Mutational analysis of immunoglobulin E-binding epitopes of β-casein and β-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera

BACKGROUND: For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding. OBJECTIVE: To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG). METHODS: Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled. RESULTS: Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition. CONCLUSION: These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.     

Genetic control of serum IgE levels: a study of low molecular weight protein tyrosine phosphatase

Protein tyrosine phosphatases (PTPases) have recently been recognized as important modulators of various signal transduction pathways in immune cells. Genetic polymorphisms have been described in genes codifying for members of this family of enzymes, and the genetics of PTPases is predicted to play an important role in the etiology of immune diseases and of their clinical variability. The low molecular weight protein tyrosine phosphatase (ACP1 or LMPTP) is one of the few PTPases with a known genetic polymorphism, and has been proposed to be associated with atopic dermatitis in a small sample from an Italian population. In this paper we describe the association of the ACP1 polymorphism with total IgE levels in two independent samples from English and Italian populations. In both the samples the mean value of serum IgE is lower among subjects carrying the BC genotype than in other ACP1 genotypes. The BC genotype is associated with the highest total ACP1 enzymatic activity. Our data suggest that one or both of the ACP1 isoforms exert an inhibitory role on some signal transduction pathway relevant for IgE hyperproduction.     

Sensitization and lung function in workers occupationally exposed to natural thickening products

BACKGROUND: Three workers engaged in the manufacture of natural thickener products (Cassia spp., guar, and tamarind flour) were occupationally sensitized to Cassia spp. Therefore, a cross-sectional study on the prevalence of sensitization among and respiratory health of the employees of this plant was conducted. METHODS AND RESULTS: Sixty-two workers (36 with high, 26 with low exposure) participated in the survey. Skin prick tests and specific IgE tests with extracts of these components revealed that 11.3% were sensitized to Cassia spp. and 9.7% to at least one species of storage mites, with a significantly higher portion of atopic subjects in the sensitized group. Overall, 55% of the subjects reported work-related symptoms (upper and lower airways, eyes, or skin). FVC % pred. was significantly lower in highly exposed workers, while RV % pred. and RV%TLC % pred. were significantly higher in this group. In the multivariate model, sensitization was not a risk factor for impairment of lung function. CONCLUSIONS: Dust exposure to flours may not only cause allergic sensitization but also induce chronic changes in lung function.     

Potentiation of in vitro synthesis of human IgE by cyclosporin A (CsA).

In this study, we investigated the modulatory effects of CsA on in vitro synthesis of IgE, IgG1 and IgG4 by human peripheral blood mononuclear cells (PBMC). In contrast to its known immunosuppressive effect, we have demonstrated that a low dose of CsA (10(-7) M, 120 ng/ml) potentiated IgE production by up to 40-fold (i.e. from 33 +/- 4.5 to 1346 +/- 290 ng/ml). This potentiation was specific for IgE since no such effect was demonstrable with IgG1 and IgG4. Potentiation of IgE synthesis by CsA in the PBMC cultures was partly due to CsA acting on T cells, as demonstrated by the addition of CsA-treated T cells to T cell-depleted cultures. However, potentiation was also demonstrable in a T cell-depleted, anti-CD40-stimulated culture (four-fold increase from 400 +/- 48 to 1606 +/- 127 ng/ml). Our data therefore suggest that there are at least two mechanisms for CsA-induced potentiation of IgE synthesis, one T cell-dependent and the other T cell-independent. The clinical implications of these findings are discussed with regard to the use of CsA in the treatment of Th2-mediated diseases.     

Whole organisms and purified cell walls compared as immunosorbents for the detection of IgE antibodies to Staphylococcus aureus

We have developed an immunoradiometric assay for IgE antibodies to Staphylococcus aureus (Staph IgE-Ab) which uses purified cell walls (PCW) from the Wood 46 strain of S. aureus as an immunosorbent. We compared Wood 46 PCW and whole organisms (WO) as immunosorbents for Staph IgE-Ab by performing tests on sera from patients with atopic dermatitis (AD) or the hyperimmunoglobulin E syndrome (hyper IgE syndrome). Sera with Staph IgE-Ab demonstrated dose-dependent binding to PCW and WO, but the ratio of specific to non-specific binding was much greater with PCW. Mean non-specific binding to WO was greater than to PCW, 5% versus 2%; and non-specific binding to WO varied directly with the serum concentration of IgE. Results of tests on patients' sera indicated that PCW are required in screening assays for Staph IgE-Ab to avoid false positive results caused by high levels of non-specific binding to WO.     

Total serum IgD is increased in atopic subjects

We have developed a sandwich-type ELISA system for measuring total IgD levels in the serum of atopics and non-atopic controls. In this ELISA system, affinity purified goat anti-human IgD was used for capture. Results were superior to those obtained with monoclonal anti-human IgD antibody. No cross-reactivity could be demonstrated to IgG, IgM, IgA or IgE. The assay showed minimal non-specific binding even with initial serum dilutions of 1:2. The results obtained were reproducible among replicates (Mean CV +/- SEM = 0.03 +/- 0.002; n = 251), between dilutions (CV = 0.08 +/- 0.006; n = 108), and between assays (CV = 0.05 +/- 0.12; n = 5). We used routine radioimmunoassay for measuring total serum IgE. Using these assays total serum IgD and IgE levels were measured in 75 atopic patients and 33 normal subjects. None of the atopics had recent immunotherapy. As expected, the geometric mean serum IgE in atopics (373 ng/ml) was significantly higher than that in normal subjects (49 ng/ml) (P less than 0.01). However, geometric mean serum IgD was also significantly higher in atopics (20.3 micrograms/ml) than that in normal subjects (8.4 micrograms/ml) (P less than 0.02). In both atopic and normal groups, mean serum IgD level did not differ significantly on the bases of age, sex or asthmatic status. Furthermore, total serum IgD was not significantly correlated with total serum IgE (r = 0.14; P = 0.14; n = 108), indicating that immunoregulatory control of the basal levels of the two isotypes is not linked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens

BACKGROUND: The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed. METHODS: A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison. RESULTS: Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE. CONCLUSIONS: The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.     

Co-stimulation of T cells via CD28 inhibits human IgE production; reversal by pertussis toxin.

In lymphocyte cultures, IgE production was achieved by stimulating T cells with anti-CD2 and IL-2. Here we show that anti-CD28, in the presence or absence of IL-2, reduces this IgE production approximately 10-fold. This inhibition of IgE production was almost completely reversed by Pertussis toxin (PT). PT had no effect on IgE production when the cells were stimulated in the absence of anti-CD28. No major effects of PT were found on IgM production. PT had no effect on purified B cells, stimulated with IL-4 and anti-CD40. In the presence of saturating amounts of rIL-4 similar results were obtained, albeit the absolute amounts of IgE produced were higher in all situations. Furthermore, PT-induced IgE production was still dependent on IL-4, as was evident from experiments in which anti-IL-4 was added to the culture. The IgE enhancing effect was dependent on the adenosine diphosphate (ADP)-ribosyltransferase activity of PT, because a mutant molecule lacking this activity was not able to restore anti-CD28-induced inhibition of IgE synthesis. Thus, we show that co-stimulation with anti-CD28 causes an inhibition of T cell-dependent IgE production by B cells, which inhibition can be specifically overcome by PT. An analysis of the molecular pathways underlying this phenomenon may contribute to our understanding of the regulation of IgE synthesis in (patho)physiological conditions.