In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization

OBJECTIVE: To explore the use of β-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized β-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized β-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to β-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized β-lactoglobulin. CONCLUSIONS: The polymerization of β-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.

[Allergic reaction of goats and cows milk proteins]

Systemic anaphylaxis test in mouse showed that the visual effect of injection of cow casein, whey, casein fraction and bovine beta-lactoglobulin was strong. Similar results were found when goat casein, Kappa-casein and beta-casein were injected. The visual effect of injection of beta-lactoglobulin and alpha[s],-casein was low. No response was found when bovine and goat alpha-Lactalbumin was injected in animals. Passive cutaneous anaphylaxis test in mouse showed that the reaction area of cow casein injection was 0.63 cm[2] in comparison with 0.19 cm[2] for goat casein. The reaction of cow beta-lactoglobulin injection was 0.12 cm[2], while no reaction was occurred when goat beta-lactoglobulin, goat and cow alpha-lactalbumin were injected. The percentage of degranulation of mast cells when treated with cow raw milk, casein, whey, beta-lactoglobulin and alpha-lactalbumin were 32.11, 100, 41.80, 90.01 and 12.73% respectively, In comparison with 14.33, 80.19, 34.73, 39.57 and 10.86% respectively for the same proteins in goat milk

[Study of the immunological cross-reactions between goats and cows milk proteins]

Immunoelectrophoresis analysis showed immunological cross reactions between goat and cow milk caseins which belong to beta-casein, however, no such reaction were observed between goat and cow beta-lactoglobulin and alpha-lactalbumin. Systemic anaphylaxis test in guinea pigs showed strong immunological reactions between goat and cow milk proteins, injection of cow milk in animal's vein, which fed on cow milk caused 100% mortality. Same results were also obtained with injection of goat milk. Passive hemagglutination test against goat and cow milk was used to estimate antibody titer in guinea pigs serum, which fed cow's milk. The obtained results showed that the highest titer was found against casein followed by beta-lactoglobulin and alpha-lactalbumin for cow milk proteins, while for goat milk proteins the highest titer was found against casein followed by alpha-lactalbumm and beta-lactoglobulin. The titer of antibodies against goat alpha[s]-casein and Kappa-casein was lower than that for cow milk, the behavior of goat and cow beta-casein was similar for both proteins

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.