Induction of systemic immunologic tolerance to beta-lactoglobulin by oral administration of a whey protein hydrolysate.

BACKGROUND: Oral administration of an antigen has been shown to suppress the specific immune response to this antigen. This approach, called oral tolerance, has been demonstrated with intact proteins in animal models for prevention of allergy and autoimmune diseases. OBJECTIVE: The purpose of this study was to determine whether oral tolerance can be induced with protein peptides. Partially hydrolyzed and extensively hydrolyzed cow's milk formulas were compared for their capacity to induce tolerance to cow's milk proteins. METHODS: Five-week-old Sprague-Dawley rats were fed cow's milk formulas ad libitum from day 1 to day 19. All animals were immunized with beta-lactoglobulin and ovalbumin on day 5 and bled on day 19. Sera were analyzed for specific IgE and IgG antibodies by ELISA and for functional IgE response by in vitro mast cell mediator (tritiated serotonin) release. In vivo modulation of intestinal mast cells was analyzed by the specific release of the rat mast cell protease II, and T-cell response was determined by tritiated thymidine incorporation into lymph node lymphocytes. RESULTS: Oral administration of a partially hydrolyzed cow's milk formula suppresses specific serum IgE and IgG anti-beta-lactoglobulin antibodies, as well as mediator release from rat mast cells and T-lymphocyte response. This suppression was shown to be antigen-specific and dose-dependent. An extensively hydrolyzed formula was unable to achieve the induction of such an oral tolerance. CONCLUSION: These results support the view that partially hydrolyzed proteins are able to induce specific oral tolerance, whereas extensively hydrolyzed proteins are not.

Milk formulae in the prevention of food allergy.

Because food allergy is frequent and severe, all possible means should be used to try to prevent its manifestations or at least to delay them until the child is older and stronger and therefore better able to follow an exclusion diet. The capacity of breast-feeding for preventing food allergy has been challenged in the past, but a consensus seems to be emerging now that breast-feeding can indeed prevent food allergy if it is started at birth and is exclusive for at least 4, and preferably 6, months. In the most "at-risk" babies the breast-feeding mother should try to eliminate the most potent allergens (eggs, fish, soya, nuts, and cow's milk) from her diet. If a substitute or a complement to breast milk is necessary, neither goat's milk nor soy milk formula are adequate. Heat treatment alone will not be sufficient to make cow's milk hypoallergenic. Only a combination of protein hydrolysis and managed heat treatment can make cow's milk hypoallergenic and retain its nutritional value. This nutritional value should be assessed by animal studies and also by studying infant growth. The hypoallergenicity of a formula can be studied in vitro and with animal tests, but only clinical trials on human infants will prove its efficacy.

Soy- and shrimp-specific IgE responses in orally and intraperitoneally immunized mice.

Female C3H/HeJ mice were immunized parenterally and/or intragastrically with two important food allergens, soy and shrimp. Although both preparations elicited specific IgE production when administered intraperitoneally, anti-shrimp titers were consistently higher. Soy or shrimp administration, i.g. with Pertussis adjuvant (intraperitoneally or intragastrically) did not induce detectable reaginic antibody. Animals treated with soy intragastrically were unable to produce a soy-specific IgE response upon intraperitoneal immunization. The combination of soy and Pertussis (intragastric) did, however, abrogate this tolerizing effect on IgE synthesis. In contrast to soy, the shrimp antigens (intragastric) did not act as tolerogens and were found to enhance subsequent reaginic responses produced by intraperitoneal immunization. These results are considered with respect to mechanisms operative in food allergy.

Ultrastructural localization of glycinin and beta-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method.

beta-Conglycinin (7 S globulin) and glycinin (11 S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7 S globulin could not be detected.

Allergenicity of whey protein: its modification by tryptic in vitro hydrolysis of the protein.

Enzymatic in vitro hydrolysis was evaluated as a possible treatment to abolish the allergenicity of whey proteins in view of their use in infant formulas. Guinea pigs without prior immunological contact (including fetal life) with cow's milk were fed various preparations of cow's milk proteins. Oral exposure to milk or untreated whey protein led to anaphylactic sensitization of the animals. In contrast, trypsin-hydrolyzed whey protein and a peptide preparation produced from the tryptic hydrolysate by ultrafiltration were devoid of sensitizing capacity by the oral route. The hydrolysate (crude or purified) was also ineffective in triggering local or systemic anaphylaxis in previously sensitized animals.

Research and development of infant formulae with reduced allergenic properties.

Infant formulae based on hydrolyzed proteins or elemental diets offer the best treatment of cow's milk allergy whenever exclusive breast-feeding is not possible. In situations with a family history of atopy, formulae using other protein sources such as soya or chicken meat can also be a good preventive measure. The food industry needs reliable research methods for the evaluation of every new option before considering clinical trials. Allergenicity of cow's milk proteins was evaluated by the induction of mouse monoclonal reagins and also by oral administration of milk preparations to mice and guinea pigs. Animals initially raised on commercial diets containing about 1% milk whey could not be sensitized. Maintenance of a milk-free diet from pregnancy until weaning, and feeding milk in a liquid form led to an optimal sensitization of the guinea pigs. These animals suffered systemic anaphylaxis and their sera sensitized skin in virgin hosts. Under optimal conditions, while giving liquid preparations to drink, a considerable proliferation of the milk flora occurred. As no mucosal alterations could be detected, primary gut damage due to infection was probably not the triggering factor for oral sensitization. Bacterial products (e.g., endotoxins, enterotoxins) could stimulate the gut response towards milk proteins, either due to an adjuvant effect or to increased mucosal permeability. Microbial contamination of milk is practically unavoidable and it can generally induce biologic activity, e.g., fresh milk regularly gives a positive limulus test for endotoxin by the time it is consumed or processed. Reduction of bacterial contaminants by milk protective factors might help to prevent oral sensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysozyme, an abomasal enzyme in the ruminants.

A strong lytic activity against Micrococcus luteus was demonstrated in abomasal secretions from calf, adult cattle, goat and sheep. This bacteriolytic activity was undetectable in other secretions. Bacteriolysis was caused by a glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) and was further characterized in the calf. This lysozyme also displayed significant chitinase activity. Immunofluorescence microscopy confirmed the secretion of lysozyme by abomasal gastric glands exclusively. Electrofocusing revealed multiple molecular forms, the predominant one (more than 80%) being characterized by Mr approx. 15,000, pH optimum 5.0, pl 7.5 and remarkable conformational stability. The lytic activity of lysozyme was ionic strength dependent and competitive inhibition was observed with both N-acetyl glucosamine and N-acetyl-muramic acid. Amino-acid analysis demonstrated common characteristics with known lysozymes, i.e. four disulphide bridges, two proline and N-terminal lysine. Structural homology between the three ruminant lysozymes was established by immunological cross-reactivity.

Calf rennet lysozyme.

A glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) was isolated from calf rennet. This lysozyme was also present in abomasal secretions from calf and adult cattle. Multiple molecular forms revealed by electrofocusing might be artefacts. The main enzyme form had Mr approx. 15 000, pH optimum 5.0, pI7.5, and a remarkable conformation stability. Competitive inhibition was observed with both N-acetylglucosamine and N-acetylmuramic acid, with apparent Ki values of 29 mM and 2.4 mM respectively. The isolated enzyme also displayed significant chitinase activity.

Treatment of infantile E. coli gastroenteritis with specific bovine anti-E. coli milk immunoglobulins.

A milk immunoglobulin concentrate (MIC) containing antibodies to enteropathogenic E. coli strains was prepared by hyperimmunisation of pregnant cows and using the milk obtained during the first 6 to 8 days of lactation. The sterile concentrate contained 70 to 80% protein and 35 to 40% immunoglobulin. The antibacterial activity was measured by bacterial passive agglutination, bacteriostatic activity in vitro, phagocytic clearance in vivo, and a protection test in mice. Though differences in titers were observed, adequate immunologic activity was demonstrated by these tests. Clinical studies were performed with 60 patients (aged 10 days to 18 months) suffering from diarrhoea with isolation of enteropathogenic E. coli. They were treated for 10 days with MIC and stool cultures were done prior to, during, and 2, 3 and 4 days after termination of treatment. Among 51 patients infected with E. coli strains incorporated in the vaccine, stool cultures became negative in 43 (84.3%) after treatment with MIC and 8 remained positive. Nine patients infected with strains O 78:K80(B-) and O 114:K--(B-)--which were not included in the vaccine used for immunisation--served as controls. Only one patient in this group became negative. If all patients receiving antibiotics for non intestinal infections during the treatment period are omitted the results remained unaltered: MIC was effective in 32 out of 38 patients (84.2%). These differences were highly significant. These results provide evidence that treatment with specific MIC is effective in eliminating enteropathogenic E. coli from the intestine.

Studies of mouse-brain-associated T-lymphocyte antigens.

Xeno- and alloantigens shared by mouse brain, thymocytes and thymus-derived lymphocytes were isolated from brain by butanol extraction, followed by Sephadex G-200 column chromatography and preparative polyacrylamide gel electrophoresis, both in the presence of SDS. The antigenic activity, as tested by inhibition of complement-mediated cytotoxicity using a xenogeneic antiserum directed against T cell-specific antigens (anti-MTLA), was enriched 50-fold during the purification procedure. Xenogeneic and allogeneic antisera which were specific for mouse T-lymphocytes, could be obtained by immunization of rabbits and mice with the partially purified antigens.