Effects of growing-up milk supplemented with prebiotics and LCPUFAs on infections in young children.

OBJECTIVE: The aim of this study was to investigate the effect of growing-up milk (GUM) with added short-chain galacto-oligosaccharides (scGOS)/long-chain fructo-oligosaccharides (lcFOS) (9:1) (Immunofortis) and n-3 long-chain polyunsaturated fatty acids (LCPUFAs) on the occurrence of infections in healthy children attending day care centres. METHODS: In a randomised double-blind controlled, parallel, multicountry intervention study, 767 healthy children, ages 11 to 29 months, received GUM with scGOS/lcFOS/LCPUFAs (the active group, n = 388), GUM without scGOS/lcFOS/LCPUFAs (the control group, n = 379), or cow's milk (n = 37) for 52 weeks. The primary outcome measure was the number of episodes of upper respiratory tract infections or gastrointestinal infections based on a combination of subject's illness symptoms reported by the parents during the intervention period. RESULTS: Children in the active group compared with the control group had a decreased risk of developing at least 1 infection (299/388 [77%] vs 313/379 [83%], respectively, relative risk 0.93, 95% confidence interval [CI] 0.87-1.00; logistic regression P = 0.03). There was a trend toward a reduction (P = 0.07) in the total number of infections in the active group, which was significant when confirmed by one of the investigators (268/388 [69%] vs 293/379 [77%], respectively, relative risk 0.89, 95% CI 0.82-0.97; P = 0.004, post hoc). More infectious episodes were observed in the cow's milk group, when compared with both GUM groups (34/37 [92%] vs 612/767 [80%], respectively, relative risk 1.15, 95% CI 1.04-1.28). CONCLUSIONS: This is the first study in children to show a reduced risk of infection following consumption of GUM supplemented with scGOS/lcFOS/n-3 LCPUFAs. The borderline statistical significance justifies a new study to confirm this finding.

Invariant natural killer T cells contribute to the allergic response in cow's milk protein-sensitized mice.

BACKGROUND: Little is known about the contribution of the invariant natural killer T (iNKT) cells in the onset of food allergy. Using a mouse model for cow's milk allergy the function of iNKT cells was investigated. METHODS: Mice were sensitized orally with casein or whey proteins. One hour before the sensitizations the mice were injected intraperitoneally with α-galactosylceramide (αGalCer) or control. One week after the last sensitization acute allergic skin reactions were measured. Furthermore, in the liver, spleen and mesenteric lymph nodes (MLN) percentages of iNKT cells were analyzed and liver lymphocyte restimulation assays were performed. RESULTS: Whey- or casein-sensitized mice treated with αGalCer showed enhanced acute allergic skin reactions. The percentage of iNKT cells in the liver of sensitized mice was reduced compared to sham-sensitized mice. αGalCer treatment was found to deplete iNKT cells in the liver of sensitized as well as sham-sensitized mice, and these hepatocytes did not respond to ex vivo restimulation with αGalCer. αGalCer treatment did not reduce iNKT cell percentages in the spleen and MLN of sham-sensitized mice but abrogated the increase in iNKT cell percentage in the spleen upon whey sensitization, whereas it enhanced the iNKT cell percentage in the MLN of casein-sensitized mice. Due to the repeated application of αGalCer, livers were functionally depleted of iNKT cells. This resulted in an increased allergic effector response which was most pronounced in whey-sensitized mice and associated with enhanced whey-specific immunoglobulin levels. CONCLUSION: iNKT cells may suppress cow's milk allergic symptoms in mice and may differentially regulate oral sensitization for casein and whey.

A potential role for CD25+ regulatory T-cells in the protection against casein allergy by dietary non-digestible carbohydrates.

Dietary non-digestible carbohydrates reduce the development of cows' milk allergy in mice. In the present study, the contribution of CD25+ regulatory T-cells (Treg) was investigated using in vivo Treg depletion and adoptive transfer studies. Mice were orally sensitised with casein and fed a diet containing 2 % short-chain galacto-, long-chain fructo- and acidic oligosaccharides (GFA) or a control diet. Donor splenocytes of mice sensitised with casein and fed the GFA or control diet were adoptively transferred to naive recipient mice, which were casein- or sham-sensitised and fed the control diet. In addition, in vivo or ex vivo CD25+ Treg depletion was performed using anti-CD25 (PC61). The acute allergic skin response upon intradermal casein challenge and casein-specific Ig were determined. Furthermore, T-helper (TH) 1 and TH2 cell numbers were analysed in the mesenteric lymph nodes. The oligosaccharide diet strongly reduced the development of the acute allergic skin response, which was abrogated by the in vivo anti-CD25 treatment. The diet enhanced the percentage of TH1 cells and tended to reduce the percentage of TH2 cells in casein-sensitised mice. Recipient mice were protected against the development of an acute allergic skin response when transferred with splenocytes from casein-sensitised GFA-fed donor mice before sensitisation. Ex vivo depletion of CD25+ Treg abrogated this transfer of tolerance. Splenocytes from sham-sensitised GFA-fed donor mice did not suppress the allergic response in recipient mice. In conclusion, CD25+ Treg contribute to the suppression of the allergic effector response in casein-sensitised mice induced by dietary intervention with non-digestible carbohydrates.

Non-digestible oligosaccharides reduce immunoglobulin free light-chain concentrations in infants at risk for allergy.

Prebiotic oligosaccharides influence the intestinal microbiota and can positively modulate the infant's immune system. It was demonstrated that a special prebiotic mixture (Immunofortis(®)) of short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) can reduce the cumulative incidence of atopic dermatitis (AD) in infants at risk for allergy as determined using the AD symptom score (SCORAD). Additionally, it was shown very recently that immunoglobulin free light-chain (Ig-fLC) might be involved in the pathophysiology of allergic disease. Increased Ig-fLC concentrations were found in patients suffering from AD, cow's milk allergy, allergic rhinitis, or asthma. In this study, the effect of supplementation of scGOS/lcFOS on the Ig-fLC plasma concentrations in infants at risk for allergy was assessed. The plasma kappa and lambda Ig-fLC concentrations were measured in a double-blind, placebo-controlled, randomized trial, in which infants at risk for developing allergic disease received a hypoallergenic whey formula containing 8 g/l of the scGOS/lcFOS mixture (n = 34) or maltodextrin as a placebo (n=40) for 6 months. After intervention, plasma samples were collected, and total plasma concentrations of lambda and kappa Ig-fLC were analyzed using ELISA. Total kappa and lambda Ig-fLC plasma concentrations were higher in infants suffering from AD when compared to infants without any sign of AD. In infants receiving the prebiotic mixture, the Ig-fLC levels were significantly lower compared to the placebo-fed infants (p<0.001). Interestingly, lambda Ig-fLC concentrations were positively correlated with total IgE (p<0.05). These data demonstrate for the first time that the specific scGOS/lcFOS mixture lowered kappa and lambda Ig-fLC plasma concentrations in infants at high risk for allergies when compared to infants receiving placebo formula. Because Ig-fLC concentrations were increased in infants suffering from AD, this may have contributed, at least in part, to the reduced incidence in AD as described previously. This suggests a possible role for Ig-fLC in the pathophysiology of AD in infants at risk for allergy development.

Oral tolerance induction by partially hydrolyzed whey protein in mice is associated with enhanced numbers of Foxp3+ regulatory T-cells in the mesenteric lymph nodes.

BACKGROUND: Hypoallergenic formulas are considered a good option for infants at risk for cow's milk allergy. The aim of this animal study was to investigate whether whey hydrolyzates (WH) have the capacity to induce oral tolerance to whey. METHODS: Whey, partial or extensive WH was given via gavages to naïve mice prior to oral whey sensitization using cholera toxin as an adjuvant. The acute allergic skin response, mouse mast cell protease-1 (mMCP-1), whey-specific IgE, IgG(1) and effector Th2-cells, Th1-cells, and Foxp3(+) regulatory T-cells were determined in the mesenteric lymph nodes (MLN). MLN cells from tolerized mice were adoptively transferred to naïve recipient mice prior to whey sensitization. RESULTS: In contrast to the extensive WH, pre-treatment of naïve mice with whey or partial WH reduced the acute allergic skin response and mast cell degranulation after whey challenge. However, only treatment with whey prevented the generation of serum-specific IgE/IgG(1) . In partial WH tolerized mice, Foxp3(+) regulatory T-cell numbers in the MLN were increased compared to whey-sensitized mice. Both whey and partial WH treatment showed a tendency toward a decreased number of effector Th2-cells. Transfer of MLN cells from tolerized mice protected recipient mice from developing an acute allergic skin response. CONCLUSION: These results show that partial WH with limited sensitizing properties reduced the effector response upon whey challenge. This effect is transferable using MLN cells and was associated with enhanced Foxp3(+) regulatory T-cell numbers in the MLN. Partial WH retained the capacity to induce active immune suppression in mice which may be relevant for allergy prevention.

Contribution of IgE and immunoglobulin free light chain in the allergic reaction to cow's milk proteins.

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of young infants. In previous murine studies it was observed that allergic sensitization to the major cow's milk allergens casein and whey led, respectively, to IgE-independent and IgE-dependent clinical responses. OBJECTIVES: In this study the involvement of immunoglobulin free light chains (Ig-fLCs) in the hypersensitivity response to cow's milk proteins was explored in mice, and Ig-fLC serum levels were determined in children affected by CMA or atopic dermatitis (AD). METHODS: Mice were orally sham, casein, or whey sensitized. Acute allergen-specific skin responses were determined, and serum immunoglobulin and Ig-fLC concentrations were measured. Ig-fLC dependency was validated by using the Ig-fLC blocker F991 in actively and passively sensitized mice. Ig-fLC serum concentrations were measured in a cohort of infants with CMA and infants with AD. RESULTS: After sensitization, no specific IgE was detectable in sera of casein-sensitized mice, whereas specific IgE levels were enhanced in whey-sensitized mice. Instead, Ig-fLC levels were increased in sera from casein-sensitized mice. Furthermore, blocking Ig-fLCs strongly diminished the allergic skin responses not only in casein-sensitized mice but also in mice transferred with splenocyte supernatants of casein-sensitized mice. In both patients with CMA and patients with AD, serum Ig-fLC concentrations were significantly enhanced. CONCLUSIONS: This study indicates that sensitization with cow's milk proteins can lead to both IgE-dependent and Ig-fLC-dependent allergic hypersensitivity responses. Also, in children affected with CMA or AD, serum Ig-fLC concentrations were increased, implying the relevance of Ig-fLC measurements in the diagnoses of human allergic disease.

Oligosaccharide-induced whey-specific CD25(+) regulatory T-cells are involved in the suppression of cow milk allergy in mice.

Dietary intervention with a unique prebiotic nondigestible carbohydrate mixture has been shown to reduce the development of allergic disease in infants at risk. In this study, the involvement of CD25(+) regulatory T-cells (Treg) in the carbohydrate-induced effects was investigated in mice orally sensitized with whey using adoptive transfer experiments. Donor mice were sensitized with whey and fed a diet containing short-chain galacto-, long-chain fructo- and acidic-oligosaccharides, or a control diet starting 2 wk before sensitization. The acute allergic skin reaction upon intradermal whey challenge was determined and whey-specific Ig were measured. Splenocytes of the donor mice were transferred to naïve recipient mice after partial ex vivo depletion of CD25(+) Treg. The prebiotic diet clearly diminished the acute allergic skin reaction (P < 0.001). Whey-sensitized recipient mice transferred with splenocytes from whey-sensitized, prebiotic-fed donor mice displayed almost complete prevention of the acute allergic skin reaction compared with mice receiving cells from sham-sensitized, prebiotic-fed donor mice (P < 0.001). Partial depletion of CD25(+) T-cells inhibited these effects (P < 0.001), although IgE sensitization was not prevented. This study indicates the involvement of whey-specific CD25(+) Treg in the suppression of the allergic effector response induced by dietary intervention with prebiotics.

Exposure of intestinal epithelial cells to UV-killed Lactobacillus GG but not Bifidobacterium breve enhances the effector immune response in vitro.

BACKGROUND: Intestinal bacteria and intestinal epithelial cells (IEC) may modulate the mucosal immune response. In this study, immune modulation by Lactobacillus GG (LGG) and Bifidobacterium breve (Bb1, Bb2) in the presence or absence of IEC was addressed in an in vitro transwell co-culture model. METHODS: UV-killed LGG,Bb1, Bb2 or Toll-like receptor (TLR) 2 or nucleotide oligomerization domain (NOD) 2 ligands were added directly to unstimulated or anti-CD3/CD28-stimulated PBMC, or applied apically to human IEC (HT-29) co-cultured with PBMC. A mixture of live bacteria was used as reference. The effect on T helper 1 (IFN-gamma, IL-12), T helper 2 (IL-13), inflammatory (TNF-alpha) and regulatory (IL-10) cytokine secretion was determined. RESULTS: Both UV-killed LGG and Bb enhanced IL-12, IFN-gamma, TNF-alpha and IL-10, and reduced IL-13 secretion when added directly to stimulated PBMC, similar to live bacteria. IEC reduced IL-13, IFN-gamma and IL-10 secretion by stimulated PBMC. Apically added LGG, TLR2 and NOD2 ligands,but not Bb, enhanced IFN-gamma, IL-12 and/or TNF-alpha secretion. Bacteria did not induce cytokine secretion when added to HT-29/unstimulated PBMC co-cultures, whereas direct incubations with PBMC did. CONCLUSION: UV-killed LGG as well as Bb supported a T helper 1 and/or regulatory phenotype when added directly to activated PBMC, similar to live bacteria. In contrast, LGG, TLR2 or NOD2 ligands - but not Bb - enhanced T helper 1 type cytokine secretion when added to IEC, while IL-10 secretion remained suppressed. Co-cultures combining IEC and PBMC may reveal differences between bacterial strains relevant for the in vivo situation.

Acute allergic skin response as a new tool to evaluate the allergenicity of whey hydrolysates in a mouse model of orally induced cow's milk allergy.

Hypoallergenic milk formulae are used for cow's milk allergic infants and may be a good option for infants at risk. Clinical studies have shown that the protein source or the hydrolysis methodology used may influence the effectiveness in infants stressing the importance of adequate pre-clinical testing of hypoallergenic formulae in an in vivo model of orally induced cow's milk allergy. This study was undertaken to introduce a new read-out system to measure the residual allergenicity of whey hydrolysates on both the sensitization and challenge phase of orally induced cow's milk allergy in mice. Mice were sensitized orally to whey or a partial whey hydrolysate (pWH) to measure the residual sensitizing capacity. To predict the residual allergenicity of hydrolysates, whey allergic mice were challenged in the ear with pWH, extensive whey hydrolysate or an amino acid-based formula. An acute allergic skin response (ear swelling at 1 h), whey-specific serum antibodies, and local MCP-1 concentrations were measured. In contrast to whey, oral sensitization with pWH did not result in the induction of whey-specific antibodies, although a minor residual skin response to whey was observed after challenge. Skin exposure to whey hydrolysates showed a hydrolysation dependent reduction of the acute allergic skin response in whey allergic mice. In contrast to whey, skin exposure to pWH did not enhance tissue MCP-1 levels. The acute allergic skin response in mice orally sensitized to cow's milk proteins reveals a new pre-clinical tool which might provide information about the residual sensitizing capacity of hydrolysates supporting the discussion on the use of hypoallergenic formulae in high risk children. This mouse model might be a relevant model for the screening of new hypoallergenic formulae aimed to prevent or treat cow's milk allergy.

Cow milk allergy symptoms are reduced in mice fed dietary synbiotics during oral sensitization with whey.

Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. The purpose of this study was to compare effects of dietary supplementation with a prebiotic mixture (Immunofortis), a probiotic strain [Bifidobacterium breve M-16V], or a synbiotic diet combining both on the outcome of the allergic response when provided during oral sensitization with whey in mice. Mice were fed diets containing 2% (wt:wt) Immunofortis and/or the B. breve M-16V (n = 6/group). The acute allergic skin response was determined by measuring ear swelling. Antigen-induced anaphylaxis was scored. Furthermore, whey-specific serum immunoglobulins and mouse mast cell protease-1 (mMCP-1) were determined. In mice fed the synbiotic mixture, the allergic skin response and the anaphylactic reaction were strongly reduced compared with whey-sensitized mice fed the control diet (P < 0.01). Immunofortis or B. breve M-16V alone were significantly less effective in reducing the allergic skin response than the synbiotic diet and did not reduce the anaphylactic reaction. The whey-specific IgE and IgG(1) responses were not affected; however, IgG(2a) was greater in all treated groups than in the control group (P < 0.05). Serum mMCP-1 concentrations, reflecting mucosal mast cell degranulation, were lower in mice fed synbiotics compared with those fed the control diet (P < 0.01). Dietary supplementation with Immunofortis, B. breve M-16V, and particularly the synbiotic mixture, provided during sensitization, reduces the allergic effector response in a murine model of IgE-mediated hypersensitivity that mimics the human route of sensitization. This model shows the potential for dietary intervention with synbiotics in reducing the allergic response to food allergens.

Acute allergic skin reactions and intestinal contractility changes in mice orally sensitized against casein or whey.

BACKGROUND: Cow's milk allergy (CMA) is characterized by hypersensitivity against casein or whey, affecting 2.5% of young infants. The pathogenesis of CMA involves IgE as well as non-IgE-mediated reactions and clinical symptoms are found in the skin, lungs and gastrointestinal tract. In this study, local and systemic immunopathology was determined in whey- or casein-allergic mice. METHODS: Mice were orally sensitized with casein or whey using cholera toxin as an adjuvant. Serum immunoglobulins and the acute allergic skin reaction (ear swelling 1 h after intradermal allergen challenge) were determined to reveal systemic hypersensitivity. Furthermore, pathophysiological changes were assessed within the intestine. RESULTS: An acute allergic skin reaction was induced in both whey- and casein-sensitized mice. In these mice, whey-specific IgE, IgG(1), IgG(2a) and casein-specific IgG(1) levels were found to be increased. In addition, the serum mouse mast cell protease-1 (mMCP-1) concentration was enhanced, reflecting mast cell degranulation. Indeed, the number of mMCP-1-positive mast cells within the colon was diminished in both whey- and casein-sensitized mice. Only in casein-sensitized mice isometric contraction of the colon was reduced, reflecting motility alterations. CONCLUSION: Mice, orally sensitized against casein or whey, revealed an allergen-specific acute allergic skin reaction. In casein-sensitized mice, hypocontractility of the colon reflected pathophysiological changes within the intestine. Allergen-induced ear swelling and intestinal contractility changes are novel parameters in animal models of CMA which may add to the search for new therapeutic strategies to relieve symptoms of CMA.

Estrogenic potency of food-packaging-associated plasticizers and antioxidants as detected in ERalpha and ERbeta reporter gene cell lines.

This study presents the estrogenic potency of 21 food-packaging-associated compounds determined for the first time, using two transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) estrogen receptor (ER) alpha and beta cell lines. Six plasticizers and three antioxidants were slightly estrogenic in the ERalpha cells. The model compounds bisphenol A and nonylphenol, one plasticizer [tris(2-ethylhexyl)trimellitate (TEHTM)], and two antioxidants (propyl gallate and butylated hydroxyanisole) were estrogenic in both ERalpha and ERbeta cells. Compared to estradiol (E2), these compounds appeared to be relatively more estrogenic in the ERbeta cells than in the ERalpha cells. Three sorbitol-based plasticizers activated neither ERalpha nor ERbeta and may be good replacements of existing plasticizers. All responses were additive with the response of E2. This indicates that they may contribute to the total effects of the pool of estrogenic compounds humans are exposed to. The estrogenic potencies of these compounds, together with the suggested beneficial effect of ERbeta-mediated responses and adverse ERalpha-mediated effects, support the importance of detecting characteristics for ERalpha and ERbeta response separately in independent models, as done in the present study.

Regulatory T-cells have a prominent role in the immune modulated vaccine response by specific oligosaccharides.

Regulatory T-cells are increasingly important in vaccine strategies. In a Flu-vaccination model the role of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) and the immune modulation by orally supplied prebiotic oligosaccharides consisting of scGOS/lcFOS/pAOS, were assessed using anti-CD25 (PC61) mediated depletion studies. As expected, in C57BL/6J mice the Flu-vaccination resulted in significantly (p<0.001) increased DTH responses when receiving scGOS/lcFOS/pAOS. In addition, increased T-bet expression of activated CD4(+) T-cells was detected compared to placebo. In vivo depletion of CD25(+) Tregs significantly (p<0.05) increased basal DTH responses, indicating the suppressive function of these CD25(+) Tregs normally present. Surprisingly, in vivo Tregs depletion diminished scGOS/lcFOS/pAOS induced immune modulation completely to control levels (p<0.05). Although no difference in number, percentage or activation of Tregs could be determined after scGOS/lcFOS/pAOS supplementation, changes in Treg function still remains to be investigated. In conclusion, CD25(+) Tregs have an important role in modulated Flu-vaccine responses induced by scGOS/lcFOS/pAOS.