Angelo Peruffo

IgE binding to soluble and insoluble wheat flour proteins in atopic and non-atopic patients suffering from gastrointestinal symptoms after wheat ingestion

Background The involvement of IgE‐mediated hypersensitivity reactions in the genesis of gastrointestinal symptoms after ingestion of foods containing wheat has been rarely reported.

Effects of wheat germ agglutinin on human gastrointestinal epithelium: Insights from an experimental model of immune/epithelial cell interaction

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only approximately 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

Temperature-dependent decay of wheat germ agglutinin activity and its implications for food processing and analysis

Abstract A recently-described immunoenzymatic (ELISA) method for the quantitative determination of biologically-active wheat germ agglutinin (WGA) in unknown samples has been applied to measure the concentration of active WGA in raw and cooked wheat-derived foodstuffs. The method exploits the binding specificity of WGA to ovalbumin as a first step followed by identification of bound lectin with polyclonal antibodies. Purified WGA was used to obtain calibration curves. Detectable amounts of WGA were found in raw foodstuffs and wheat flours, whilst variable amounts of agglutinin were found in wholemeal pasta probably as a consequence of thermal inactivation during food processing. The thermal gradient gel electrophoresis (TGGE) technique was therefore applied to analyse the thermal stability of WGA. The biological activity of WGA decreased as a function of heating temperatures and time of exposure to thermal treatment in an S-shaped fashion with an inflection point around 65 °C. As a consequence, WGA might represent a biochemical “indicator” allowing one to determine the thermal treatment undergone by wheat-derived foods during processing.

Active soybean lectin in foods: quantitative determination by ELISA using immobilised asialofetuin

A recently described immunoenzymatic method for the quantitative determination of biologically active lectins in unknown samples has been adapted to measure the concentration of active soybean lectin (SBA) in foodstuffs. The method was developed by using purified SBA to build up reference standard curves and to determine the specificity and the sensitivity of the assay. Detectable amounts of soybean lectin were found in soy-based edible products such as hamburger, milk and sprouts and they have been compared to those determined in standard hemagglutination tests. Particular attention has been given to SBA quantitation in soy sprouts as a function of germination time. SBA was not degraded during germination although the hamagglutination activity of sprout extracts rapidly decreased. SBA expression was modulated during the first 10 days of germination, a time period spanning that used to produce soy-sprouts for alimentary purposes.

Egg-matrix for large-scale single-step affinity purification of plant lectins with different carbohydrate specificities.

Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.

Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study

Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freunds adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis.

Fully reversible procedure for silver staining improves densitometry of complex mixtures of biopolymers resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Due to its high sensitivity, silver staining is a widely popular method for the revelation of biopolymers separated by both native and denaturing electrophoresis. A step‐by‐step method for the destaining and restaining of overdeveloped/overloaded silver‐stained bands is described that is applicable to both proteins and nucleic acids. The procedure significantly improves densitometric analysis of gels that have been silver stained with either commercial kits or solutions made in‐house. The method permits reproducible densitometry of silver‐stained gels and allows quantification of both main and minor components in complex mixture of molecules resolved on the same gel slab. All steps may be interrupted and are readily reversible, allowing for facile densitometric analyses and photographic recording under optimized conditions. Furthermore, common artifacts such as differential staining of the two gel surfaces, localized uneven yellow‐ochre background, and the presence of fold marks and fingerprints can be easily removed.