Human milk contains large amounts of free oligosaccharides (HMOs). HMOs have been shown to exert antiinflammatory properties, and evidence for their immunomodulatory effects is increasing. The purpose of this study was to evaluate influences of two human breast milk-derived oligosaccharide samples (neutral and acidic oligosaccharides), and of a low-molecularweight fucoidan on cytokine production and activation of cord blood mononuclear cells. Cord blood mononuclear cells from randomly chosen healthy newborns were co-cultured with the oligosaccharide samples. By means of flow cytometry, intracellular cytokine production (d 20) and surface marker expression of T cells (d 5) were measured. In vitro-induced Ig levels were quantified nephelometrically (total IgG1) and by ELISA (total IgE) in the supernatant of cell cultures. The acidic oligosaccharide fraction increased the percentage of interferon-␥ producing CD3ϩCD4ϩ and CD3ϩCD8ϩ cells (p Ͻ 0.05) and the IL-13 production in CD3ϩCD8ϩ cells (p Ͻ 0.05). In acidic oligosaccharide cultures, CD25ϩ expression on CD3ϩCD4ϩ cells was significantly elevated (p Ͻ 0.05). Low-molecular-weight fucoidan induced IL-4 production in CD3ϩCD4ϩ T cells (p Ͻ 0.05) and IL-13 production in CD3ϩCD8ϩ T cells (p Ͻ 0.05), whereas interferon-␥ production remained unaffected in both T-cell populations. Ig production (total IgE and total IgG1) remained unaffected. Human milk-derived oligosaccharides and plant-derived oligosaccharides affect the cytokine production and activation of cord blood derived T cells in vitro. Therefore, oligosaccharides and, in particular, acidic oligosaccharides may influence lymphocyte maturation in breast-fed newborns. The postnatal period is a crucial time for the maturation of the immune system. At birth, T-lymphocytes exhibit a Th2-profile, characterized by a limited ability to produce cytokines. Throughout the first months after birth these Th2-skewed responses are modified into low-level immunity, predominantly expressing Th1-cytokines and IgG-antibodies, particularly of the IgG1 subclass (1,2).Human milk is a biologic fluid containing large amounts of free oligosaccharides. HMOs represent the third largest solid component (after lactose and lipids) in breast milk, occurring at a concentration of 20 -23 g/L in colostrum and 12-14 g/L in mature milk (3). HMOs are very resistant to enzymatic hydrolysis (4,5), indicating that these oligosaccharides must display essential nonnutritive functions.Throughout the last decade, numerous studies demonstrated the ability of HMOs to inhibit pathogens acting as receptors for microbes (6,7). Only a few reports on direct effects on immune function have been published so far. Human milk oligosaccharide structures like lacto-N-fucopentaose III (LNFPIII) and lacto-N-neotetraose (LNneoT) showed an effect on murine IL-10 production (8). It is further discussed that human milk is involved in the generation of antiinflammatory mediators that suppress Th1-type and inflammatory responses in mice (9). 0031-3998/04/5604-0536 PEDIATRIC RES...

Allergies are increasing, and despite deeper insights into the immunologic basis of these diseases, preventive measures are not yet efficient. As the induction of allergic diseases is often triggered in early childhood, perinatal or prenatal preventive strategies would be beneficial. We investigated the transfer of inhalant and nutritive allergens across the human placenta. For this purpose, the maternal side of a placental cotyledon was perfused in vitro with an allergen-containing medium, and a specific ELISA was used to detect the allergens on the fetal side. Both allergens evaluated, birch pollen major allergen Bet v1 and the milk allergen beta-lactoglobulin, could be shown to cross the placenta. The nutritive allergen beta-lactoglobulin was not only transferred across the placenta in all eight experiments, but was also detectable within the first minutes of perfusion. The peak allergen concentration on the fetal side could be increased by addition of human immunoglobulin. For the inhalant allergen Bet v1, transfer was observed in two of 10 placental experiments, and only if human immunoglobulin was added. A pulsatility wave with a frequency of 30 -35 min suggested an active transfer mechanism. We conclude that allergens are actively and selectively transferred across the placenta. Therefore, controlled maternal allergen exposure might offer new ways to induce tolerance to specific allergens in the fetus. There is increasing evidence that the prevalence of allergic diseases is currently rising at an unprecedented rate (1-3). It is also becoming clear that this rise is largely restricted to developed countries, a phenomenon that is widely believed to be linked to a western lifestyle (4). Eliciting agents, preferentially environmental allergens, are predominantly found in indoor environments. Recently, concern has been raised as to any priming effects of exposure to environmental influences even before birth; that is, through the placenta.Allergen-specific T cell proliferation in cord blood cells has been demonstrated by many groups and for many different allergens (5-15). Inhalant and nutritive allergens in the form of crude extracts [birch pollen (5, 7), house dust mite (8 -10, 15), timothy grass pollen (5, 7), cat fur (5, 7), BLG (5, 7, 13, 16), alpha-casein (13), beta-casein (13), kappa-casein (13), BSA (5-7, 13), and ovalbumin (5, 7, 11, 15, 16)] and in the form of recombinant allergens [Lol p1 (9), Der p1 (9), Bet v1 (14), and Phl p1 (14)] has been studied. A line of evidence now supports the hypothesis that fetal T cells are exposed during gestation to maternally derived allergens. These allergens may be ingested or inhaled by the mother (14). The time at which maternofetal allergen exposure takes place in the course of a pregnancy is less clear. The current knowledge suggests early times during gestation, presumably around wk 20 of gestation (5, 14), Szépfalusi et al. unpublished experiments).The occurrence of prenatal T cell priming and its possible impact on later allergic status has been the subject...

Allergen-specific cord blood reactivity may be attributed to low levels of allergens crossing the human placenta and providing the fetus with the necessary stimulus for T cell priming.

These findings provide direct evidence for the release of tiny amounts of nutritive allergens from placental tissue indicating diaplacental allergen transfer and fetal exposure to nutritive allergens in vivo.

In vitro/ex vivo trans-trophoblastic and trans-placental allergen transfer is shown with an accumulation of most of the allergen in placental tissues, potentially explaining the missing direct dose-response relationship between prenatal (maternal) allergen exposure and allergen-specific cellular reactivity in cord blood.

The mode of inactivation of probiotic bacteria may profoundly affect their immune-modulatory properties to the point of reversal of effects in in vitro human intestinal epithelial-like cell cultures (Caco-2). To further investigate the influence of inactivation treatment on cytokine production, three probiotic strains were evaluated-live, heat-inactivated, and formalin-inactivated strains-for their impact on interleukin (IL) 6, IL-8, and IL-10 production in Caco-2-leucocyte cocultures. The tested bacteria induced strain-specific production of IL-6, IL-8, and IL-10. No suppressive effects on cytokine synthesis were observed. Live microorganisms seemed to be slightly more potent inducers of cytokine production than nonviable strains, but differences to inactivated bacteria were not statistically significant. Our results indicate that heat and formalin treatments of probiotic microorganisms are equivalent inactivation methods in terms of induction of IL-6, IL-8, and IL-10 production in Caco-2-peripheral blood mononuclear cell cocultures and do not invert immune-modulatory effects.

Objective: The mechanisms by which nutritive allergens are transported from mother to fetus and the ensuing immunological response are incompletely understood. We investigated the role of different allergen concentrations in influencing the diaplacental allergen transport in preterm and term placentas. Method: Twenty-seven human term placentas and 12 preterm placentas were dually perfused in vitrofor up to 4 h by adding alternately two different nutritive allergens, β-lactoglobulin (BLG) or ovalbumin (OVA), at four different allergen concentrations (0.02, 0.2, 2 and 20 mg/ml) to the maternal perfusate medium. Allergen concentrations in fetal venous outflow samples collected during perfusion were measured by using specific ELISAs. Results: Perfusion of increasing allergen concentrations via the maternal circulation resulted in a concentration-dependent increase of fetal allergen uptake in all term and preterm placentas. A mean maternal-to-fetal ratio of 20,000/1 and 3,000/1 for BLG, and 40,000/1 and 5,000/1 for OVA was found in term and preterm placentas, respectively. Preterm placentas (27–36 weeks of gestation) were found to favor the diaplacental passage of nutritive allergens compared with placentas at term (>36 weeks of gestation). Conclusion: Maternal-to-fetal allergen transport occurs in a dose-dependent and molecular weight-dependent manner with clear accentuation in preterm placentas.

Immunoglobulin E (IgE)-mediated immediate-type allergic reactions to hyaluronidase have been observed in children with central nervous system (CNS) tumors. Glucocorticoids, used as therapy for brain edema, are discussed controversially as T helper 2 (Th2) stimulatory factors. In this study we investigated the role of glucocorticoids on a Th2 cytokine-promoting effect in children with CNS tumors. Peripheral blood mononuclear cells (PBMCs) from: 29 children suffering from malignant brain tumors, of whom 23 received short-term glucocorticoid treatment (for 3-4 days) during the course of chemotherapy; 18 children with nephrotic syndrome or renal transplantation receiving long-term glucocorticoid treatment; and 13 healthy children, were incubated with phytohemagglutinin (PHA) and/or anti-CD28 monoclonal antibody (mAb) and, in a second approach, with hyaluronidase. The concentrations of Th cell-mediated cytokines - interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) - were measured in supernatants. The IL-4 production of PBMCs incubated with PHA/anti-CD28 mAb from children with repeated co-administration of glucocorticoids, hyaluronidase, and cytostatic drugs (median: 249.9 pg/ml; range: 234.4-261.7) was significantly higher (p < 0.0001) than IL-4 production of PBMC from children of all the other groups (median: 86.18; range: 16.0-212.5). There was no significant difference in the levels of IL-10 and IFN-gamma within the groups. PBMCs stimulated only with hyaluronidase failed to produce detectable levels of cytokines. The results of this study indicate that repeated co-administration of glucocorticoids and hyaluronidase (a neo-antigen) enhance IL-4 production in vitro and thus may induce the production of specific IgE antibodies in children immunocompromised with cytostatic drugs. Hyaluronidase itself does not stimulate in vitro IL-4 synthesis in PBMCs of children receiving cytostatic drugs.