Infant milk formulas differ regarding their allergenic activity and induction of T-cell and cytokine responses.

BACKGROUND: Several hydrolyzed cow's milk (CM) formulas are available for avoidance of allergic reactions in CM-allergic children and for prevention of allergy development in high-risk infants. Our aim was to compare CM formulas regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T-cell proliferation, and cytokine secretion. METHODS: A blinded analysis of eight CM formulas, one nonhydrolyzed, two partially hydrolyzed (PH), four extensively hydrolyzed (EH), and one amino acid formula, using biochemical techniques and specific antibody probes was conducted. IgE reactivity and allergenic activity of the formulas were tested with sera from CM-allergic patients (n = 26) in RAST-based assays and with rat basophils transfected with the human FcεRI, respectively. The induction of T-cell proliferation and the secretion of cytokines in Peripheral blood mononuclear cell (PBMC) culture from CM allergic patients and nonallergic individuals were assessed. RESULTS: Immune-reactive α-lactalbumin and ß-lactoglobulin were found in the two PH formulas and casein components in one of the EH formulas. One PH formula and the EH formula containing casein components showed remaining IgE reactivity, whereas the other hydrolyzed formulas lacked IgE reactivity. Only two EH formulas and the amino acid formula did not induce T-cell proliferation and proinflammatory cytokine release. The remaining formulas varied regarding the induction of Th2, Th1, and proinflammatory cytokines. CONCLUSION: Our results show that certain CM formulas without allergenic and low proinflammatory properties can be identified and they may also explain different outcomes obtained in clinical studies using CM formulas.

Casein hydrolysate diet controls intestinal T cell activation, free radical production and microbial colonisation in NOD mice.

AIMS/HYPOTHESIS: Dietary and microbial factors and the gut immune system are important in autoimmune diabetes. We evaluated inflammatory activity in the whole gut in prediabetic NOD mice using ex vivo imaging of reactive oxygen and nitrogen species (RONS), and correlated this with the above-mentioned factors. METHODS: NOD mice were fed a normal diet or an anti-diabetogenic casein hydrolysate (CH) diet. RONS activity was detected by chemiluminescence imaging of the whole gut. Proinflammatory and T cell cytokines were studied in the gut and islets, and dietary effects on gut microbiota and short-chain fatty acids were determined. RESULTS: Prediabetic NOD mice displayed high RONS activity in the epithelial cells of the distal small intestine, in conjunction with a proinflammatory cytokine profile. RONS production was effectively reduced by the CH diet, which also controlled (1) the expression of proinflammatory cytokines and colonisation-dependent RegIIIγ (also known as Reg3g) in ileum; (2) intestinal T cell activation; and (3) islet cytokines. The CH diet diminished microbial colonisation, increased the Bacteroidetes:Firmicutes ratio, and reduced lactic acid and butyric acid production in the gut. CONCLUSIONS/INTERPRETATION: Epithelial RONS production and proinflammatory T cell activation appears in the ileum of NOD mice after weaning to normal laboratory chow, but not after weaning to an anti-diabetogenic CH diet. Our data suggest a link between dietary factors, microbial colonisation and mucosal immune activation in NOD mice.

Neonatal supplementation of processed supernatant from Lactobacillus rhamnosus GG improves allergic airway inflammation in mice later in life.

BACKGROUND: Oral supplementation with probiotic bacteria can protect against the development of allergic and inflammatory diseases. OBJECTIVE: The aim of this study was to investigate potential immunomodulatory and allergy-protective effects of processed Lactobacillus rhamnosus GG (LGG)-derived supernatants early in life in neonatal mice. METHODS: In vitro, RAW264.7 mouse macrophages were stimulated with viable LGG, LGG-derived supernatants, prepared from different growth phases, and different size fractions thereof, and pro- and anti-inflammatory cytokine production was analysed. Supernatant fractions were also treated with protease, DNAse or carbohydrate-digesting enzymes to define the nature of immunomodulatory components. In vivo, neonatal Balb/c mice were orally supplemented with differentially processed LGG supernatants. Starting at 4 weeks of age, a protocol of ovalbumin-induced acute allergic airway inflammation was applied and protective effects of processed LGG supernatants were assessed. RESULTS: Incubation of RAW264.7 cells with LGG-derived supernatants significantly increased TNFα and IL-10 production. These effects were not restricted to a particular molecular size fraction. Treatment with protease, but not with DNAse or carbohydrate-digesting enzymes, completely abolished the immunomodulatory activities. Incubation of TLR/NOD-transfected cells with LGG-derived supernatants revealed that recognition and signalling of bioactive components is mediated by TLR2 and NOD2. In vivo supplementation of newborn mice with processed LGG-derived supernatants resulted in pronounced protective effects on the allergic inflammatory response as reflected by reduced eosinophil numbers, modified T helper cell cytokine production, significantly less lung inflammation and reduced goblet cell numbers in comparison with sham-treated controls. CONCLUSION: LGG-derived supernatants exert immunomodulatory activities, and neonatal administration of specifically processed supernatants may provide an alternative to viable probiotics in reducing allergic inflammatory responses.

Potential mechanisms explaining why hydrolyzed casein-based diets outclass single amino acid-based diets in the prevention of autoimmune diabetes in diabetes-prone BB rats.

BACKGROUND: It remains controversial whether avoidance of dietary diabetogenic triggers, such as cow's milk proteins, can prevent type 1 diabetes in genetically susceptible individuals. Here, different extensive casein hydrolysates (HC) and single amino acid (AA) formulations were tested for their effect on mechanisms underlying autoimmune diabetes pathogenesis in diabetes-prone BioBreeding rats. Intestinal integrity, gut microbiota composition and mucosal immune reactivity were studies to assess whether these formulations have differential effects in autoimmune diabetes prevention. METHODS: Diabetes-prone BioBreeding rats received diets in which the protein fraction was exchanged for the different hydrolysates or AA compositions, starting from weaning until the end of the experiment (d150). Diabetes development was monitored, and faecal and ileal samples were collected. Gut microbiota composition and cytokine/tight junction mRNA expression were measured by quantitative polymerase chain reaction. Cytokine levels of ileum explant cultures were measured by ELISA, and intestinal permeability was measured in vivo by lactulose-mannitol assay. RESULTS: Both HC-diet fed groups revealed remarkable reduction of diabetes incidence with the most pronounced effect in Nutramigen®-fed animals. Interestingly, AA-fed rats only showed delayed autoimmune diabetes development. Furthermore, both HC-fed groups had improved intestinal barrier function when compared with control chow or AA-fed animals. Interestingly, higher IL-10 levels were measured in ileum tissue explants from Nutramigen®-fed rats. Beneficial gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) were found associated especially with HC-diet interventions. CONCLUSIONS: Casein hydrolysates were found superior to AA-mix in autoimmune diabetes prevention. This suggests the presence of specific peptides that beneficially affect mechanisms that may play a critical role in autoimmune diabetes pathogenesis.

Lipid ratios representing SCD1, FADS1, and FADS2 activities as candidate biomarkers of early growth and adiposity.

BACKGROUND: Altered lipid metabolism in early life has been associated with subsequent weight gain and predicting this could aid in obesity prevention and risk management. Here, a lipidomic approach was used to identify circulating markers for future obesity risk in translational murine models and validate in a human infant cohort. METHODS: Lipidomics was performed on the plasma of APOE*3 Leiden, Ldlr-/-.Leiden, and the wild-type C57BL/6J mice to capture candidate biomarkers predicting subsequent obesity parameters after exposure to high-fat diet. The identified candidate biomarkers were mapped onto corresponding lipid metabolism pathways and were investigated in the Cambridge Baby Growth Study. Infants' growth and adiposity were measured at 0-24 months. Capillary dried blood spots were sampled at 3 months for lipid profiling analysis. FINDINGS: From the mouse models, cholesteryl esters were correlated with subsequent weight gain and other obesity parameters after HFD period (Spearman's r≥0.5, FDR p values <0.05) among APOE*3 Leiden and Ldlr-/-.Leiden mice, but not among the wild-type C57BL/6J. Pathway analysis showed that those identified cholesteryl esters were educts or products of desaturases activities: stearoyl-CoA desaturase-1 (SCD1) and fatty acid desaturase (FADS) 1 and 2. In the human cohort, lipid ratios affected by SCD1 at 3 months was inversely associated with 3-12 months weight gain (B±SE=-0.31±0.14, p=0.027), but positively with 12-24 months weight and adiposity gains (0.17±0.07, p=0.02 and 0.17±0.07, 0.53±0.26, p=0.04, respectively). Lipid ratios affected by SCD1 and FADS2 were inversely associated with adiposity gain but positively with height gain between 3-12 months. INTERPRETATION: From murine models to human setting, the ratios of circulating lipid species indicating key desaturase activities in lipid metabolism were associated with subsequent body size increase, providing a potential tool to predict early life weight gain.

Microarray and allergenic activity assessment of milk allergens.

BACKGROUND: Cow's milk is one of the most common causes of food allergy affecting approximately 2.5% of infants in the first years of their life. However, only limited information regarding the allergenic activity of individual cow's milk allergens is available. OBJECTIVE: To analyse the frequency of IgE reactivity and to determine the allergenic activity of individual cow's milk allergens. METHODS: A nitrocellulose-based microarray, based on purified natural and recombinant cow's milk allergens was used to determine IgE reactivity profiles using sera from 78 cow's milk-sensitized individuals of varying ages. The allergenic activity of the individual allergens was tested using patients' sera for loading rat basophil leukaemia cells (RBL) expressing the α-chain of the human receptor FcεRI. RESULTS: Using the microarray and the RBL assay, cow's milk allergens were assessed for frequency of IgE recognition and allergenic activity. Moreover, the RBL assay allowed distinguishing individuals without or with mild clinical reactions from those with severe systemic or gastrointestinal symptoms as well as persons who grew out cow's milk allergy from those who did not. CONCLUSIONS: Component-resolved testing using milk allergen microarrays and RBL assays seems to provide useful additional diagnostic information and may represent a basis for future forms of prophylactic and therapeutic strategies for cow's milk allergy.

Antioxidants and polyunsaturated fatty acids in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Oligodendrocyte damage and subsequent axonal demyelination is a hallmark of this disease. Different pathomechanisms, for example, immune-mediated inflammation, oxidative stress and excitotoxicity, are involved in the immunopathology of MS. The risk of developing MS is associated with increased dietary intake of saturated fatty acids. Polyunsaturated fatty acid (PUFA) and antioxidant deficiencies along with decreased cellular antioxidant defence mechanisms have been observed in MS patients. Furthermore, antioxidant and PUFA treatment in experimental allergic encephalomyelitis, an animal model of MS, decreased the clinical signs of disease. Low-molecular-weight antioxidants may support cellular antioxidant defences in various ways, including radical scavenging, interfering with gene transcription, protein expression, enzyme activity and by metal chelation. PUFAs may not only exert immunosuppressive actions through their incorporation in immune cells but also may affect cell function within the CNS. Both dietary antioxidants and PUFAs have the potential to diminish disease symptoms by targeting specific pathomechanisms and supporting recovery in MS.

Polyunsaturated fatty acid supplementation stimulates differentiation of oligodendroglia cells.

Dietary polyunsaturated fatty acids (PUFAs) have been postulated as alternative supportive treatment for multiple sclerosis, since they may promote myelin repair. We set out to study the effect of supplementation with n-3 and n-6 PUFAs on OLN-93 oligodendroglia and rat primary oligodendrocyte differentiation in vitro. It appeared that OLN-93 cells actively incorporate and metabolise the supplemented PUFAs in their cell membrane. The effect of PUFAs on OLN-93 differentiation was further assessed by morphological and Western blot evaluation of markers of oligodendroglia differentiation: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), zonula occludens-1 (ZO-1) and myelin-associated glycoprotein (MAG). Supplementation of the OLN-93 cells with n-3 and n-6 PUFAs increased the degree of differentiation determined by morphological analysis. Moreover, CNP protein expression was significantly increased by gamma-linolenic acid (GLA, 18:3n-6) supplementation. In accordance with the OLN-93 results, studies with rat primary oligodendrocytes, a more advanced model of cell differentiation, showed GLA supplementation to promote oligodendrocyte differentiation. Following GLA supplementation, increased numbers of proteolipid protein (PLP)-positive oligodendrocytes and increased myelin sheet formation was observed during differentiation of primary oligodendrocytes. Moreover, increased CNP, and enhanced PLP and myelin basic protein expression were found after GLA administration. These studies provide support for the dietary supplementation of specific PUFAs to support oligodendrocyte differentiation and function.

Abrogation of IFN-gamma mediated epithelial barrier disruption by serine protease inhibition.

The intestinal barrier function is often impaired in a variety of diseases including chronic inflammatory bowel disease. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex. IFN-gamma has been widely studied for its effect on barrier function and tight junction structures but its mode of action remains unclear. Since the claudin family of tight junction proteins is proposed to be involved in barrier maintenance we studied the effect of IFN-gamma on claudin expression in relation to epithelial barrier function. Cycloheximide and protease inhibitors were used to study mechanisms of IFN-gamma mediated barrier disruption. Intestinal epithelial cells were exposed to IFN-gamma and permeability was evaluated by horse radish peroxidase (HRP) and 4 kD FITC-dextran fluxes. Occludin and claudin-1, -2, -3, and -4 tight junction protein expression was determined by Western blotting. Occludin and claudin-2 protein expression was dramatically reduced after IFN-gamma exposure, which correlated with increased permeability for HRP and FITC-dextran. Interestingly, cleavage of claudin-2 was observed after incubation with IFN-gamma. Serine protease inhibitor AEBSF completely abrogated IFN-gamma mediated barrier disruption which was associated with preservation of claudin-2 expression. Moreover, IFN-gamma induced loss of barrier integrity was found to affect claudin-2 and occludin expression through different mechanisms. Since inhibition of serine protease activity abrogates IFN-gamma mediated barrier disruption this may be an important target for therapeutic intervention.

Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofibroblasts.

BACKGROUND: The mucus layer protects the gastrointestinal mucosa from mechanical, chemical, and microbial challenge. Mucin 2 (MUC-2) is the most prominent mucin secreted by intestinal epithelial cells. There is accumulating evidence that subepithelial myofibroblasts regulate intestinal epithelial cell function and are an important source of prostaglandins (PG). PG enhance mucin secretion and are key players in mucoprotection. The role of bacterial fermentation products in these processes deserves further attention. AIMS: We therefore determined whether the effect of short chain fatty acids (SCFA) on MUC-2 expression involves intermediate PG production. METHODS: Both mono- and cocultures of epithelial cells and myofibroblasts were used to study the effects of SCFA on MUC-2 expression and PG synthesis. Cell culture supernatants were used to determine the role of myofibroblast derived prostaglandins in increasing MUC-2 expression in epithelial cells. RESULTS: Prostaglandin E(1) (PGE(1)) was found to be far more potent than PGE(2) in stimulating MUC-2 expression. SCFA supported a mucoprotective PG profile, reflected by an increased PGE(1)/PGE(2) ratio in myofibroblast supernatants and increased MUC-2 expression in mono- and cocultures. Incubation with indomethacin revealed the latter to be mediated by PG. CONCLUSIONS: SCFA can differentially regulate PG production, thus stimulating MUC-2 expression in intestinal epithelial cells. This mechanism involving functional interaction between myofibroblasts and epithelial cells may play an important role in the mucoprotective effect of bacterial fermentation products.

A coculture model mimicking the intestinal mucosa reveals a regulatory role for myofibroblasts in immune-mediated barrier disruption.

The pathogenesis of Crohn's disease involves a mucosal inflammatory response affecting the barrier function of the gut. Myofibroblasts directly underlining the intestinal epithelium may have a regulatory role in immune-mediated barrier disruption. A coculture system of T84 epithelial and CCD-18Co myofibroblasts was established in order to mimic the in situ spatial interactions between these cell types and to evaluate their role in barrier: integrity. Lamina propria mononuclear cells (LPMC) were introduced in co- and monocultures. Effects of immune cells on barrier integrity was determined by measuring resistance and permeability for macromolecules. Introduction of LPMC in both culture systems caused a time-dependent decrease in barrier integrity. This was found to be less pronounced in cocultures indicating a regulatory role for mesenchymal cells. The effects were also found to depend on the route of LPMC stimulation. Additional analyses suggested that the regulatory role of myofibroblasts in barrier integrity involves production of growth factors.