunotherapy might be caused by IgG4 acting as sensitizing antibodies. This explanation, however, is opposed by the lack of relation to systemic side effects.

Notes: The IgE-response was evaluated by skin prick test, bronchial provocation test and RAST in a 1-year placebo-controlled double-blind immunotherapy study. Eleven adult asthmatics were treated with a Cladosporium allergen preparation and 11 comparable patients received histamine placebo. The bronchial sensitivity (PC20) decrease 〉 0.5 log step in 8/11 (73%) Cladosporium-treated versus 3/11 (27%) in the placebo group. Corresponding figures for skin prick test sensitivity was 10/11 (91%) and 1/11 (9%) respectively. Circulating IgE showed a temporary boost in the Cladosporium group and then values approaching the pretreatment value. Only minimal and insignificant changes were found in the placebo-treated patients. Changes in IgE-reactivity were not related to allergen dose, clinical efficacy or to the occurrence of side effects. Some interrelation between changes in skin prick test, bronchial provocation test and RAST was found indicating a differentiated effect of immunotherapy on various IgE compartments. In spite of the pathogenetic role of IgE in allergic diseases, changes in IgE-reactivity do not seem directly involved in the mechanisms underlying the clinical efficacy of immunotherapy but might be of importance in a complex interaction with other immunological parameters.

Notes: Dose-response curves of histamine- and allergen-induced wheal areas were evaluated in seven normals (defined as negative skin prick test (SPT) to inhalant allergens and no clinical signs of allergy), seven latent allergies (positive SPT without allergic symptoms), and 20 manifest allergies (positive SPT and allergic symptoms). Three concentrations of histamine HCl (1, 10 and 100 mg/ml) and three 10-fold concentrations of nine inhalant allergens (birch, timothy, mugwort, horse, dog, cat, house dust mite, Cladosporium and Alternaria) in concentrations 1,000 10,000 and 100,000 BU/ml were used and linear regression was performed on the skin reactions. Only tests with an SD% 〈 40%, a log slope 〉 0.1, and a correlation coefficient 〉 0.95 were accepted. In normals a significantly higher concentration of histamine was needed to elicit a wheal reaction of 2 mm2 (end-point) compared with allergies. Likewise, normals had a significantly higher slope i.e. steeper dose-response curve of histamine than manifest allergies. The slope of the allergen-induced wheal area was significantly higher than the histamine slope. No relation between corresponding slope of histamine and allergens was found (Rho = 0.15). The skin sensitivity equivalent to histamine calculated as the allergen concentration eliciting a wheal equal to histamine showed a median increase of 5–6 fold in allergen concentration by a 10-fold increase of histamine concentration. The highest correlation between the wheal area of a single allergen concentration and the skin sensitivity was found for allergen concentration 100,000 BU. Based on these results it is recommended that biological standardization of allergen extracts is based on quantitative skin titration of at least three 10-fold concentrations of both histamine and allergens and the biological activity (HEP unit) re-defined from histamine HCl 10 mg/ml.

Notes: Thirty-two patients with previous systemic allergic reaction to yellow jacket stings were randomly allocated to three groups receiving immunotherapy with different preparations of yellow jacket venom: 1) extract adsorbed to aluminium hydroxide (Alutard®-SQ), 2) Pharmalgen® extract or 3) non-adsorbed extract from Allergologisk Laboratorium (ALK aq.). Regular examinations showed a decrease in skin prick test size in nearly all patients. Specific IgE-antibody (RAST and CRIE scores) showed a similar, but not significant tendency to decrease in all three groups. Specific IgG-antibody increased considerably in the Alutard group only; after 2 years, however, no difference could be detected between the three groups. During dose increase, patients treated with ALK aq. generally had smaller local reactions to injections than those treated with Pharmalgen. Few systemic reactions occurred in all three groups. Nineteen patients treated for 21/2–31/2 years were challenged in-hospital with stings from yellow jackets. No systemic and only minor local reactions occurred. Consequently, with the dose regimens applied all three extracts seem effective even though no common changes in either specific IgE or IgG could be demonstrated.

Notes: Thirty-three adult asthmatic patients suspected of mould allergy were investigated by in vitro and in order to tests in order to establish a specific diagnosis of asthma caused by the mould species Cladosporium. The patients were evaluated by daily symptom scores in the peak Cladosporium season, bronchial provocation test (BPT), skin prick test (SPT), RAST, histamine release from basophil granulocytes (HIST), and crossed radio-immunoelectrophoresis (CRIE), and the results were scored as negative (score 0), equivocal (score 1) or positive (score 2). Based on daily symptom scores and the result of BPT the patients were classified as being manifest allergic (asthma) to Cladosporium (positive allergy), inconclusive or negative. Positive allergy was defined as asthma symptoms oscillating with the spore concentration and a BPT score 2 (positive at allergen concentration 〈 10,000 BU). Negative allergy was defined as NO asthma symptoms and a negative BPT (score 0) and inconclusive in the case of symptoms and BPT sum of score 1-3. According to the classification a final diagnosis (positive or negative) could be established in 85% of the patients. “False positive” tests were found: for BPT in 27%, SPT 18%, RAST 0%, HIST 18%, and CRIE 0%. The corresponding figures for “false negative” were; BPT 0%, SPT 0%, RAST 27%, HIST 18%, and CRIE 23%. The relative risk of being allergic in spite of a negative test result was 0 % for BPT and SPT, and 25-30 % with RAST, HIST, and CRIE. In the case of positive test the risk was 90-100%. Excluding BPT, SPT was found to be the optimal single test to predict/rule out clinical allergy. A stepwise combination of positive SPT and positive RAST was found exclusively in patients clinically evaluated as positive, and does not call for an additional BPT. Using a potent allergenic extract a negative SPT excluded clinically important allergy. The primary conclusion of the study, however, is that the final diagnosis of Cladosporium asthma could not be based on a positive BPT alone (due to “false positive”), but only on a combination of clinical symptoms during the spore season and a positive BPT.

Notes: The reproducibility of repeated quantitative skin prick test titrations was evaluated in seven asthmatic patients tested 2–4 times within 6 weeks. The skin sensitivity was estimated as endpoint titration and histamine equivalent reaction using both 1 and 10 mg/ml histamine dihydrochloride. The histamine equivalent reaction had a significantly higher reproducibility compared to endpoint titration, with a coefficient of variation less than 10% for histamine 10 mg/ml. Further, using this histamine concentration, the deviation in repeated testing was for practical purposes less than 0.5 log step. The same constancy in skin sensitivity was found in one patient tested 3 times and then retested another 3 times 6 months later by a different tester. The results indicate that the quantitative skin prick test and histamine-estimated skin sensitivity have a clinical application in the assessment of pharmacologically or immunotherapy-induced changes in the releasability of skin mast cells.