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Notes: Windelborg Nielsen B, Lind P, Hansen B, Reimert CM, Nansen P, Schiotz PO. Immune responses to nematode exoantigens: sensitizing antibodies and basophil histamine release.High levels of IgE and eosinophilia are found in both allergy and helminth infections, but allergic symptoms are rare in naturally acquired helminth infections. The interrelation of specific IgE antibodies and in vitro basophil histamine release (HR) induced by exoantigens from the larval stages (L-2/L3) of the nematodes Toxocara canis and Ascaris suum was examined in 148 patients visiting an outpatient clinic for parasitic diseases. The antigen sensitivity of the basophils was found to be dependent not only on the absolute amount of antigen-specific IgE present in patient plasma, but also on the ratio between specific and total IgE. Thus, large HR was observed in some patients in response to helminth antigens despite low levels of both specific and total IgE content in plasma. Patients with eosinophilia showed greater IgE-mediated HR than the other patients examined. In contrast, only five patients showed HR after challenge with anti-IgG4, despite the presence of high levels of antigen-specific IgG4 and IgGl in all patients showing specific IgE antibodies.

Notes: Nine adult patients suffering from severe atopic dermatitis (AD) and increased total serum IgE were treated with recombinant human interferon-alpha 2A (Roferon-AR; IFN-α-2A) 3 × 106 units daily for 21 d to study the effect upon serum IgE, basophil histamine release (HR), eosinophil-derived proteins in serum, and clinical symptoms. The skin disease was so severe that all patients needed topical treatment with glucocorticosteroid cream. Changes were not observed in total serum IgE or in eosinophil-derived proteins, the latter being increased in six of nine patients. A significant reduction in basophil HR was found in six of nine patients after anti-IgE and concanavalin A (Con A) stimulation, but not after A23187 stimulation. The clinical skin score was reduced in eight of nine patients at the end of therapy, but disease activity returned to pretreatment levels within 3 weeks despite continued topical treatment. rIFN-α-2A was well tolerated with few clinical side-effects, and all patients completed the study. The short-term therapy using IFN-α-2A neither brought a sustained clinical remission nor reduced total serum IgE or eosinophil-derived proteins. However, a significant reduction in IgE-receptor-mediated basophil HR was observed in six of nine patients.

Notes: Background: The diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) patients may be difficult to establish because ABPA shares many characteristics with coexisting atopy or other lung infections in these patients. This study aimed to evaluate the sensitivity and specificity of various paraclinical parameters in the diagnosis of ABPA in patients with CF. Methods: Accumulated data from a 5-year period in 238 CF patients were used to divide patients into two groups designated the ABPA group (n=26) and the non-ABPA group (n=35). Patients in both groups were colonized with Aspergillus fumigatus, but only the ABPA group consistently demonstrated specific IgE antibodies and specific precipitins. Patients without A. fumigatus colonization were not assigned to either of these groups (n=177). By this selection as the true diagnosis, 10 patients were selected from the ABPA group and 10 patients from the non-ABPA group. Results: The groups were comparable as to age, sex, lung function (P=0.6), and presence of chronic Pseudomonas aeruginosa infection (P〉0.1). No significant difference between the groups in unspecific atopic parameters such as eosinophil count (P=0.9) or eosinophil cationic protein (ECP) in sputum, plasma, or serum (P=0.9, P=0.59, and P=0.9, respectively) was demonstrated. Total IgE was significantly higher in the ABPA group (P〈0.01). The groups were comparable in skin prick test (SPT) positivity to a standard panel of aeroallergens (pollen, dander, molds, and mites) (P〉0.2). Statistically significantly higher levels in the ABPA group were demonstrated in specific IgE to Af. (P〈0.05), SPT positivity to Af. (P〈0.02), and Af. precipitins (P〈0.05). Histamine release (HR) to Af. tended to be higher (P=0.075) in the ABPA group. Specific IgE to Af. was determined by Magic Lite (ML), CAP, and Maxisorp (in-house RAST). The CAP level was one to two classes higher than the ML level; however, the results were comparable (r=0.66, P〈0.005). IgE to Af. measured by CAP was the test which offered the highest positive predictive value (PPV) and negative predictive value (NPV). Optimal diagnostic cutoff levels for the diagnosis of ABPA were determined: class 2 for HR to Af., 200 kU/l for total IgE, and 3.5 (titer) for precipitating antibodies to Af., and class 2 for IgE to Af. (by CAP System). Conclusions: Unspecific atopy markers were of limited value for the diagnosis of ABPA. Patients with ABPA do not seem to be more atopic to other aeroallergens than non-ABPA patients. The most valid parameters for the diagnosis of ABPA in CF are SPT to Af., IgE to Af. in combination with precipitating antibodies to Af., and/or total IgE.

Notes: A simple microtiter assay for eosinophil activation is described. The assay used 1000–4000 eosinophils/microtiter well, and the design allows for a separate or simultaneous quantitative assessment of eosinophil adhension to protein-coated microtiter wells and degranulation after stimulation with eosinophil-activating factors. The number of adherent eosinophils is quantified indirectly by expressing the amount of eosinophil cationic protein (ECP) extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the cells added to the wells. Degranulation is quantified in the same way by expressing the amount of ECP or eosinophil protein X (EPX) released to the supernatant in percentage of the total amount of ECP or EPX. Known eosinophil-activating agents such as PMA, interleukin (IL)-3, IL-5, GM-CSF, and platelet-activating factor (PAF) all induced a time- and does-dependent adhension to albumin-coated wells, whereas L_PAF did not. KInetic experiments showed that most adhesion occurred within the first 15–30 min, reaching a plateau around 60 min. After prolonged incubation, a decline in adhesion was detected. GM-CSF-induced adhesion was completely inhibited by incubation with monoclonal antibodies directed against the common β2-chain (CD18) of the LFA-1, Mac-1, p150,95 complexes. Monoclonal antibodies against CD11a, CD11b, CD11c, VLA-4 ALFA, ICAM-1, VCAM-1, Sialyl-Lem, ELAM-1, and LECAM had no inhibitory effect. Simultaneous monitoring of adhesion and degranulation after stimulation of eosinophils in albumincoated wells with either PMA or GM-CSF showed that adhesion always preceded degranulation. Replacing the albumin coating of the microtiter wells with IgG or secretory IgA augmented both the spontaneous and the PMA- or GM-CSF-stimulated responses. In conclusion, the assay allows dynamic evaluation of eosinophil activationa dn can be used to assess soluble eosinophil-activating factors as well as to study eosinophil activation by solidphase-bound proteins.

Notes: Abstract. Objectives: Potentially harmful leukocyte- and platelet-derived bioactive substances are accumulated extracellularly during storage of different blood products. Therefore, we studied the effect of prestorage leukocyte filtration on concentrations of bioactive substances in whole blood (WB) and saline-adenine-glucose-mannitol (SAGM) erythrocyte suspension during storage.¶Methods: Ten units of WB and 10 units of SAGM blood from 20 blood donors were stored at +4 °C for 24h. Subsequently, half of every unit was leukocyte-reduced by filtration. The 40 half units (20 filtered and 20 unfiltered) were stored at +4 °C for further 34 days. Samples were collected from all 40 half blood units on day 1, 21 and 35. Total content and extracellular concentration of myeloperoxidase (MPO), eosinophil cationic protein (ECP), histamine and plasminogen activator inhibitor-1 (PAI-1) was analysed by ELISA or RIA methods.¶Results: In unfiltered WB, the total content of all 4 substances decreased during storage, and extracellular concentrations increased significantly and storage time dependently. Similarly, this was also seen with MPO and ECP in unfiltered SAGM blood. Prestorage filtration of WB resulted in a significant reduction of total content and of extracellular concentrations of all 4 substances as well. Additionally, storage time dependent extracellular accumulation was prevented for all substances. Prestorage filtration of SAGM blood significantly reduced total content and extracellular concentrations of MPO and ECP and prevented storage time dependent extracellular accumulation. Filtered SAGM blood contained significantly lower concentrations of all analysed substances compared to filtered WB.¶Conclusion: Prestorage leukocyte filtration reduces total content of leukocyte- and platelet-derived bioactive substances and prevents the storage time dependent extracellular accumulation of these substances in WB and the partly accumulation in SAGM blood.¶