Allergen extraction: Factors influencing immunogenicity and sensitivity of immunoassays.

Food allergy prevalence is increasing worldwide, therefore there is a high demand for reliable tests to correctly diagnose this disease. Knowledge of proteins allergenicity and how they react both in the body and in diagnostic tests is necessary to adequately assess the potential immunogenicity of both natural foods and those produced through biotechnological processes. Thus, our aim was to analyze the factors that influence the protein extraction of foods in terms of, immunogenicity and immunoassays sensitivity. Peanut proteins were extracted using four distinct extraction buffers with different pH values (physiological saline, tris buffer, borate buffer with and without ß-mercaptoethanol), the protein concentration was determined by the Lowry method and polyacrylamide electrophoresis (SDS-PAGE) was used to compare the protein profile of each extract. The immunogenicity of each extract was verified by sensitizing two mouse strains (Balb/c and C57Bl/6) with a solution containing 100 µg of the extracted proteins and was determined by ELISA. Results show that extraction with the distinct buffers resulted in protein solutions with different yields and profiles. The immunogenicity of the different extracts also demonstrated distinct patterns that varied depending on the extraction methods, mouse strain and in vitro test. Immunoreactivity varied in accordance with the protein extract used to coat the microtitration plates. In conclusion, the protein profile in the extracts is critically influenced by the salt composition and pH of the extraction buffers, this in turn influences both in vivo immunogenicity and in vitro immunoreactivity.

Induction of food tolerance is dependent on intestinal inflammatory state.

Food allergies are usually managed by food avoidance. Hidden allergens in food, due to cross-contamination and/or allergenic additives added during production, place an important concern in today's increasing food allergy cases worldwide. Previous studies showed that the introduction of unacquainted food components, in an inflamed intestine, results in sensitization to this food. Thus, our aim was to evaluate the kinetics of multiple food allergy induction. Adult male C57BL/6 mice were divided into five groups, four of which were submitted to an intestinal inflammation induction protocol to peanuts. Egg white (OVA) diluted 1:5 v/v in distilled water was instilled by gavage 6h-before (PRIOR), concomitant (AT) and 6h-after (DURING) the onset of the peanut challenge diet. Positive control (POS CONT) and NEG CONT received saline per gavage. Finally, animals were challenged with subcutaneous injections of OVA. Results showed no changes in diet intake were observed. Anti-OVA polyisotypic IgG antibody titers significantly increased in AT. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in AT. Histomorphometrically, AT and DURING were classified as Infiltrative and Partial Destruction stages. PRIOR was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. Together, our results confirm that the introduction of unfamiliar food only a few hours before the initiation of a gut inflammation process is able to induce oral tolerance, however the introduction of a dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.

Comparison of two techniques for a comprehensive gut histopathological analysis: Swiss Roll versus Intestine Strips.

Assessing the gut mucosa milieu is important to grade the inflammatory process in conditions such as food hypersensitivity, allergy, gut parasitosis, etc. However, the gastrointestinal tract comprises a challenging system to evaluate, due to its thin tubular structure and mucosa, which suffer fast autolysis after death. Irrespective of the preferred inflammatory score system, it is important to choose the technique that will render the best tissue analysis. Thus, our aim was to compare two of the most frequently used methods to collect, process and analyze gut segments, the Swiss Roll and the Intestinal Strips. Normal C57Bl/6 mice were randomly assigned to Rolls or Strips group. After an overdose of anesthetics, segments of the duodenum, jejunum and ileum were collected and prepared accordingly for histological processing and analysis. Our results show the villi in the Rolls tend to be shorter and wider than those in the Strips in the duodenum and jejunum but not the ileum. No significant differences were observed in intra-epithelial lymphocytes and goblet cells counts. Finally, we staged each segment using our histomorphometric classification system, which revealed that although all animals presented a normal intestinal mucosa, those assigned to the Rolls group had their mucosa staged in the Infiltrative Stage while the Strips group had their mucosa staged as Normal. In conclusion, Swiss Rolls might be desirable for a wider assessment of the intestine, as it allows large segments to be analyzed at once, while Strips are better suited when detailed evaluation of each villus is intended.