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.

Taro Lectin Can Act as a Cytokine-Mimetic Compound, Stimulating Myeloid and T Lymphocyte Lineages and Protecting Progenitors in Murine Bone Marrow.

Taro (Colocasia esculenta) corm is traditionally consumed as a medicinal plant to stimulate immune responses and restore a health status. Tarin, a taro lectin, is considered responsible for the immunomodulatory effects of taro. In the present study, in order to investigate the effects of tarin on bone marrow hematopoietic population, murine cells were stimulated with tarin combined with a highly enriched conditioned medium containing either IL-3 or GM-CSF. Cells challenged with tarin proliferated in a dose-dependent manner, evidenced by the increase in cell density and number of clusters and colonies. Tarin exhibited a cytokine-mimetic effect similar to IL-3 and GM-CSF, increasing granulocytic cell lineage percentages, demonstrated by an increase in the relative percentage of Gr-1+ cells. Tarin does not increase lymphocytic lineages, but phenotyping revealed that the relative percentage of CD3+ cells was increased with a concomitant decrease in CD19+ and IL-7Rα+ cells. Most bone marrow cells were stained with tarin-FITC, indicating non-selective tarin binding, a phenomenon that must still be elucidated. In conclusion, taro corms contain an immunomodulatory lectin able to boost the immune system by promoting myeloid and lymphoid hematopoietic progenitor cell proliferation and differentiation.

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.

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.

Comparison between digital and optical microscopy: Analysis in a mouse gut inflammation model.

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.

Seroreactivity to Anisakis spp. in the perinatal period.

BACKGROUND: This study had sought to assess the seroreactivity to the fish nematode Anisakis spp. in a puerperal population, as well as to ascertain whether a correlation exists between maternal and cord blood levels. METHODS: Blood samples were obtained from puerperal women and cord blood to measure specific anti-Anisakis antigen IgG and IgE by ELISA. Non-parametric tests were used to compare two or more independent and related samples. RESULTS: Of the 99 maternal serum samples assessed, 21 were positive on ELISA (21.2%). There were no significant differences in the mean ranks of IgG optical density levels between women who ate fish and those who did not (p = 0.456), those who ate raw fish and those who did not (p = 0.479), or between those who had allergic complaints and those who did not (p = 0.431). CONCLUSION: Transplacental passage of antibodies occurred, leading to moderate correlation between maternal and cord blood serum levels.

Subchronic toxicity and anti-HSV-1 activity in experimental animal of dolabelladienetriol from the seaweed, Dictyota pfaffii.

This study examined in rats the subchronic toxicity and anti- HSV-1activity after oral administration of dolabelladienetriol (D1), a diterpene isolated from the seaweed Dictyota pfaffii. In subchronic toxicity (SCT) tests, female rats received D1 by gavage 15 mg/kg/day (n = 5) for 50 days, and general behavior, death, hematological, biochemical and histological changes in the liver, kidney, stomach, and duodenum were determined. For the anti-HSV-1 activity, female mice were infected and treated orally with a dose of 20 mg/kg (n = 5) twice a day with D1 and any lesions in the skin were then recorded for 18 days. Dolabelladienetriol in SCT did not significantly change behavior, body weight, hematological or biochemical profiles. The liver and kidneys, however, showed some alterations in rats treated with D1, similar to those in rats treated with ACV, while the other tissues had no significant changes. The anti-HSV-1 activity of D1 had a similar efficacy to the ACV drug control in mice. Our results showed that D1 has potential commercial development as a new HSV-1drug.

Principal component analysis of factors for sensitization to Anisakis spp. in postpartum women.

INTRODUCTION: Immunoreactivity to Anisakis spp. is believed to be associated with frequency of fish intake. The objective of this study was to evaluate, using principal component analysis, the main factors potentially involved in reactivity to these nematodes in postpartum women. METHODS: Retrospective study conducted on a database of 309 postpartum women. All completed a structured questionnaire and had blood samples collected for ELISA analysis of specific immunoglobulins against total Anisakis spp. antigens and assessment of reactivity. Parametric and nonparametric tests were used to assess factors for sensitization in the reactive and nonreactive groups, and a principal component analysis was performed. A Pearson correlation matrix with varimax rotation was used to assess the variables of interest (place of residence, age, number of prenatal visits, type of birth facility, fish intake and frequency, raw fish intake, fish handling, history of allergies). RESULTS: After exclusions, samples from 203 women were assessed. Of these, 52 (25.6%) were reactive for anti-Anisakis IgG. Most women claimed not to handle fish (n = 121) and eat fish only sporadically (n = 71). Significant differences in age were seen between the reactive and nonreactive groups (p = 0.001). The first two components explained 32.55% and 38.94% of variances in the nonreactive and reactive groups respectively. The adjusted matrix assigned greater probabilistic weight to weekly intake frequency (0.804), followed by raw fish intake (0.759), with differences in relation to the nonreactive group. CONCLUSION: Correlation matrices revealed a direct relationship between seroreactivity to Anisakis spp. and frequency of fish intake in a sample of postpartum women.

Association between immunoreactivity to Anisakis spp. antigens and high-risk pregnancy.

Numerous factors contribute to perinatal risk, many of which remain undefined. This study sought to determine the frequency of fish intake in postpartum women, and to establish a relationship between the rates of immunoreactivity for antigens from Anisakis spp. and high-risk pregnancy. In this prospective noninterventional study, a structured questionnaire was administered and serum was collected from postpartum women at two perinatal centers (a high-risk birth unit [HRBU] and a low-risk birth unit [LRBU]) in the Niteroi municipality of Brazil. Anisakis species-specific IgG and IgE were measured by ELISA. The chisquared test was performed, and odds ratios (ORs) with their 95% confidence intervals were estimated. The t-test or Mann-Whitney test was applied to continuous, normally distributed variables. In total, 309 women (170 from HRBU, 139 from LRBU) between 24.8 and 26.7 years old with a median of 6 to 8 prenatal visits were enrolled. Women in the two units exhibited differences in some variables, including prenatal care (p = 0.01), maternal and fetal risk (p = 0.00; OR = 6.17), and gestational age (p = 0.00), but no differences in fish consumption (p = 0.29), frequency of fish intake (p = 0.40), allergic symptoms (p = 0.51), or frequency of anti-Anisakis reactivity (p = 0.22). Logistic regression analysis revealed that only age was independently associated with postpartum anti-Anisakis reactivity. This study confirmed a low prevalence of fish intake and suggested that Anisakis spp. had no impact on high-risk pregnancies among this postpartum study population.

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.

Cross-sectional study of serum reactivity to Anisakis simplex in healthy adults in Niterói, Brazil.

Although the incidence of anisakiasis is rising worldwide, its frequency is still unknown in Brazil. The aim of this study was to verify immunoreactivity to Anisakis simplex antigens in healthy adults and determine its possible relationship with frequency of fish consumption and allergy symptoms. A prospective cross-sectional study was carried out with 67 volunteers recruited from a military facility in Niterói, Brazil. The subjects completed a structured questionnaire and serum titers of specific anti-Anisakis IgE and IgG antibodies were measured. The association between frequency of fish intake and IgE reactivity was evaluated by Fisher's exact test. Almost all subjects (97.0%, 65/67) that consumed seafood; 64.6% (42/65) ate fish at least once weekly. Of all seafood consumers, 56.9% (37/65) reported allergy symptoms, being gut allergies most often cited (35.5%). IgE seroreactivity to Anisakis simplex was found in 20.9% of subjects (14/67), with 13.4% (9/67) reacting exclusively to somatic antigen, 3.0% (2/67) exclusively to excretory/secretory antigens and 4.5% (3/67) to both antigens. There was a significant association between frequency of fish consumption and positive serology (p = 0.019). An immunoblot assay for Anisakis antigens showed different positive bands for IgG. The direct relationship between ELISA reactivity and frequency of fish intake and absence of association with allergy symptoms suggests previous contact with Anisakis simplex antigens.

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).

A technique for the intra-gastric administration of live larvae of Anisakis simplex in mice.

To understand the mechanisms of infection and to attempt to simulate human infection by the Anisakidae family, many in vivo experimental approaches have been developed. The aim was to develop and present a technique for the induction of an oral infection through the use of an intra-gastric gavage of live Anisakis simplex in mice. A commercial pediatric gastric tube (No. 4) was cut longitudinally to produce a 3-cm slit at the distal end where the larva was placed to then be administered to the stomach of the mouse. There were no abnormal clinical complications before, during or after the procedure. In conclusion oral infection through the direct delivery of larvae in the stomach is simple and effective.

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.

Induction of an antigen specific gut inflammatory reaction in mice and rats: a model for human Inflammatory Bowel Disease

Food allergy is an adverse reaction that occurs in susceptible people when they eat sensitizing foods and is one of the causes of Inflammatory Bowel Disease (IBD). The effort to understand the induction process of these diseases is important as IBD is increasing worldwide, including in Brazil. The aim of this study was to develop an experimental antigen specific inflammatory process of the gut of mice and rats, using peanut seeds. Animals were immunized with peanut protein extract before their exposure to the in natura peanut seeds. Results showed that systemic immunization with peanut protein extracts rendered significantly higher antibody titers than control groups and that immunized animals submitted to a challenge diet containing peanuts presented time dependent alterations of the gut similar to celiac disease. In conclusion, results suggested that this experimental model was a convenient tool to study the evolution of alterations in chronic antigen specific gut inflammatory process.

A alergia alimentar consiste em uma reação adversa que ocorre em pessoas susceptíveis quando ingerem alimentos sensibilizantes, sendo uma das causas das Doenças Inflamatórias Intestinais (IBD). O objetivo deste estudo foi desenvolver um protocolo experimental de indução de um processo inflamatório intestinal antígeno-específico em camundongos e ratos. Foi escolhida para a indução deste processo a semente de amendoim. Os animais foram imunizados com o extrato protéico previamente à exposição com a semente in natura. Nossos resultados mostram que a imunização sistêmica com extratos protéicos de amendoim ocasiona títulos significativamente maiores de anticorpos quando comparado ao grupo controle e que os animais imunizados submetidos ao desafio com a dieta contendo exclusivamente amendoim apresentam alterações intestinais tempo-dependente similares àquelas observadas na doença celíaca. Os resultados obtidos sugerem que este modelo experimental constitui uma ferramenta conveniente para avaliar as alterações no processo inflamatório intestinal crônico antígeno-específico

The serum D-xylose test as a useful tool to identify malabsorption in rats with antigen specific gut inflammatory reaction.

The inappropriate immune response to foods, such as peanut, wheat and milk may be the basis in the pathogenesis of enteropathies like coeliac and Crohn disease, which present small intestinal malabsorption. A number of recent studies have utilized d-xylose absorption as an investigative tool to study small intestinal function in a variety of clinical settings. Thus, the aim of this experimental study was to evaluate the intestinal absorption of D-xylose in an antigen-specific gut inflammatory reaction rat model. Animals of the experimental group were inoculated with peanut protein extract before their exposure to a challenge diet containing exclusively peanut seeds to induce the gut inflammatory reaction caused by peanut allergy. Our results show that systemic inoculation with peanut protein extract renders significantly higher antibody titres (5.085 +/- 0.126 units) (P < 0.0001) than control rats (0.905 +/- 0.053 units) and that the antibody titres correlate positively to an inflammatory alteration of the gut morphology (P < 0.0001). Animals pertaining to the experimental group showed an intestinal absorption of D-xylose lower than control rats (P < 0.0001). We also observed that D-xylose absorption correlates negatively with IgG titres and positively with morphometric parameters (Pearson correlation). In conclusion, the use of serum D-xylose test was useful to identify the presence of small intestinal malabsorption in our antigen specific gut inflammatory reaction rat model.

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.