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Gut-associated intestinal lymphoid tissue, the largest secondary lymphoid organ in the human body, constantly samples antigens from the gut lumen, presenting as a default response the activation of TCD4 FOXP3 regulatory T cells that secrete a profile of anti-inflammatory cytokines maintaining gut homeostasis denominated from an immunological perspective as mucosal tolerance. However, when antigens are sampled in an inflammatory setting, the immune response may either be protective, in the case of harmful pathogens, or cause further inflammatory reactions as in food allergy, inflammatory bowel diseases, coeliac disease or food protein-induced enterocolitis syndrome. Therefore, there is a need for accurate and consistent experimental models. However, a drawback in comparing these models is the lack of a classification system similar to that which is already used for humans. Thus, the aim of this work was to propose a classification system of the small intestinal histomorphology in experimental mice. To do this we used a mouse antigen-specific gut inflammation model developed by our research group. Duodenum sections stained with haematoxylin-eosin and Alcian blue were scanned using the APERIO scanning system and analysed with the ImageScope software. The evaluated parameters were villus area, villus height and width, enterocyte count, mononuclear intra-epithelial leucocyte and goblet cell counts, and architectural and cellular ratios. Food-sensitized animals challenged with a diet containing the corresponding food allergen presented, as for humans, time-dependent shortened and widened villi accompanied by leucocyte infiltrate and loss of goblet cells. With these data, we were able to establish a classification system for experimental intestinal inflammation in mice thus permitting better comparisons among and between experiments than has been possible previously.

Abstract. Virtual microscopy is currently widely used for various purposes, such as teaching, archiving, collaborations and research. Although the cost of this technique has reduced, it continues to be expensive for the majority of laboratories in developing countries. The Graduate Program in Pathology at the Federal Fluminense University (Niterói, Brazil) has acquired equipment for virtual microscopy. However, this novel method faced prejudice, as students and technicians were skeptical about its reliability. Thus, the aim of the current study was to evaluate whether virtual microscopy is a reliable method of analysis for our research. Thus, a mouse gut inflammation model developed by our research group was used in the present study. Analysis was performed using optical microscopy and digital imaging using the APERIO scanning system and the ImageScope ® software. Intestinal epithelial cells (IECs), intra epithelial leucocytes (IEL), and villi number and area were evaluated. No significant differences were observed in villi number, IEC and IEL; however, the villi area was significantly smaller when measured using the computer. Thus, the present study indicates that virtual microscopy is a trustworthy method for research purposes.

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Because of the high social impact of Food allergy, it is of great importance to correctly diagnose this disease using reliable tests. Knowledge of the allergenicity properties of proteins, how they react 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 (physiological saline, tris buffer, borate buffer with and without β-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 C57/BL6) with solution containing 100μg of the extracted proteins and 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 to 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.

BACKGROUND: Food allergies are usually managed by food avoidance. Hidden allergens in food, due to crosscontamination and/or allergenic additives added during production, place an important concern in today's increasing food allergy cases worldwide. Previous studies showed that introduction of new 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. METHODS: 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 (EXP-1), concomitant (EXP-2) and 6h-after (EXP-3) 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: No changes in diet intake were observed. Anti-OVA total IgG antibody titers significantly increased in EXP-2. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in EXP-2. Histomorphometrically, EXP-2 and EXP-3 were classified as Infiltrative and Partial Destruction stages. EXP-1 was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. CONCLUSION: The introduction of a new food only a few hours before the initiation of a gut inflammation is able to induce oral tolerance, however the introduction of a new dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.

BACKGROUND: 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 introduction of new 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. METHODS: 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 (EXP-1), concomitant (EXP-2) and 6h-after (EXP-3) 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: No changes in diet intake were observed. Anti-OVA total IgG antibody titers significantly increased in EXP-2. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in EXP-2. Histomorphometrically, EXP-2 and EXP-3 were classified as Infiltrative and Partial Destruction stages. EXP-1 was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. CONCLUSION: The introduction of a new food only a few hours before the initiation of a gut inflammation is able to induce oral tolerance, however the introduction of a new dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.