Rodolphe Fritsché

Induction of systemic immunologic tolerance to β-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 cows milk formulas were compared for their capacity to induce tolerance to cows milk proteins. METHODS Five-week-old Sprague-Dawley rats were fed cows 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 cows 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.

Cow's Milk Protein Allergy and Possible Means for Its Prevention

Mother’s milk is the ideal nutrition for neonates because it best ensures healthy shortand long-term development. It enhances immune functions and is hypoallergenic, and the overall composition helps to establish bifidogenic gut flora.1–3 Despite the use of modern infant formulae, exclusively breast-fed infants tend to be somewhat healthier than formula-fed infants in not only developing countries but also industrialized countries.4–7 Atopic diseases in infants and children have a prevalence of about 35% and are among the most important morbidity factors in industrialized countries.8–10 Moreover, their incidence is growing and in recent decades has roughly doubled in Western societies.11 The European Allergy White Paper has emphasized this increase with an overwhelming summary of epidemiologic data from throughout Europe.12 Double-blind, placebo-controlled food challenge has shown that general food allergies occur in about 5% to 10% of the overall infant and small-child population.13,14 In most studies, cow’s milk protein allergy (CMPA) is the most common in young infants, with a 2% to 6% incidence.15–17 During childhood, the frequency of egg allergy increases and peanut allergy seems to be a common problem in children in the United States.12–14,44,49 In exclusively breast-fed infants, the incidence of CMPA is only about 0.5%, perhaps up to 1.5% at the most.18,19 However, about 20% of infants with elevated risks of allergy will develop CMPA during their first year of life if fed on cow’s milk protein.20,21 Beyond the well-known genetic factors,22 which are not thought to have increased within a generation, contemporary research has focused on environmental differences between the past and present and between Western and non-Western lifestyles.23 Current theories have suggested a probable association between allergies and Western lifestyles, which tend to be characterized by families with few children, better-educated women having their first child at a later age, less crowded homes, and increased standards of hygiene.24 Two new theories regarding the effects of Western lifestyles on the increasing incidence of allergies are the hygiene and gut-flora hypotheses. The hygiene hypothesis is based on a switch in the balance between the T-helper cell 1 and 2 (Th1 and Th2) cytokine systems. Under normal conditions, Th1 cytokines and especially interferon-g downregulate Th2 lymphocytes and thus the production of Th2 cytokines, especially interleukin (IL)–4. If the Th1 cytokine system is insufficiently stimulated by viral and bacterial infections, the balance may be lost in favor of the Th2 system.25–27 The same is true if neonates have reduced interferon-g production.89,90 According to this hypothesis, because Th2 cytokines such as IL-4 are responsible for increased immunoglobulin (Ig)–E reaction, this upregulation of the Th2-system increases the likelihood of an IgE-mediated allergic reaction. Regarding other factors that can influence the balance between cytokines Th1 and Th2 and be influenced by nutrition, it is noteworthy that prostaglandin E2 (PGE2) has a suppressive effect on interferon-g secretion but not on the production of IL-4 or IL-5, whose production increases as a result.144 The production of PGE2 is influenced through the quantity of linoleic acid consumed and then converted into arachidonic acid, which in turn is a precursor of PGE2. A change in diet to a higher intake of v-6 fatty acids and a reduced intake of v-3 fatty acids, as it is seen nowadays, therefore should lead to an increased activity of PGE2 and a polarisation toward Th2-type immunity.145 The gut-flora hypothesis assumes that normal intestinal flora play a major role in the early development of the immune system and oral tolerance.28 Lipopolysaccharides that are produced by gram-negative bacteria such as enterobacteria play an active role in inducing oral tolerance.29 Recent studies from Scandinavia have shown that, during the first few months after birth, colonization of the neonatal intestinal flora switches from the usual gram-negative to gram-positive bacteria.30 The delayed colonization of the intestine with enterobacteria can drastically reduce exposure of the developing immune system to lipopolysaccharides, which suggests a negative effect on the development of oral tolerance.29 Further, recent findings have shown that lactobacilli may have the capacity to degrade cow’s milk proteins and modify their immunomodulatory activity.31 Thus, probiotics that favor the establishment of immunopositive lactobacilli and bifidus bacteria may play a major role in future allergy-prevention programs.

Determination of Cow Milk Formula Allergenicity in the Rat Model by in vitro Mast Cell Triggering and in vivo IgE Induction

The allergenicity of cow milk formulas was assessed in vitro by their ability to trigger prelabelled 3H-serotonin release from normal rat mast cells sensitized passively with rat reaginic anti-cow milk protein antisera. It was found that enzymatically hydrolyzed milk formulas had 1,000 (BEBAHA) to over 100,000 (ALFARE) times lower mast cell-triggering capacities than a standard milk formula (NAN). In vivo, these hydrolyzed formulas induced 100 (BEBAHA) to 10,000 (ALFARE) times less IgE antibodies than the standard milk formula.

Animal models in food allergy: assessment of allergenicity and preventive activity of infant formulas.

Food allergies occur in about 5-10% of the overall infant and small-child population. Cows milk protein allergy (CMPA) is the most common in young infants, with a 2-4% incidence. When breastfeeding is not possible, hypoallergenic (HA) cows milk based formulas are usually given during the first months of life for prevention of CMPA. Depending on primary (sensitization) or secondary (triggering) prevention, the requested quality of HA formulas may be different. Besides in vitro methods, in vivo and ex vivo animal models are helpful in assessing residual allergenicity and the preventive effect of HA formulas. The sensitizing capacity of a formula can be examined by either the parenteral rat (IgE), the guinea pig (IgG1a mediated) or the oral mouse (IgE) models. The triggering IgE mediated allergenicity is tested by a parenteral rat model with oral gavage for intestinal mast cell protease (RMCPII) release. These animal models are also used for testing the oral tolerance inducing capacities of formulas. Together with cellular in vitro assays, animal models are very helpful in predicting allergenicity and the tolerogenic potential of HA infant formulas.

Oral tolerance elicited in mice by β-lactoglobulin entrapped in biodegradable microspheres

Oral administration of antigen is known to be appropriate for some vaccine purposes as well as oral tolerance induction. In the present study, oral administration of beta-lactoglobulin (BLG) loaded poly(D,L-lactide-co-glycolide) (D,L-PLG) microspheres induced tolerance was evaluated. A single feeding of 5 micrograms of encapsulated BLG tolerized BALB/c mice to subsequent BLG parenteral challenge, suppressing the specific humoral, intestinal and cellular responses. The tolerogenic efficient dose was then reduced 10,000 times, compared to oral administration of soluble BLG. This suggests that loading food proteins into D,L-PLG microspheres might be a potential tool for inducing oral tolerance with allergens.

Immunoglobulin E suppression and cytokine modulation in mice orally tolerized to beta-lactoglobulin.

This study was designed to confirm the tolerogenic properties of β‐lactoglobulin in a mouse model and to assess specific oral tolerance induction in humoral and cellular compartments. BALB/c mice were fed β‐lactoglobulin (BLG) or whey proteins at different ages and subsequently intraperitoneally challenged 5 days later with both BLG and a non‐specific antigen, ovalbumin (OVA). Three weeks later, oral tolerance induction was analysed in CMP‐fed, versus saline‐fed mice, by measuring specific seric and intestinal antibody responses, delayed‐type hypersensitivity (DTH), specific splenocyte proliferation, and cytokine secretion patterns. Three‐week‐old mice fed high doses of either whey proteins or BLG (respectively 3 mg/g or 5 mg/g of body weight) were found to achieve oral tolerization. At humoral and mucosal levels, anti‐BLG immunoglobulin E (IgE) were suppressed in these groups when compared with saline fed mice. With respect to cellular responses, systemic DTH and lymphocyte proliferation to BLG were also inhibited in CMP‐fed mice. Weaning time was determined to be the best period for oral tolerance induction. Kinetic analyses showed however, that a minimum of 2 weeks was required for oral tolerance detection. Finally, cytokine profiles indicated a reciprocal decrease of interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) versus an increase of IL‐10 and transforming growth factor‐β (TGF‐β) secretions in tolerized mice. Taken together, these results clearly showed that oral administration of high doses of cows’ milk proteins can induce significant hyposensitization in mice, in a specific inhibition of T helper 1 (Th1) lymphocytes with the participation of suppressor cytokines.

Specific effects of denaturation, hydrolysis and exposure to Lactococcus lactis on bovine beta-lactoglobulin transepithelial transport, antigenicity and allergenicity

Background Food allergy in developed countries represents a growing concern as reflected by epidemiological studies, indicating that up to 4% of the overall population is affected. Reduction of symptoms takes place following eviction or processing of some allergens. However, it cannot be predicted which structural changes will be associated with significant effects on the allergenicity.

In Vitro Determination of the Allergenic Potential of Technologically Altered Hen’s Egg

Hens egg allergy represents one of the most common and severe IgE-mediated reactions to food in infants and young children. It persists, however, in many cases also lifelong. Therefore, the aim of this study was the detailed analysis of a technological process used to reduce the allergenic potential of hens egg. The investigation focused on the pasteurized egg as starting material, intermediate, and final products of a nine-step manufacturing process performed for use of eggs in convenience products appropriate for allergic individuals. The steps consisted of a combination of various heat treatments and enzymatic hydrolyses. The alterations were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, enzyme allergosorbent test (EAST) inhibition, and mass spectrometry. Thereby it could be demonstrated that the allergenic potential of the raw material was reduced from step to step, and despite the known stability against heat and proteolysis of certain egg proteins, the total allergenic potential was finally below 1/100 that of the starting material without a significant change in texture and flavor as evaluated in various products.

INDUCTION OF ORAL TOLERANCE TO COW'S MILK PROTEINS IN RATS FED WITH A WHEY PROTEIN HYDROLYSATE

Animal models have shown that oral administration of an antigen induces a specific immunologic tolerance to this antigen. Induction of such an oral tolerance has been well documented with intact proteins. The purpose of our study was to determine whether oral tolerance to cows milk proteins (CMP) can be induced also with protein peptides contained in either partially hydrolysed or extensively hydrolysed cows milk formulas. Five-week-old Sprague-Dawley rats were fed cows milk formulas ad libitum before they were parenterally challenged with CMP. The modulation of specific IgE anti-CMP antibodies and intestinal mast cell stimulation were measured. Results showed that preventive feeding of rats with a partially hydrolysed cows milk formula suppresses specific IgE anti-CMP antibodies as well as IgE mediated mediator release (rat mast cell protease RMCPII) from intestinal mast cells. An extensively hydrolysed cows milk formula was unable to achieve the induction of such an oral tolerance. Our experiments show that selected peptides of CMP may induce a specific oral tolerance to CMP.

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

ABSTRACT Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cows milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.