A histomorphometric classification system for normal and inflamed mouse duodenum-Quali-quantitative approach.

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.

Maternal immunomodulation of the offspring's immunological system.

The mother's and the offspring's immunological system are closely related thus one can influence the other. This hypothesis drove our aim to study the impact of the mother's immunological status over the immunological response of their offspring. For this, female mice tolerant or allergic to peanuts were exposed or not to a challenge diet containing peanuts during the gestation-lactation period (TEP/AEP; TNEP/ANEP, respectively). After weaning the offspring was submitted to the peanut allergy or peanut tolerization protocol and then challenged with a peanut diet. Our results showed that when the offspring is submitted to the allergy induction protocol, they behave differently depending on their mother's immunological status. Offspring born to TEP mothers produced the lowest antibody titters while those born to AEP mothers produced the highest antibody titters compared to mice born to TNEP and ANEP. On the other hand when the offspring was submitted to the tolerization protocol all groups presented low antibody titers with no significant difference between groups, independent of the mothers immunological status and/or contact with peanuts during the gestation-lactation period. The analysis of the histological profile of the offspring correlates well to the serological response. In other words, offspring born to TEP mothers and submitted to the allergy induction protocol presented a normal histological profile, while the offspring born to AEP mothers produced the worst gut inflammation. These results indicate that mothers, exposed to the antigen (by the oral route) during gestation, actively influence the immune response of their offspring. This work sheds some light on the importance of the immunomodulation induced by dietary antigens during gestation and their influence on the immunological response of their offspring. However, more work is needed to elucidate the molecular and cellular components of this regulatory phenomenon.

Therapeutic dose of Ginkgo biloba extract 761 may alter the urine excretion of Wistar rats

Wistar rats (n=20) were divided in two groups: G1 received 2 mg/kg of GBE (Ginkgo biloba extract 761), whereas G2 received the same volume of a sodium chloride solution (0.9%), both for 10 days. After a 7-day interval, the treatment was repeated for 8 days. Urine volume and food and water intake were measured daily during this protocol. Histological assessments were performed. No significant difference (p>0.05) was observed in food and water intake of animals during treatment with GBE. Animals who received GBE had a smaller urine volume and increase of weight with a significance difference (p<0.05) during the first and second exposure period. No histological alteration was observed in tissues, except for the kidney of the experimental group, which revealed a higher concentration of red cells in the glomerulus with a strong staining for Vascular Endothelial Growth Factor (VEGF). The introduction of GBE (therapeutic dose) in health rats may promote alterations in the physiology of the kidney, but no sufficient to modify the glomerulus architecture, including at ultra structural level (electron microscopy).

Evaluation of the acute toxicity of dolabelladienotriol, a potential antiviral from the brown alga Dictyota pfaffii, in BALB/c mice

Dolabelladienotriol is a product extracted from the brown marine alga Dictyota pfaffii from Brazil that has been shown to have antiviral activity and low cytotoxicity. Our studies have evaluated the acute toxicity of dolabelladienotriol in BALB/c mice for ten days after administration of a single dose. Among the parameters considered were behavior, weight, biochemical and histological analyses of blood samples taken at three different times (Bs.0, Bs.1 and Bs.2) and optical microscopic examination of organs like liver, kidney, stomach and small intestine. Mice deaths were not observed at any dose during the ten day period. There were some changes in the biochemical analysis results for urea nitrogen (BUN) and alanine aminotransferase (ALT), but the changes were not significantly different from the reference levels of the animals before administration of the substance. Histological analyses of tissues were very similar for all animals. The alterations in liver and kidney tissues did not affect the animals´ behavior at any concentration, not even at 50 mg/kg, where the most significant changes in tissues were seen. This study indicates that dolabelladienotriol has low toxicity in administered dose range.

Food allergy/hypersensitivity: antigenicity or timing?

Mechanisms involved in the induction of oral tolerance (OT) or systemic immunization through the oral rout are still poorly understood. In our previous studies, we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. Conversely, if immunized with peanut proteins prior to a challenge diet (CD) containing peanuts they develop chronic inflammation of the gut. Our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen-specific gut inflammation. Adult, female C57BL/6J mice were divided in control (C) and experimental (E) groups. C1-C3 received peanut protein immunization, animals of the control groups C4 were sham immunized, and control group C5 received ovalbumin (OVA) immunization. The experimental group was immunized with peanut protein extract. Before initial exposure to a 30-day peanut containing CD, the experimental group was divided into 5 groups (E1-E5). OVA feeding began 7 days prior CD (E1) on day 0 (E2), 7 (E3), 14 (E4) and 21 (E5) during CD. Our results show that oral exposure to a novel protein (OVA) in the absence of gut inflammation (E1) leads to low levels of systemic antibody (Ab) titers, comparable to tolerant animals. Conversely, as off initial induction of inflammation, groups submitted to OVA (OT) protocol develop increasingly higher systemic Ab titers similar to animals of the immune control group. In conclusion, our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet.

Diet selection in immunologically manipulated mice.

Diet selection is a complex problem that animals in wildlife have to deal with daily. In their natural environment, these animals meet a great variety of foods some of which they are able and prepared to eat, yet, not all of it is eaten. In addition to the biological factors, some of which we shall discuss deeper in this paper, an important factor in food preference is social contact. Alterations in the physiology of mammals can have profound effects on the choice or preference for certain foods. On the other hand the decline of taste and smell perception in the elderly, the degree of food restriction, the sensorial properties of foods (such as presentation, taste, and smell) can be considered factors that influence feeding behavior leading to aversion. Many species, including man, learn to associate nausea with taste, and as a consequence avoid its specific intake, which has been shown to be persistent. Conditioned taste aversion is a form of associative learning in which animals display an aversion to the taste of a food that has previously been paired with illness. Our group has investigated the pattern of ingestion of foods that are frequently eaten by mice in wildlife and are potentially allergenic to humans in order to study the immunological consequences to these foods such as oral tolerance and inflammatory processes of the gut. We have chosen two seeds, peanuts (Arachis hypogea) and cashew nuts (Anacardium occidentale), as our source of antigens as the first is considered to be one of the most potent food allergens and for the second there seems to be very little allergy in the human setting. We used male and female, normal, adult CBA/J, A/J, C57BL/6 and Balb/c mice 2-3 months old and hybrid (C57Bl/6xBalb/c) F1, (Balb/cxC57Bl/6) F1), (C57Bl/6xDBA2) F1 mice. Food preference appeared to be strain-specific. Animals tolerized to a determined seed, then immunized with its protein extract and re-exposed to the seed in natura alter their feeding pattern. We suggest that diet selection, a multi-factorial event, is influenced by genetic factors such as the MHC and the immunological status of the animal.