Immunoglobulin-E-binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins.

BACKGROUND: At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. OBJECTIVE: To characterize the epitopes of different wheat kernel allergens: α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. METHODS: Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. RESULTS: Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit. CONCLUSION AND CLINICAL RELEVANCE: The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations.

The crystal structure of oxylipin-conjugated barley LTP1 highlights the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins.

The barley lipid transfer protein (LTP1) adducted by an alpha-ketol, (9-hydroxy-10-oxo-12(Z)-octadecenoic acid) exhibits an unexpected high lipid transfer activity. The crystal structure of this oxylipin-adducted LTP1, (LTP1b) was determined at 1.8A resolution. The covalently bound oxylipin was partly exposed at the surface of the protein and partly buried within the hydrophobic cavity. The structure of the oxylipidated LTP1 emphasizes the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins when compared to the other members of the family. The plasticity of the hydrophobic cavity and increase of its surface hydrophobicity induced by the oxylipin account for the improvement of the lipid transfer activity of LTP1b. These observations open new perspectives to explore the different biological functions of LTPs, including their allergenic properties.

Dehydrodimers of Ferulic Acid in Maize Grain Pericarp and Aleurone: Resistance Factors to Fusarium graminearum.

ABSTRACT The relationship between the primary cell wall phenolic acids, dehydrodimers of ferulic acid, and maize grain resistance to Fusarium graminearum, the causal agent of gibberella ear rot, was investigated. Concentrations of dehydrodimers of ferulic acid were determined in the pericarp and aleurone tissues of five inbreds and two hybrids of varying susceptibility and in a segregating population from a cross between a resistant and susceptible inbred. Significant negative correlations were found between disease severity and diferulic acid content. Even stronger correlations were observed between diferulic acid and the fungal steroid ergosterol, which is an indicator of fungal biomass in infected plant tissue. These results were consistent over two consecutive field seasons, which differed significantly for temperature and rainfall during pollination, the most susceptible stage of ear development. No correlation was found between the levels of these phenolics and deoxynivalenol levels. This is the first report of in vivo evidence that the dehydrodimers of ferulic acid content in pericarp and aleurone tissues may play a role in genotypic resistance of maize to gibberella ear rot.

Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol.

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.

Fungal growth and fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and Spain.

Fungi of the genus Fusarium are common fungal contaminants of maize and are also known to produce mycotoxins. Maize that has been genetically modified to express a Bt endotoxin has been used to study the effect of insect resistance on fungal infection of maize grains by Fusarium species and their related mycotoxins. Maize grain from Bt hybrids and near-isogenic traditional hybrids was collected in France and Spain from the 1999 crop, which was grown under natural conditions. According to the ergosterol level, the fungal biomass formed on Bt maize grain was 4-18 times lower than that on isogenic maize. Fumonisin B(1) grain concentrations ranged from 0.05 to 0.3 ppm for Bt maize and from 0.4 to 9 ppm for isogenic maize. Moderate to low concentrations of trichothecenes and zearalenone were measured on transgenic as well as on non-transgenic maize. Nevertheless, significant differences were obtained in certain regions. The protection of maize plants against insect damage (European corn borer and pink stem borer) through the use of Bt technology seems to be a way to reduce the contamination of maize by Fusarium species and the resultant fumonisins in maize grain grown in France and Spain.

Toxigenic potential of Fusarium culmorum strains isolated from French wheat.

Sixty F. culmorum strains were isolated from wheat grains collected from different wheat-growing areas in France and from different cultivars. The isolates were grown on autoclaved wheat grain to assess their ability to produce trichothecenes and zearalenone. Fungal biomass was evaluated through the ergosterol grain content. All the isolates produced zearalenone (0.39-1660 mg kg(-1)). Thirty-five of the 60 F. culmorum produced nivalenol (0.11-11.7 mg kg(-1)), 12 of 60 produced fusarenone X (0.05-8.42 mg kg(-1)), five of 60 produced 15-acetyldeoxynivalenol (0.48-27.7 mg kg(-1)), 13 of 60 produced 3-acetyldeoxynivalenol (0.07-21.0 mg kg(-1) and 24 of 60 produced deoxynivalenol (0.92-51.9 mg kg(-1)). According to the results, the distribution of the different chemotypes as well as the high and the low mycotoxin-producing Fusarium strains could not be associated to geographical origin.