Detection of Serum-Specific IgE by Fluoro-Enzyme Immunoassay for Diagnosing Type I Hypersensitivity Reactions to Penicillins

Diagnosis of type I hypersensitivity reactions (IgE-mediated reactions) to penicillins is based on clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP^® (Thermo-Fisher, Waltham, MA, USA) is the most widely used commercial method for detecting drug-specific IgE (sIgE). In this study, we aimed to analyze the utility of ImmunoCAP^® for detecting sIgE to penicillin G (PG) and amoxicillin (AX) in patients with confirmed penicillin allergy. The study includes 139 and 250 patients evaluated in Spain and Italy, respectively. All had experienced type I hypersensitivity reactions to penicillins confirmed by positive STs. Additionally, selective or cross-reactive reactions were confirmed by DPTs in a subgroup of patients for further analysis. Positive ImmunoCAP^® results were 39.6% for PG and/or AX in Spanish subjects and 52.4% in Italian subjects. When only PG or AX sIgE where analyzed, the percentages were 15.1% and 30.4%, respectively, in Spanish patients; and 38.9% and 46% in Italian ones. The analysis of positive STs showed a statistically significant higher percentage of positive STs to PG determinants in Italian patients. False-positive results to PG (16%) were detected in selective AX patients with confirmed PG tolerance. Low and variable sensitivity values observed in a well-defined population with confirmed allergy diagnosis, as well as false-positive results to PG, suggest that ImmunoCAP^® is a diagnostic tool with relevant limitations in the evaluation of subjects with type I hypersensitivity reactions to penicillins.     

Salicylaldehyde Suppresses IgE-Mediated Activation of Mast Cells and Ameliorates Anaphylaxis in Mice

Mast cells (MCs) play key roles in IgE-mediated immunoresponses, including in the protection against parasitic infections and the onset and/or symptoms of allergic diseases. IgE-mediated activation induces MCs to release mediators, including histamine and leukotriene, as an early response, and to produce cytokines as a late phase response. Attempts have been made to identify novel antiallergic compounds from natural materials such as Chinese medicines and food ingredients. We herein screened approximately 60 compounds and identified salicylaldehyde, an aromatic aldehyde isolated from plant essential oils, as an inhibitor of the IgE-mediated activation of MCs. A degranulation assay, flow cytometric analyses, and enzyme-linked immunosorbent assays revealed that salicylaldehyde inhibited the IgE-mediated degranulation and cytokine expression of bone-marrow-derived MCs (BMMCs). The salicylaldehyde treatment reduced the surface expression level of FcεRI, the high affinity receptor for IgE, on BMMCs, and suppressed the IgE-induced phosphorylation of tyrosine residues in intercellular proteins, possibly Lyn, Syk, and Fyn, in BMMCs. We also examined the effects of salicylaldehyde in vivo using passive anaphylaxis mouse models and found that salicylaldehyde administration significantly enhanced the recovery of a reduced body temperature due to systemic anaphylaxis and markedly suppressed ear swelling, footpad swelling, and vascular permeability in cutaneous anaphylaxis.     

Anti-allergic effect of lactic acid bacteria isolated from seed mash used for brewing sake is not dependent on the total IgE levels

Lactic acid bacteria (LAB) in fermented foods have attracted considerable attention recently as treatment options for allergic diseases, the incidence of which has been increasing worldwide. Five strains of LAB isolated from kimoto, the traditional seed mash used for brewing sake, were screened for the ability to suppress IgE-mediated hypersensitivity reaction. Leuconostoc mesenteroides and Lactobacillus sakei, the normal microflora in kimoto, significantly suppressed the reaction, but the contaminant Lactobacillus curvatus did not. Next, we examined the effect of L.?sakei LK-117 on atopic dermatitis in the NC/Nga mouse model. LK-117 supplementation significantly reduced the development of atopic dermatitis-like skin lesions in a manner independent of the IgE plasma levels. In the in vitro intestinal model constructed using the human intestinal epithelial cell line Caco-2 and murine macrophage cell line RAW 264.7, treatment with L.?sakei LK-117, but not L.?curvatus, significantly upregulated TNF-α production from RAW264.7 cells. This result indicated that L.?sakei on the apical side affected the macrophages on the basolateral side, and this organism may have the ability to improve allergy symptoms mediated by the intestinal immune system.     

Prophylactic effect of Lactobacillus oral vaccine expressing a Japanese cedar pollen allergen

Lactic acid bacteria (LAB) represent an attractive delivery vehicle for oral allergy vaccine because of their safety as a food microorganism as well as their potent adjuvant activity triggering anti-allergic immune response. Here, we report the generation of recombinant LAB expressing a major Japanese cedar pollen allergen Cry j 1 (Cry j 1-LAB), and their prophylactic effect in vivo. To facilitate heterologous expression, the codon usage in the Cry j 1 gene was optimized for the host LAB strain Lactobacillus plantarum by the recursive PCR-based exhaustive site-directed mutagenesis. Use of the codon-optimized Cry j 1 cDNA and a lactate dehydrogenase gene fusion system led to a successful production of recombinant Cry j 1 in L. plantarum NCL21. We also found that oral vaccination with the Cry j 1-LAB suppressed allergen-specific IgE response and nasal symptoms in a murine model of cedar pollinosis.     

Peanut polyamines may be non‐allergenic

Polyamines such as putrescine, spermidine and spermine have been implicated in preventing food allergies in early life, but they have also been reported to be able to bind to immunoglobulin E (IgE) antibodies in vitro (ie they are possibly allergenic). The objective of this study was to determine if polyamines bind in vitro to IgE antibodies from a pooled serum of five peanut‐allergic individuals. Levels of polyamines were also determined by ion‐exchange chromatography. Indirect and inhibition enzyme immunosorbent assays (ELISAs) were used to determine the IgE binding or allergenic properties of polyamines. Results showed that, of the three polyamines, spermidine was predominant in peanuts. In both indirect and inhibition ELISAs, IgE antibodies did not bind to the polyamines. It was concluded that polyamines from peanuts, unlike peanut proteins, are not allergenic or an additional threat to patients who are allergic to peanuts. Published in 2005 for SCI by John Wiley & Sons, Ltd.     

Cross-reactivity between peanut and lupin proteins

Peanut-allergic individuals may also react to lupin, which, for this reason, has been included in the EU list of food allergens. Since there is not yet any general consensus on the major allergen/s in lupin, the objective of this investigation was to compare the reactivity of the main lupin proteins towards the IgE of the sera of allergic patients. Both Lupinus albus and Lupinus angustifolius were investigated. ELISA’s, Western blotting and mass spectrometry, including also de novo sequencing of the unknown lupin proteins, were used for identifying the IgE-binding proteins. Significant differences in the protein reactivities were observed. In particular, there was a direct relationship between the level of peanut-specific IgE’s and the cross-reactivity to lupin proteins; also the reactivity of each serum appeared to be unique. Although numerous lupin proteins bind IgE’s, our data suggest a predominant contribution of α-conglutin in the reactivity of both L. albus and L. angustifolius.     

Immunoreactive properties of peptide fractions of cow whey milk proteins after enzymatic hydrolysis

Commercial whey protein concentrate (WPC) was hydrolysed with either Alcalase 2.4 FG (Novo Nordisk), or papain (Sigma) (in one‐step process) or with two enzymes (in two‐step process) to determine the changes in the immunoreactivity of α‐lactalbumin and β‐lactoglobulin. Enzymatic hydrolysis of WPC was performed by pH‐stat method. Hydrolysates were analysed using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, immunoblotting and size‐exclusion chromatography (SE‐HPLC). Immunoreactive properties of peptide fractions separated from the hydrolysates by fast protein liquid chromatography (FPLC) were determined using dot‐immunobinding and enzyme‐linked immunosorbent assay (ELISA) methods. Finally the sensory analysis was used to confirm organoleptic changes resulting from the application of different enzymes. The ‘two‐step’ process was observed to be the most effective however allergenic epitopes were still present, as it was found by ELISA with anti‐α‐la and anti‐β‐lg antibodies. The addition of papain as the second enzyme in the hydrolysis process contributed to the improvement of the sensory properties of WPC hydrolysate as compared with the Alcalase hydrolysate. Alcalase‐papain partially hydrolysated WPC can be found a promising base for production of the tolerogenic formula.     

Reducing the allergenic capacity of peanut extracts and liquid peanut butter by phenolic compounds

Phenolic compounds are known to form soluble and insoluble complexes with proteins. The objective of this study was to determine if phenolics such as caffeic, chlorogenic and ferulic acids form insoluble and irreversible complexes with major peanut allergens, and if such complexation reduces immunoglobulin E (IgE) binding. After adding each of the phenolics to peanut extracts and liquid peanut butter, the soluble materials were analysed by SDS–PAGE and inhibition ELISA. Results showed that addition of the phenolics precipitated most of the major peanut allergens, Ara h 1 and Ara h 2, and that complexation was irreversible. IgE binding was reduced approximately 10- to 16-fold. We concluded that reducing IgE binding by phenolics is feasible. The research, if proven by clinical studies, could lead to the development of less allergenic liquid peanut-based products.     

Allergen sequence databases

A number of specialized databases have been developed to facilitate studies of human allergens. These include molecular databases focused on protein sequences and structures, informational databases focused on clinical, biochemical and epidemiological data related to protein allergens, a database on allergen nomenclature, and other knowledge bases or informational websites that are peripherally-related to research on allergens. Examples of each type of databases are listed and described briefly in this review. Database construction and maintenance and their impact on database quality and usefulness are also discussed.