concanavalin-a and Food-Hypersensitivity

Few chronic food protein models have described the relationship between allergenicity and the molecular structure of food protein after physical processing. The effect of γ-radiation on the structure of food protein was measured by fluorescence, circular dichroism and microcalorimetry. BALB/c mice were intraperitoneally sensitized and then given non-irradiated and irradiated Con-A by daily gavage for 28days. The tendency to form insoluble amorphous aggregates and partially unfolded species was observed after irradiation. The administration of non-irradiated and irradiated samples at low-dose significantly increased weight loss as well as plasma levels of eotaxin in animals repeatedly exposed to Con-A. Significant lymphocytic infiltrate filling completely the stroma of microvilli and tubular glands was observed in the small intestinal of the group given Con-A irradiated at a low dose. This phenotype was not observed in animals treated with Con-A irradiated at a high dose.

Topics: Administration, Oral; Animals; Calorimetry, Differential Scanning; Circular Dichroism; Concanavalin A; Disease Models, Animal; Dose-Response Relationship, Radiation; Female; Food Hypersensitivity; Gamma Rays; Intestine, Small; Lymphocytes; Mice; Mice, Inbred BALB C; Microvilli; Protein Conformation; Weight Loss

Oral tolerance (OT) is being studied with great interest because of its therapeutic potential in allergy and autoimmunity. In the present study, two mouse strains with extreme phenotypes of OT susceptibility (TS) or resistance (TR) to ovalbumin (OVA) were used to demonstrate whether the tr and ts genes, cumulated during 18 generations of bi-directional genetic selection, influence expression of immunobiological traits in naive or antigen-gavaged TR/TS mice. The difference in anti-OVA titres was 2048-fold between OVA-gavaged TS and TR mice. Tolerance susceptibility to OVA gavage in individuals from a (TS x TR)F(2) population was 24% high-susceptibility, 62% low-susceptibility and 14% non-tolerant. Different antigens, unrelated to OVA, were tested by gavage and TS mice were generally susceptible while TR mice were resistant. The stability of TS and TR phenotypes was not affected by the use of strict protocols of intraperitoneal immunization or feeding over 30 consecutive days. The levels of interleukin-2 (IL-2), IL-4, interferon-gamma and IL-10 cytokines evaluated in concanavalin A-stimulated spleen cells from naive mice and in OVA-stimulated spleen cells from OVA-gavaged mice were higher in TS mice. Interleukin-10 was up-regulated in OVA-gavaged TS mice and down-regulated in TR mice. In naive mice, the percentage of CD4(+) CD25(+) and CD4(+) Foxp3(+) spleen cells and IL-10 expression by CD4(+) cells was significantly higher in TS mice. These results indicate that regulation of IL-10 expression could be an important factor contributing to the mechanisms controlling OT susceptibility, and that the OT responses of TR and TS individuals strongly correlate with their innate potential to secrete this cytokine.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Antibody Formation; Antigens; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cell Count; Cell Proliferation; Concanavalin A; Crosses, Genetic; Cytokines; Female; Food Hypersensitivity; Forkhead Transcription Factors; Genes, Dominant; Immune Tolerance; Immunity, Humoral; Immunoglobulin G; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Lipopolysaccharides; Lymphocyte Activation; Male; Mice; Mice, Inbred Strains; Ovalbumin; Phenotype; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Vaccination

There is a growing need for the development of methods to characterize the allergenic properties of novel proteins, particularly those expressed by transgenic crop plants. Hence, there is considerable interest in the development of suitable animal models for this purpose. The production of specific IgE antibody has been reported following sensitization with food allergen via oral or systemic (intraperitoneal) routes of exposure. We have characterized cytokine profiles induced by intradermal treatment of BALB/c strain mice with a purified peanut allergen, Arachis hypogea lectin. Mice were exposed to peanut lectin by intradermal administration and the cytokine responses in the lymph node draining the site of exposure analyzed at the secreted protein level by enyzme-linked immunosorbent assay (ELISA) and cytokine mRNA level by ribonuclease protection assay (RPA). Exposure to peanut lectin, under conditions that induced robust IgE antibody titers, was found to be associated with a T helper 2 (Th2)-type cytokine expression profile at both the mRNA and secreted protein levels. Culture of naïve lymph node cells with peanut lectin failed to stimulate marked proliferation or cytokine production, confirming this protein is not mitogenic for mouse lymphocytes. Furthermore, the expression of Th2 cytokines was associated with the effector/memory CD62L- cell population. Similar treatment with a non-allergenic protein, potato acid phosphatase, failed to induce Th2 cytokine expression. These data demonstrate that exposure of mice to peanut allergen results in the selective stimulation of a Th2-type response.

Topics: Animals; Cell Fractionation; Concanavalin A; Cytokines; Female; Food Hypersensitivity; Immunoglobulin E; Injections, Intradermal; L-Selectin; Lymph Nodes; Mice; Mice, Inbred BALB C; Mitogens; Nuclease Protection Assays; Peanut Agglutinin; RNA, Messenger; Thy-1 Antigens

In this study we evaluated antigen-specific in vitro responses of peripheral blood lymphocytes to lipopolysaccharide (LPS)-depleted food allergens in children who reacted to food challenge (cow's milk or hen's egg) with a deterioration of their atopic dermatitis (AD). Some of the children showed immediate symptoms (urticaria, bronchial asthma or gastrointestinal symptoms) as well. The proliferation of casein-stimulated lymphocytes from children reacting to cow's milk (age 0.7-5.9 years) was significantly higher (P < 0.01) than the proliferation of lymphocytes from 15 children with AD without milk allergy (age: 2.1-9.1 years). Twenty-eight T-cell clones (TCC) were established from the blood of three children sensitized to cow's milk and hen's egg who reacted to double-blind, placebo-controlled oral food challenge both with a deterioration of AD and with immediate symptoms. Surprisingly, 16 of 28 casein- or ovalbumin-specific TCC were CD8+. All TCC produced high amounts of IFN-gamma upon stimulation with concanavalin A. In addition, 75% of the CD4+ TCC and 44% of the CD8+ TCC secreted IL-4. Our results indicate that: (i) food-specific proliferation of blood lymphocytes can be detected in patients with clinically relevant food allergy with LPS-depleted allergens in vitro and (ii) circulating food-specific lymphocytes are CD4+ and CD8+ T cells with the capacity of producing both type 1 and type 2 cytokines.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Child; Child, Preschool; Concanavalin A; Dermatitis, Atopic; Double-Blind Method; Eggs; Food Hypersensitivity; Humans; Immunologic Tests; Infant; Interferon-gamma; Interleukin-4; Lymphocyte Activation; Milk; Milk Hypersensitivity; T-Lymphocytes

The lymphoblastic responses of peripheral blood lymphocytes to mitogens were analyzed in 36 infants and children aged 1-28 months. The patients had confirmed or suspected cow's milk protein intolerance (CMPI). Immediately after a positive provocation test 12 out of 14 patients with repeatedly confirmed CMPI showed more than 30% decrease of their lymphoblastic responses to pokeweed mitogen and concanavalin A compared with their initial values. In 10 infants the response to phytohemagglutinin was similarly decreased. In 4 out of 12 infants with previously confirmed CMPI, but with no reaction at challenge, similar decreases were seen to some of the mitogens. In patients with suspected CMPI, but with no obvious reaction to the challenge with cow's milk, generally an increase of the lymphoblastic responses was seen compared with the initial values.

Topics: Animals; Cattle; Concanavalin A; Food Hypersensitivity; Humans; Immunity, Cellular; Infant; Lymphocyte Activation; Milk; Phytohemagglutinins; Pokeweed Mitogens

Crude peanut protein fractions from raw and roasted peanuts were examined in the RAST with 10 sera from patients showing clinical peanut sensitivity. The radioactive uptake results, which were generally high, did not reveal any distinguishable pattern. Two commercially available peanut proteins, peanut lectin and phospholipase D, gave poor RAST responses. Three purified peanut proteins, alpha-arachin, conarachin I, and concanavalin A-reactive glycoprotein, all gave significant RAST results that were generally lower than those obtained with the crude extracts. The extent of RAST inhibition obtained with these materials was inversely related to their abundance in the total peanut protein. Crossed immunoelectrophoresis with extracts from raw and roasted peanut indicated the presence of 22 and 10 anodically migrating antigens, respectively. Sixteen IgE binding antigens were revealed for raw peanut and seven for roasted peanut after incubation with a mixed serum from the 10 patients in crossed radioimmunoelectrophoresis (CRIE) using 125I-labeled anti-IgE. CRIE plates treated with individual serum samples showed that all the patients had specific IgE for the major antigen peak, which has been tentatively identified as alpha-arachin. This major storage protein of peanut, which is known to be particularly heat resistant; may be of greater clinical significance than its apparently low RAST activity would seem to indicate.

Topics: Adolescent; Adult; Allergens; Antigens, Plant; Arachis; Child; Child, Preschool; Concanavalin A; Counterimmunoelectrophoresis; Electrophoresis, Polyacrylamide Gel; Female; Food Hypersensitivity; Glycoproteins; Humans; Immunoglobulin E; Lectins; Male; Phospholipase D; Plant Lectins; Plant Proteins; Radioallergosorbent Test; Sodium Dodecyl Sulfate

beta-Lactoglobulin (BLG) is clinically the most frequent allergen in cow's milk protein hypersensitivity (CMPH). We therefore assessed its in vitro blastogenic effect on lymphocytes of patients suffering from CMPH. Twenty-two patients aged from 1 to 20 months were investigated. beta-Lactoglobulin produced a significantly (p less than 0.00003) higher blastogenic response in lymphocytes of patients with CMPH (mean stimulation index 7.7 +/- 0.7 SEM) than in 26 age-matched controls (2.7 +/- 0.4). This response was age related, being most marked in infants up to 5 months of age. Lymphocyte proliferation following stimulation with BLG can be a useful in vitro test in the diagnosis of CMPH. Because of this augmented lymphocyte response, we considered the possibility of a related defect in immune suppression. Nonspecific concanavalin A-induced suppressor cell activity was assessed in the above patient and control populations. Concanavalin A-induced suppressor cell activity was significantly (p less than 0.05) reduced in patients with CMPH. This too was most marked in the first few months of life. We suggest that the development of CMPH may be due to delayed maturation of a suppressor cell population.

Topics: Concanavalin A; Food Hypersensitivity; Humans; Infant; Infant, Newborn; Lactoglobulins; Lymphocyte Activation; Milk Proteins; T-Lymphocytes, Regulatory