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.

Introgression of peanut smut resistance from landraces to elite peanut cultivars (Arachis hypogaea L.).

Smut disease caused by the fungal pathogen Thecaphora frezii Carranza & Lindquist is threatening the peanut production in Argentina. Fungicides commonly used in the peanut crop have shown little or no effect controlling the disease, making it a priority to obtain peanut varieties resistant to smut. In this study, recombinant inbred lines (RILs) were developed from three crosses between three susceptible peanut elite cultivars (Arachis hypogaea L. subsp. hypogaea) and two resistant landraces (Arachis hypogaea L. subsp. fastigiata Waldron). Parents and RILs were evaluated under high inoculum pressure (12000 teliospores g-1 of soil) over three years. Disease resistance parameters showed a broad range of variation with incidence mean values ranging from 1.0 to 35.0% and disease severity index ranging from 0.01 to 0.30. Average heritability (h2) estimates of 0.61 to 0.73 indicated that resistance in the RILs was heritable, with several lines (4 to 7 from each cross) showing a high degree of resistance and stability over three years. Evidence of genetic transfer between genetically distinguishable germplasm (introgression in a broad sense) was further supported by simple-sequence repeats (SSRs) and Insertion/Deletion (InDel) marker genotyping. This is the first report of smut genetic resistance identified in peanut landraces and its introgression into elite peanut cultivars.

Recombinants from the crosses between amphidiploid and cultivated peanut (Arachis hypogaea) for pest-resistance breeding programs.

Peanut is a major oilseed crop worldwide. In the Brazilian peanut production, silvering thrips and red necked peanut worm are the most threatening pests. Resistant varieties are considered an alternative to pest control. Many wild diploid Arachis species have shown resistance to these pests, and these can be used in peanut breeding by obtaining hybrid of A and B genomes and subsequent polyploidization with colchicine, resulting in an AABB amphidiploid. This amphidiploid can be crossed with cultivated peanut (AABB) to provide genes of interest to the cultivar. In this study, the sterile diploid hybrids from A. magna V 13751 and A. kempff-mercadoi V 13250 were treated with colchicine for polyploidization, and the amphidiploids were crossed with A. hypogaea cv. IAC OL 4 to initiate the introgression of the wild genes into the cultivated peanut. The confirmation of the hybridity of the progenies was obtained by: (1) reproductive characterization through viability of pollen, (2) molecular characterization using microsatellite markers and (3) morphological characterization using 61 morphological traits with principal component analysis. The diploid hybrid individual was polyploidized, generating the amphidiploid An 13 (A. magna V 13751 x A. kempff-mercadoi V 13250)4x. Four F1 hybrid plants were obtained from IAC OL 4 × An 13, and 51 F2 seeds were obtained from these F1 plants. Using reproductive, molecular and morphological characterizations, it was possible to distinguish hybrid plants from selfed plants. In the cross between A. hypogaea and the amphidiploid, as the two parents are polyploid, the hybrid progeny and selves had the viability of the pollen grains as high as the parents. This fact turns the use of reproductive characteristics impossible for discriminating, in this case, the hybrid individuals from selfing. The hybrids between A. hypogaea and An 13 will be used in breeding programs seeking pest resistance, being subjected to successive backcrosses until recovering all traits of interest of A. hypogaea, keeping the pest resistance.

Induced systemic resistance and symbiotic performance of peanut plants challenged with fungal pathogens and co-inoculated with the biocontrol agent Bacillus sp. CHEP5 and Bradyrhizobium sp. SEMIA6144.

Synergism between beneficial rhizobacteria and fungal pathogens is poorly understood. Therefore, evaluation of co-inoculation of bacteria that promote plant growth by different mechanisms in pathogen challenged plants would contribute to increase the knowledge about how plants manage interactions with different microorganisms. The goals of this work were a) to elucidate, in greenhouse experiments, the effect of co-inoculation of peanut with Bradyrhizobium sp. SEMIA6144 and the biocontrol agent Bacillus sp. CHEP5 on growth and symbiotic performance of Sclerotium rolfsii challenged plants, and b) to evaluate field performance of these bacteria in co-inoculated peanut plants. The capacity of Bacillus sp. CHEP5 to induce systemic resistance against S. rolfsii was not affected by the inoculation of Bradyrhizobium sp. SEMIA6144. This microsymbiont, protected peanut plants from the S. rolfsii detrimental effect, reducing the stem wilt incidence. However, disease incidence in plants inoculated with the isogenic mutant Bradyrhizobium sp. SEMIA6144 V2 (unable to produce Nod factors) was as high as in pathogen challenged plants. Therefore, Bradyrhizobium sp. SEMIA6144 Nod factors play a role in the systemic resistance against S. rolfsii. Bacillus sp. CHEP5 enhanced Bradyrhizobium sp. SEMIA6144 root surface colonization and improved its symbiotic behavior, even in S. rolfsii challenged plants. Results of field trials confirmed the Bacillus sp. CHEP5 ability to protect against fungal pathogens and to improve the yield of extra-large peanut seeds from 2.15% (in Río Cuarto) to 16.69% (in Las Vertientes), indicating that co-inoculation of beneficial rhizobacteria could be a useful strategy for the peanut production under sustainable agriculture system.

Prevalence and factors associated to peanut allergy in Mexican school children.

BACKGROUND: In our country, the prevalence and the factors associated to peanut allergy are unknown, a health problem that has been emerging worldwide. OBJECTIVE: To establish the prevalence and the factors that are associated to peanut allergy amongst school children. METHODS: This is a population-based cross-sectional study. We included 756 children aged 6-7 years. The children's parents were questioned about their peanut intake habits. A structured questionnaire was applied, it included questions regarding peanut intake; family and personal history of asthma; rhinitis; and atopic dermatitis. Allergic reactions to peanuts were registered as: probable, convincing and systematic. The statistical analyses included logistical regression models to look for associated factors. RESULTS: Males were 356/756 (47.1%). Peanut allergy prevalence: probable reaction: 14/756 (1.8%), convincing reaction: 8/756 (1.1%) and systemic reaction: 3/756 (0.4%). Through multivariate analysis, the presence of symptoms of allergic rhinitis (OR=4.2 95% CI 1.3-13.2) and atopic dermatitis (OR=5.2; 95% CI 1.4-19.5) during the previous year, showed significant association to probable peanut reaction. The former year, the presence of atopic dermatitis was the only variable that was substantially associated to a convincing reaction (OR=7.5; 95% CI 1.4-38.4) and to a systematic reaction (OR=45.1; 95% CI 4.0-510.0), respectively. CONCLUSIONS: The reported prevalence of peanut allergy was consistent with that found in previous studies; symptoms of allergic rhinitis and atopic dermatitis were identified as associated factors to peanut allergy.

Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6.

BACKGROUND: Cross-linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE-binding epitopes of peanut allergens have been defined, little is known about conformational IgE-binding epitopes. OBJECTIVE: To identify clinically relevant conformational IgE epitopes of the two most important peanut allergens, Ara h 2 and Ara h 6, using phage peptide library. METHODS: A phage 12mer peptide library was screened with allergen-specific IgE from 4 peanut-allergic patients. Binding of the mimotopes to IgE from a total of 29 peanut-allergic subjects was measured by ELISA. The mimotope sequences were mapped on the surface areas of Ara h 2 and Ara h 6 using EpiSearch. RESULTS: Forty-one individual mimotopes were identified that specifically bind anti- Ara h 2/Ara h 6 IgE as well as rabbit anti-Ara h 2 and anti-Ara h 6 IgG. Sequence alignment showed that none of the mimotope sequences match a linear segment of the Ara h 2 or Ara h 6 sequences. EpiSearch analysis showed that all the mimotopes mapped to surface patches of Ara h 2 and Ara h 6. Eight of the mimotopes were recognized by more than 90% of the patients, suggesting immunodominance. Each patient had distinct IgE recognition patterns but the recognition frequency was not correlated to the concentration of peanut specific IgE or to clinical history. CONCLUSIONS: The mimotopes identified in this study represent conformational epitopes. Identification of similar surface patches on Ara h 2 and Ara h 6 further underscores the similarities between these two potent allergens.

Peanut allergy in Mexican children: what is the effect of age at first consumption?

Studies suggest that children who start solid foods early are at risk for developing food allergies. Herein, we evaluated the effects of the introduction of peanuts to the diets of children on emerging peanut allergies. Children with allergic rhinitis and asthma were enrolled in the present study and evaluated in four stages. In the first stage, a clinical history was completed for all participants. In the second stage, skin tests were conducted to detect the sensitization to peanuts. In the third stage, the parents were interviewed about the peanut-eating habits of their children. In the fourth stage, children with a convincing history of allergy or a positive peanut skin test result were subjected to an open oral food challenge (OOFC). Three hundred children in four groups were included, 58.2% of the subjects were male, and the mean age was 7.3±3.9 years. The median age of first exposure to peanuts in patients with peanut allergies was greater than that in children without peanut allergies (2 years versus 1 year; p=0.009). The multivariate analysis, including only those children subjected to the OOFC, revealed that the consumption of peanuts after the age of ≥2 years is a risk factor for developing a peanut allergy (odds ratio=8.0, 95% confidence interval 1.3-50.0, p=0.026). The results of the present study showed that the late introduction of peanuts to children increases the risk of developing a peanut allergy.

New hybrids from peanut (Arachis hypogaea L.) and synthetic amphidiploid crosses show promise in increasing pest and disease tolerance.

The primary gene pool of the cultivated peanut (Arachis hypogaea L., allotetraploid AABB) is very narrow for some important characteristics, such as resistance to pests and diseases. However, the Arachis wild diploid species, particularly those from the section Arachis, still have these characteristics. To improve peanut crops, genes from the wild species can be introgressed by backcrossing the hybrids with A. hypogaea. When diploid species whose genomes are similar to those of the cultivated peanut are crossed, sterile hybrids result. Artificially doubling the number of chromosomes of these hybrids results in fertile synthetic polyploids. The objectives of this study were: 1) to obtain progenies by crossing amphidiploids with the cultivated peanut, and 2) to characterize these two groups of materials (amphidiploids and progenies) so that they may be efficiently conserved and used. Using morphological, molecular, and pollen viability descriptors we evaluated one cultivar of A. hypogaea (IAC 503), eight synthetic amphidiploids, and the progenies resulting from four distinct combinations of crossing between IAC 503 and four amphidiploids.

[Anaphylaxis secondary to prick-to-prick tests to foods and its risk factors]. / Anafilaxia secundaria a pruebas cutáneas prick-to-prick para alimentos y sus factores de riesgo.

The diagnosis of food allergy requires a proper anamnesis and diagnostic testing with skin prick tests with fresh foods and/or standardized allergen, or specific IgE tests. The risk of systemic reactions is of 15-23 per 100,000 skin tests performed by prick method, specifically anaphylaxis at 0.02%. This paper reports the case of four patients, who while performing prick to prick test with fresh food presented anaphylactic reaction. Implicated foods were fruits of the Rosaceae, Anacardiaceae and Caricaceae families. The severity of anaphylaxis was: two patients with grade 4, one patient grade 2 and one grade 3, all with appropriate response to drug treatment. The risk factors identified were: female sex, personal history of atopy, previous systemic reaction to Hymenoptera venom, prior anaphylaxis to prick tests to aeroallergens. We found that a history of positive skin test for Betulla v, can be a risk factor for anaphylaxis in patients with oral syndrome. During testing prick to prick with food anaphylaxis can occur, so it should be made with aerial red team on hand. The history of positivity Betulla v is an additional risk factor in these patients.

El diagnóstico de alergia alimentaria requiere una anamnesis adecuada y la realización de pruebas diagnósticas, las pruebas cutáneas con alimentos en fresco, con alergenos estandarizados, o con ambos; las pruebas de IgE específica para alimentos son útiles. El riesgo de reacciones sistémicas por pruebas cutáneas por punción es de 15 a 23 por cada 100,000 y el de anafilaxia es de 0.02%. Comunicamos el caso de cuatro pacientes que sufrieron anafilaxia durante la realización de prueba Prick-to-Prick con alimentos frescos. Los alimentos implicados fueron frutas de las familias Rosaceae, Anacardiaceae y Caricaceae. En dos pacientes la anafilaxia fue de grado 4, en una grado 2 y en otra grado 3, todas con adecuada respuesta al tratamiento farmacológico. Los factores de riesgo fueron: sexo femenino, antecedente personal de atopia, reacción sistémica previa a veneno de himenópteros y anafilaxia previa con pruebas por punción para aeroalergenos. Las cuatro pacientes tuvieron síndrome de alergia oral y 50% tenía antecedente de prueba por punción positiva a betuláceas. Durante la realización de pruebas Prick-to-Prick para alimentos los pacientes pueden presentar anafilaxia, por lo que deben realizarse en un área que cuente con equipo rojo. El antecedente de síndrome de alergia oral se observó en todos los casos y la mitad de las pacientes tuvieron positividad a betuláceas, estos antecedentes pueden ser factores de riesgo adicional de anafilaxia dura te la realización de pruebas Prick-to-Prick para alimentos.

The value of specific IgE to peanut and its component Ara h 2 in the diagnosis of peanut allergy.

BACKGROUND: To avoid unnecessary oral food challenges, which are time consuming, stressful, and risky, improved in vitro diagnostic methods for food allergy such as component resolved diagnostics are still under investigation. OBJECTIVE: To investigate the role of whole peanut- and peanut-component (Ara h 1, Ara h 2, Ara h 3, Ara h 6 and Ara h 8)-specific IgE levels in the diagnostic procedure of peanut allergy as well as the diagnostic properties of peanut-specific IgG and IgG4. METHODS: Sixty-one children underwent oral peanut challenge tests for diagnostic purposes irrespective of their peanut-specific IgE levels. Peanut-specific serum IgE, IgG, and IgG4 levels were determined by ImmunoCAP FEIA and specific IgE against individual peanut proteins by Immuno Solid-phase Allergen Chip. RESULTS: Thirty-four of 61 patients (56%) had a peanut allergy. No significant difference was observed for peanut-specific IgG or peanut-specific IgG4 levels between patients who were allergic and tolerant patients, whereas peanut-specific IgE was significant higher in patients who were allergic than in tolerant patients (P < .005). Twenty-five of 61 children had peanut-specific IgE above a previously proposed cutoff level of 15 kUA/L; however, 7 of these 25 children (28%) were clinically tolerant. Ara h 2-specific IgE was significantly lower in tolerant than in patients with allergies (P < .0001). Interestingly, 94% of the patients with peanut allergies showed IgE-binding to Ara h 2. Unfortunately, 26% of the sensitized but tolerant patients have shown IgE binding to Ara h 2 too. CONCLUSIONS: Neither the level of specific IgE to peanut nor to Ara h 2 was able to clearly distinguish patients with clinical relevant peanut allergy from those who were clinical tolerant in our population. As expected, peanut-specific IgG and IgG4 did not improve the diagnostic procedure.

Sensibilización al maní en pacientes venezolanos con enfermedades atópicas y/o urticaria / Sensitization to peanut in Venezuelan patients with atopic diseases and / or uticaria

La sensibilización y las manifestaciones alérgicas al maní se han incrementado últimamente a nivel mundial, constituyendo el mismo la causa principal de anafilaxia por alimentos. Como la prevalencia de alergia al maní varía de acuerdo a las regiones nos propusimos evaluar, en una etapa preliminar, la sensibilización al maní por pruebas cutáneas (“skin prick test”) en pacientes venezolanos atópicos y/o con urticarias que acudieron a la Consulta ambulatoria de Alergía del Instituto de Inmunología. El 5,4 % de los pacientes manifestó algún tipo de manifestación cutánea o respiratoria al ingerir maní. Se demostró sensibilización al maní por pruebas cutánea en el 6,5 % de los pacientes. Sin embargo, un porcentaje pequeño (2 %) de ellos mostró, en conjunto, pruebas cutáneas positivas y síntomas a la ingesta del maní. Ningún paciente refirió síntomas severos tras la ingestión de maní. La mayoría de los pacientes con pruebas positivas al maní, también mostraron pruebas positivas a otros alimentos. Estos resultados concuerdan con la percepción de los médicos venezolanos de una baja frecuencia de reacciones adversas, especialmente graves, a la ingesta de maní en nuestro país

Peanut allergy and sensitization incidence has increased world wide to become the first cause of food anaphylaxis. Since the prevalence of peanut allergy changes according to geographical areas, the aim of the study was to assess, in a preliminary report, peanut allergy incidence by skin prick test in atopic Venezuelan patients with atopy and or urticaria from the outpatient allergy clinic of the Institute of Immunology. Cutaneous or respiratory manifestations after peanut ingestion was observed in 5.4 % of the patients studied. Cutaneous test was positive in 6.5 % of patients. In the other hand, a small group (2 %), showed positive skin test along with symptoms after peanut ingestion. None of the patients had severe reactions. Most of the patients with peanut positive skin test were positive to other food allergens. These results are in accordance with the general clinical perception of small frequency of adverse reaction, specially the most serious ones, to peanut ingestion in our country

Identification of candidate genome regions controlling disease resistance in Arachis.

BACKGROUND: Worldwide, diseases are important reducers of peanut (Arachis hypogaea) yield. Sources of resistance against many diseases are available in cultivated peanut genotypes, although often not in farmer preferred varieties. Wild species generally harbor greater levels of resistance and even apparent immunity, although the linkage of agronomically un-adapted wild alleles with wild disease resistance genes is inevitable. Marker-assisted selection has the potential to facilitate the combination of both cultivated and wild resistance loci with agronomically adapted alleles. However, in peanut there is an almost complete lack of knowledge of the regions of the Arachis genome that control disease resistance. RESULTS: In this work we identified candidate genome regions that control disease resistance. For this we placed candidate disease resistance genes and QTLs against late leaf spot disease on the genetic map of the A-genome of Arachis, which is based on microsatellite markers and legume anchor markers. These marker types are transferable within the genus Arachis and to other legumes respectively, enabling this map to be aligned to other Arachis maps and to maps of other legume crops including those with sequenced genomes. In total, 34 sequence-confirmed candidate disease resistance genes and five QTLs were mapped. CONCLUSION: Candidate genes and QTLs were distributed on all linkage groups except for the smallest, but the distribution was not even. Groupings of candidate genes and QTLs for late leaf spot resistance were apparent on the upper region of linkage group 4 and the lower region of linkage group 2, indicating that these regions are likely to control disease resistance.

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.

B cells are involved in the modulation of pathogenic gut immune response in food-allergic enteropathy.

Food enteropathies involve uncontrolled or hypersensitivity reactions to ingested nutrients and may result in IgE and T-helper type 2 (Th2) responses as in food allergy. However, the precise role of B cells in the development of food enteropathies remains uncertain. In this work, we used B cell-deficient mice (B KO) and a model of peanut sensitization to examine the involvement of B lymphocytes in the pathogenesis of food allergy. Results showed that priming of wild-type (WT) mice with peanut proteins induced specific IgG1 and IgE responses in serum, with edema, tissue destruction, epithelial exulceration and inflammatory infiltrate in the gut of sensitized and challenged (S + Peanut) WT animals. In contrast, there was no sera immunoglobulin detection and absence of tissue destruction in the gut of B KO mice, which presented moderate inflammatory infiltrate and villous enlargement after peanut challenge. These animals presented marked decrease in IL-4 and TNF-alpha and high levels of IL-10, TGF-beta, IL-12p40 and IFN-gamma mRNA in the gut. Moreover, the expression of CCL5, CCL11 and CXCL1 was reduced in the gut of B KO mice, in contrast to elevated messages of CCL2 or similar detection of Th1-related chemokines in S + Peanut WT mice. Finally, we provided evidence that B cells are necessary to the development of food-related enteropathies and induction of gut inflammation during allergic reactions to food.

Development and use of single nucleotide polymorphism markers for candidate resistance genes in wild peanuts (Arachis spp).

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid of recent origin, with an AABB genome and low genetic diversity. Perhaps because of its limited genetic diversity, this species lacks resistance to a number of important pests and diseases. In contrast, wild species of Arachis are genetically diverse and are rich sources of disease resistance genes. Consequently, a study of wild peanut relatives is attractive from two points of view: to help understand peanut genetics and to characterize wild alleles that could confer disease resistance. With this in mind, a diploid population from a cross between two wild peanut relatives was developed, in order to make a dense genetic map that could serve as a reference for peanut genetics and in order to characterize the regions of the Arachis genome that code for disease resistance. We tested two methods for developing and genotyping single nucleotide polymorphisms in candidate genes for disease resistance; one is based on single-base primer extension methods and the other is based on amplification refractory mutation system-polymerase chain reaction. We found single-base pair extension to be an efficient method, suitable for high-throughput, single-nucleotide polymorphism mapping; it allowed us to locate five candidate genes for resistance on our genetic map.

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.

Modulation of mucosal immunity in a murine model of food-induced intestinal inflammation.

BACKGROUND: Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. OBJECTIVE: To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens. METHODS: C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses. RESULTS: Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gammadelta+ and CD4+CD25+Foxp3+ cells in Peyer's patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut. CONCLUSION: A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.

IgE food sensitization in infants with eczema attending a dermatology department.

OBJECTIVES: Because community-based studies, which report IgE food sensitization (IgE-FS) in more than 80% of infants with moderate atopic eczema, may be influenced by referral bias, we assessed the prevalence of IgE-FS in a cohort of infants with moderate atopic eczema attending a dermatology department clinic. STUDY DESIGN: Consecutive infants (n = 51, 39 males; median age, 34 weeks; range, 20 to 51 weeks) with moderate atopic eczema referred to a university-affiliated dermatology department were studied prospectively. Clinical history and eczema severity were documented. IgE-FS was assessed by the skin prick test (SPT; n = 51) and food-specific serum IgE antibodies (CAP-FEIA test; n = 41). IgE-FS was diagnosed if the SPT or CAP-FEIA level exceeded the >95% predictive reference cutoff for positive food challenges. RESULTS: Based on SPT, 44 of 51 infants (86%; 95% confidence interval [CI] = 74% to 94%) had IgE-FS (cow's milk, 16%; egg, 73%; peanut, 51%). Using age-specific 95%-predictive cutoff values, CAP-FEIA identified 34 of 41 infants (83%; 95% CI = 68% to 93%) with IgE-FS (cow's milk, 23%; egg, 80%). Forty-six (90%) infants had IgE-FS to at least 1 food item by either SPT or CAP-FEIA. CONCLUSIONS: Atopic eczema was found to be closely associated with IgE-FS in infants attending a dermatology department.