Suppression of T-cell activation by Lactobacillus rhamnosus GG-degraded bovine casein.

Earlier data indicate that Lactobacillus rhomnosus GG ATCC 53103 (L. GG), a commensal intestinal bacterial strain, promotes the degradation of proteins in the gut in vivo, and bovine casein hydrolysed with L. GG-derived proteases suppresses lymphocyte proliferation in vitro. The present study aimed to evaluate the effect of L. GG-degraded bovine casein on T-cell activation, i.e. IL-2 mRNA expression and protein kinase C (PKC) translocation. To this end, Northern blot analyses for IL-2 mRNA expression and PKC assays with and without L. GG-degraded casein were carried out on T cells isolated from 11 healthy adults. Cell cultures in 8-11 experiments contained 1 mg ml(-1) bovine casein in degraded or undegraded form in the presence of a mitogen, i.e. phorbol 12,13-dibutyrate plus calcium ionophore (PBDu + A23187) or anti-CD3. Also IL-2, IL-4 and IFN-gamma syntheses were determined in 24-h culture supernatants. IL-2 mRNA expression was reduced in experiments with L. GG-degraded casein. In parallel, the IL-2 concentration in PBDu + A23187-stimulated culture supernatants, expressed as geometric means (95% confidence interval), decreased from 15,892 (7174-35,203) pg ml(-1) to 4744 (2095-10,742) pg ml(-1) when containing L. GG-degraded casein. L. GG-degraded casein inhibited PKC translocation, the action resembling that of PKC inhibitor, RO31-8220. These results extend previous data on L. GG-degraded casein, showing in vitro the suppression of T-cell activation.

Probiotics: effects on immunity.

The gastrointestinal tract functions as a barrier against antigens from microorganisms and food. The generation of immunophysiologic regulation in the gut depends on the establishment of indigenous microflora. This has led to the introduction of novel therapeutic interventions based on the consumption of cultures of beneficial live microorganisms that act as probiotics. Among the possible mechanisms of probiotic therapy is promotion of a nonimmunologic gut defense barrier, which includes the normalization of increased intestinal permeability and altered gut microecology. Another possible mechanism of probiotic therapy is improvement of the intestine's immunologic barrier, particularly through intestinal immunoglobulin A responses and alleviation of intestinal inflammatory responses, which produce a gut-stabilizing effect. Many probiotic effects are mediated through immune regulation, particularly through balance control of proinflammatory and anti-inflammatory cytokines. These data show that probiotics can be used as innovative tools to alleviate intestinal inflammation, normalize gut mucosal dysfunction, and down-regulate hypersensitivity reactions. More recent data show that differences exist in the immunomodulatory effects of candidate probiotic bacteria. Moreover, distinct regulatory effects have been detected in healthy subjects and in patients with inflammatory diseases. These results suggest that specific immunomodulatory properties of probiotic bacteria should be characterized when developing clinical applications for extended target populations.

Interleukin-10 generation in atopic children following oral Lactobacillus rhamnosus GG.

Oral Lactobacillus rhamnosus GG ingestion for 5 days to 4 weeks has been shown to alleviate clinical symptoms of gastrointestinal inflammation and atopic dermatitis. To determine whether oral Lactobacillus rhamnosus GG may act by generating immunosuppressive mediator in atopic children. Lactobacillus rhamnosus GG (ATCC 53103) at a daily dose of 2 x 1010 cfu was added for 4 weeks to the diets of nine children (mean age, 21 months) with atopic dermatitis. Blood and faecal samples were collected before supplementation and at early (2 weeks) and late stage (4 and 8 weeks from the beginning). The concentrations of interleukin-6 (IL-6), IL-10, IL-12, tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) in sera, as well as the production of IL-2, IL-4, IL-10 and IFNgamma in mitogen-induced peripheral blood mononuclear cells, were assessed. Secretory IgA and TNFalpha were also determined in faeces. The serum IL-10 concentration differed significantly between before, early and late samples (P < 0.001) due to the elevation of serum IL-10 in the later phase of oral Lactobacillus rhamnosus GG ingestion. The enhancement of IL-10 production in mitogen-induced cultures preceded the rise in serum IL-10. The enhanced IL-10 generation in vivo substantiates the anti-inflammatory properties of specific probiotic bacteria strains, and provides an additional reason for considering such treatments for patients with intestinal inflammation.

Probiotics in the management of atopic eczema.

BACKGROUND: Over the last two decades the incidence of allergic diseases has increased in industrialized countries, and consequently new approaches have to be explored. OBJECTIVE: The potential of probiotics to control allergic inflammation at an early age was assessed in a randomized double-blind placebo-controlled study. METHODS: A total of 27 infants, mean age 4.6 months, who manifested atopic eczema during exclusive breast-feeding and who have had no exposure to any infant or substitute formula were weaned to probiotic-supplemented, Bifidobacterium lactis Bb-12 or Lactobacillus strain GG (ATCC 53103), extensively hydrolysed whey formulas or to the same formula without probiotics. The extent and severity of atopic eczema, the growth and nutrition of infants, and concentrations of circulating cytokines/chemokines and soluble cell surface adhesion molecules in serum and methyl-histamine and eosinophilic protein X in urine were determined. RESULTS: The SCORAD score reflecting the extent and severity of atopic eczema was 16 (7-25) during breast-feeding, median (interquartile range). After 2 months, a significant improvement in skin condition occurred in patients given probiotic-supplemented formulas, as compared to the unsupplemented group; chi(2) = 12.27, P = 0.002. SCORAD decreased in the Bifidobacterium lactis Bb-12 group to 0 (0-3.8), and in the Lactobacillus GG group to 1 (0.1-8.7), vs unsupplemented 13.4 (4.5-18.2), median (interquartile range), in parallel with a reduction in the concentration of soluble CD4 in serum and eosinophilic protein X in urine. CONCLUSION: The results provide the first clinical demonstration of specific probiotic strains modifying the changes related to allergic inflammation. The data further indicate that probiotics may counteract inflammatory responses beyond the intestinal milieu. The combined effects of these probiotic strains will guide infants through the weaning period, when sensitization to newly encountered antigens is initiated. The probiotic approach may thus offer a new direction in the search for future foods for allergy treatment and prevention strategies.

Late onset reactions to oral food challenge are linked to low serum interleukin-10 concentrations in patients with atopic dermatitis and food allergy.

BACKGROUND: Aberrant cytokine production in vitro has been associated with atopic disease. No study has as yet been made of the circulating cytokine profiles in atopic patients with food allergy in response to oral allergen challenge. OBJECTIVE: To assess the effect of oral allergen challenge on the serum cytokine concentrations in patients with atopic dermatitis and food allergy. METHODS: Serum concentrations of interleukin (IL)-10, transforming growth factor beta 1, IL-1ra, IL-6, IL-5, IL-4 and interferon (IFN)-gamma were measured before and after double-blind, placebo-controlled food challenges (DBPCFC) (n = 73). Before DBPCFC, combined skin prick and patch testing was performed for cow milk, egg, soybean and cereals, and production of IFNgamma, IL-4, IL-10 and tumour necrosis factor alpha (TNFalpha) was determined in supernatants of cultures of peripheral blood mononuclear cells (PBMCs) stimulated by cow milk. RESULTS: The oral food challenge triggered immediate onset exanthematous reactions in 22 cases and late onset eczematous reactions in 29. The late-reacting cases had more positive skin patch test and negative skin prick test reactivities with allergenic food, and they had lower serum IL10 concentrations than immediate-reacting cases. In challenge-positive cases, IL-10 concentrations increased from 2.9 (0.1-5.04) pg/mL to 3. 9 (1.2-8.3) pg/mL in response to DBPCFC, P = 0.05, median (interquartile ranges), but not in those tolerant to cow milk. PBMCs of patients with cow milk allergy but not of those tolerant to cow milk generated TNFalpha in response to cow milk in vitro. CONCLUSION: These results indicate that oral allergen challenge in atopic patients with food allergy triggers systemic release of IL-10. Patients with late onset reactions were found to have lower serum IL-10 concentrations than their immediate-reacting counterparts. Considering that IL-10 is an inhibitory cytokine of delayed-type hypersensitivity, low IL-10 in late-reacting patients may explain the high frequency of their positive skin patch tests combined with negative skin prick tests.

Antiproliferative effects of homogenates derived from five strains of candidate probiotic bacteria.

Unheated and heat-treated homogenates were separately prepared from candidate probiotic bacteria, including Lactobacillus rhamnosus GG, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus thermophilus. We compared the phytohemagglutinin-induced proliferation of mononuclear cells in the presence of homogenates and in the presence of a control containing no homogenate by assessing thymidine incorporation in cell cultures. All homogenates suppressed proliferation, whether the enzymatic activity was inactivated or not inactivated by heating. When the proliferation assays were repeated with cytoplasmic and cell wall extracts derived from the homogenate of L. rhamnosus GG, the cytoplasmic extract but not the cell wall extract was suppressive. These findings indicate that candidate probiotic bacteria possess a heat-stable antiproliferative component(s). These bacteria may be used to generate microbiologically nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.

Dietary fatty acids and allergy.

The increase in the prevalence of atopic diseases has recently been linked to altered consumption of polyunsaturated fatty acids (PUFAs). As typical Western diets contain almost 10 times more linoleic acid (18:2 omega-6) than alpha-linolenic acid (18:3 omega-3), it is the metabolism of the former that predominates. Subsequently produced arachidonic acid-derived eicosanoids alter the balance of T-helper cells type 1 and type 2 thus favouring the production of immunoglobulin (Ig)E. In atopic subjects, the impact of this excessive eicosanoid production may be further strengthened as a result of changes in cyclic nucleotide metabolism exacerbated by substrate availability. Dietary omega-3 fatty acids can have marked influence on both specific and nonspecific immune responses in modifying eicosanoid production and replacing omega-6 fatty acids in cell membranes. Therefore, it is concluded that careful manipulation of dietary PUFAs may play a key role in the successful management of inflammation associated with atopic diseases.

Probiotics reinforce mucosal degradation of antigens in rats: implications for therapeutic use of probiotics.

The effects of probiotics, administered with different diets, i.e., unhydrolyzed or hydrolyzed dietary antigens, on macromolecular degradation in the gut mucosa were studied. Rat pups were divided into five feeding groups at the age of 14 d. In addition to maternal milk, the milk group was gavaged daily with cows' milk and the hydrolysate group with extensively hydrolyzed whey formula, while controls received sterile saline. In addition to these diets, the milk-GG group and the hydrolysate-GG group were given probiotic bacteria, Lactobacillus GG ATCC 53103 (10(10) colony-forming units per day). At 21 d, the absorption of macromolecules, horseradish peroxidase and beta-lactoglobulin across patch-free jejunal segments was studied in Ussing chambers. The degree of macromolecular degradation was studied by means of HPLC gel filtration. The absorption rate of intact horseradish peroxidase differed among the feeding groups (P = 0.038). This was due to the high median (interquartile range) absorption of intact horseradish peroxidase (ng x h-1 x cm-2) in the milk group [255 (14-1332)] and supplementation with L. GG in the milk-GG group [35 (8-233)] restoring the status to the control level [22 (0-116)]. A parallel effect was seen in the hydrolysate group [100 (9-236)] vs. the hydrolysate-GG group [1 (0- 13)]. A gel filtration study confirmed that larger molecules were absorbed across the mucosa in the milk group compared to the other groups. The absorption of degraded horseradish peroxidase differed between the feeding groups (P = 0. 005). L. GG had a distinct effect when administered with unhydrolyzed, native protein vs. hydrolyzed protein: it increased absorption of degraded horseradish peroxidase in the milk-GG group [7310 (4763-8228)] vs. the milk group [3726 (2423-5915)], while reducing it in the hydrolysate-GG group [2051 (1463-2815)] vs. the hydrolysate group [4573 (3759-9620)]. Our results showed that probiotics not only restore aberrant macromolecular transport, but they also have a specific effect on mucosal degradation depending on dietary antigen: adjuvant-like properties (unhydrolyzed antigen) and immunosuppressive-like properties (hydrolyzed antigen). The antigenicity of the diet therefore should be taken into consideration, when introducing novel probiotic functional foods for the management of gastrointestinal disorders.

Elimination diet in cow's milk allergy: risk for impaired growth in young children.

OBJECTIVE: The objective of this study was to evaluate the nutritional impact of therapeutic elimination diets and to identify risk factors predisposing infants with food allergy to poor growth. STUDY DESIGN: We studied 100 children (mean age 7 months) with atopic dermatitis and challenge-proven cow's milk allergy and evaluated their growth during the symptomatic period before diagnosis and during the therapeutic elimination diet. RESULTS: Clinical control of symptoms was achieved in all patients. The mean length SD score and weight-for-length index of patients decreased compared with those in healthy age-matched children, p < 0.0001 and p = 0.03, respectively. Low serum albumin was present in 6% of the patients, 24% had an abnormal urea concentration, and 8% had a low serum phospholipid docosahexaenoic acid. The delay in growth was more pronounced in a subgroup of patients with early onset than in those with later of symptoms (F = 6.665, p < 0.0001). The duration of breast-feeding correlated positively with the sum of n-3 polyunsaturated fatty acids (r = 0.39, p = 0.001) and with the relative amount of docosahexaenoic acid (r = 0.36, p = 0.002). CONCLUSION: A delicate balance exists between the benefits and the risks of elimination diets.

Oral cow milk challenge abolishes antigen-specific interferon-gamma production in the peripheral blood of children with atopic dermatitis and cow milk allergy.

BACKGROUND: A major feature of atopic dermatitis (AD) is the propensity to generate IgE to environmental antigens. Despite extensive information on IgE dysregulation in AD, the nature of immune responses to ingested allergens is poorly characterized. OBJECTIVE: To determine the clinical and immunological responses to food in AD patients. METHODS: To characterize the type and timing of clinical reactions to oral cow milk, 83 AD patients aged 2 to 60 months were subjected to double-blind placebo-controlled food challenge (DBPCFC). IFN-gamma and IL-4 production by their peripheral blood mononuclear cells (PBMC) was determined before and after DBPCFC. RESULTS: Of 50 patients positive to DBPCFC. 46% manifested exanthematous-type immediate-onset reactions and 54% eczematous-type late-onset reactions. In either group, the production of IL-4 and IFN-gamma by Concanavalin A-stimulated PBMC was comparable before and after DBPCFC. For immediate-reacting patients, the median IFN-gamma production by milk-stimulated PBMC was 11.5 (4.2-17.2) pg/ml as against 2.3 (0.2-5.7) pg/ml by unstimulated PBMC, P = 0.0008 before DBPCFC, and 4.6 (2.8-10.3) pg/ml. vs 4.2 (1.7-9.0) pg/mL, p = 0.40, correspondingly after DBPCFC. CONCLUSION: Before DBPCFC, immediate-reacting but not late-reacting patients were found to be capable of allergen-specific IFN-gamma production in vitro, indicating the heterogeneity in AD patients. After DBPCFC, the IFN-gamma generation abolished, indicating the effect of oral allergen exposure on IFN-gamma producing responses of AD patients.

IFN-gamma enhances macromolecular transport across Peyer's patches in suckling rats: implications for natural immune responses to dietary antigens early in life.

BACKGROUND: The capacity to generate (interferon-gamma) IFN-gamma, a potent immunoregulatory and inflammatory cytokine, is low in neonates and deficient in patients with food allergy. METHODS: We investigated the effect of IFN-gamma on antigen transport in the gut. In experiment I rat pups were randomized into two groups at the age of 14 days i.e., before gut maturation: Group IFN was given intraperitoneally recombinant rat IFN-gamma on days 14, 16, 18, 20. In experiment II, rats were randomized into two groups at the age of 26 days, i.e., after gut maturation: Group IFN received the IFN-gamma treatment on days 26, 28, 30, 32. Controls in both experiments received sterile saline. The absorption of horseradish peroxidase (HRP) across jejunal segments with and without Peyer's patches was studied in Ussing chambers on days 21 and 33 for experiments I and II, respectively. RESULTS: In experiment I, the absorption of intact HRP across both types of segments was significantly increased in Group IFN compared to controls. The mean (95% confidence interval) rate of degraded HRP absorption across patch-containing segments in Group IFN was significantly greater than in controls, 4420 (3162-6179) ng.h-1.cm-2 in comparison to 1550 (633-3790) ng.h-1.cm-2; F = 8.96, p = 0.009. CONCLUSION: IFN-gamma increases macromolecular transport before gut maturation particularly across Peyer's patches. This Peyer's patch-targeted effect can be important eliciting mucosal immune responses against dietary antigens early in life and aiding their immune exclusion.

Suppression of lymphocyte proliferation in vitro by bovine caseins hydrolyzed with Lactobacillus casei GG-derived enzymes.

BACKGROUND: Processing of proteins in the gut and activation of T-cell suppression leads to systemic hyporesponsiveness to ingested protein antigens. OBJECTIVE: The study was designed to determine whether lactobacilli, a major part of human intestinal microflora, can contribute to degradation of food antigens in the gut and modify their immunoactivities. METHODS: Lymphocyte transformation tests were carried out in healthy adults to determine the mitogen-induced lymphocyte proliferative responses to bovine caseins hydrolyzed with pepsin and trypsin and to bovine caseins additionally hydrolyzed with enzymes derived from Lactobacillus casei strain GG (ATCC 53103). RESULTS: In experiments done with caseins hydrolyzed with pepsin and trypsin, beta- and alpha(s1)-caseins significantly suppressed the proliferation of lymphocytes at 0.1 and 10 micrograms/ml, respectively, when compared with corresponding control cultures without these hydrolysates. In contrast, kappa-casein significantly stimulated the proliferation of lymphocytes at 10 micrograms/ml. In experiments done with caseins additionally hydrolyzed with L. casei GG-derived enzymes, there was one consistent effect on lymphocyte proliferation: suppression by alpha(sl)-, beta-, and kappa-caseins at 0.1, 10, and 1000 micrograms/ml, respectively. CONCLUSIONS: Hydrolysis of caseins with L. casei GG-derived enzymes generates molecules with suppressive effects on lymphocyte proliferation. In addition, intestinal bacteria can be beneficial in the downregulation of hypersensitivity reactions to ingested proteins in patients with food allergy.

Down-regulation of anti-CD3 antibody-induced IL-4 production by bovine caseins hydrolysed with Lactobacillus GG-derived enzymes.

A prerequisite for systemic hyporesponsiveness to dietary antigens is their processing in the gut. This study investigated whether bovine caseins degraded by enzymes of an intestinal bacterial strain, Lactobacillus GG (ATCC 53103), could regulate the cytokine production by anti-CD3 antibody-induced peripheral blood mononuclear cells of 14 atopic patients, aged 5-29 (mean, 16) months. Purified casein up-regulated the interleukin-4 and interferon-gamma production, P = 0.008 and P = 0.008, respectively. Conversely, Lactobacillus GG-degraded casein down-regulated the interleukin-4 production, P = 0.003, with no effect on interferon-gamma. These results indicate that intestinal bacteria may modify immunomodulatory properties of native food proteins and introduce a promising tool to provide protection from potentially harmful dietary antigens at a young age.

Efficacy and safety of hydrolyzed cow milk and amino acid-derived formulas in infants with cow milk allergy.

OBJECTIVE: To determine the antigenicity, nutritional adequacy, and growth-promoting efficacy of protein hydrolysate or amino acid-derived formulas in infants with cow milk allergy. STUDY DESIGN: Several protein hydrolysate or amino acid-derived formulas were graded for beta-lactoglobulin content and skin reactivity in 74 atopic children with cow milk allergy proved by a double-blind, placebo-controlled challenge. A randomized, prospective follow-up study of 9 months included 22 infants with a mean age of 6 months (95% confidence interval, 4 to 7), who were fed an extensively hydrolyzed whey formula (group We), and 23 infants with a mean age of 17 (95% confidence interval, 4 to 7) months, who were given an amino acid-derived formula (group AA). RESULTS: Both formulas were clinically and biochemically tolerated. The mean concentration of essential amino acids in plasma was lower in group We but higher in group AA compared with values for breast-fed control infants (p = 0.001). There was a different trend between the groups in weight (p = 0.09) and length (p = 0.006). Growth was promoted in group AA during the follow-up; it was constant during the first months, followed by a gradual decline in rate in group We. In both groups, atopic eczema improved significantly and progressively, and a downward trend was found in serum total and milk-specific IgE concentrations, proving the efficacy of both formulas. CONCLUSIONS: Extensively hydrolyzed formulas are safe and effective for most infants; an amino acid-derived formula may be preferable for infants with multiple food allergies, especially for the maintenance of normal growth.