Probiotics have a different immunomodulatory potential in vitro versus ex vivo upon oral administration in children with food allergy.

BACKGROUND: Previous studies suggest that administration of probiotics in vitro can stimulate regulatory and Th1 immune responses. We studied both the in vitro immunological effects of probiotics and the ex vivo immunological effects after oral administration of probiotics in children with food allergy, a Th2-mediated disease. METHODS: Thirteen children were enrolled. Probiotics (n = 7) or placebo (n = 6) were orally administered during 3 months. At baseline and after 1 and 3 months, peripheral blood mononuclear cells were stimulated with crude peanut extract, anti-CD3, or anti-CD40 and IL-4 in the presence (in vitro response) or absence (ex vivo response) of probiotics. The proliferation and production of IFN-gamma, IL-5, IL-13, IL-10, TNF-alpha, IL-6 and IgE were analyzed. Sensitization to peanut, cow's milk and hen's egg was determined before and after treatment. RESULTS: The in vitro addition of probiotics to peripheral blood mononuclear cell cultures resulted in enhanced proliferation and production of IFN-gamma, IL-10 and TNF-alpha. After oral treatment, proliferation in the presence of probiotics increased, whereas in vitro IgE production decreased in the probiotics group compared to baseline. The ex vivo production of IL-10, TNF-alpha and IL-6 tended to decrease. Th1 and Th2 cytokines were not altered. Sensitization remained unchanged. CONCLUSION: Probiotics enhanced the production of Th1 and regulatory cytokines in vitro. Oral administration of probiotics resulted in a slightly decreased ex vivo production of IL-10, TNF-alpha and IL-6. This indicates that probiotics have a different potential to modulate the immune response in vitro versus ex vivo.

The interleukin-10 inducing effect of transforming growth factor-beta on human naive CD4+ T cells from cord blood is restricted to the TH1 subset.

Transforming growth factor (TGF-beta) seems to play a role in the regulation of immune responses, mainly by its suppressive function towards cells of the immune system. However, both in mice and human, conflicting data are published on the capacity of TGF-beta to induce interleukin (IL)-10 secretion in both naive and skewed T cell populations. Our aim was to test the IL-10-inducing capacity of TGF-beta in both naive and skewed cord blood mononuclear cells (CBMCs) and elucidate the mechanism by which TGF-beta exerts its effect. Therefore, naive CBMCs and CBMCs during skewing under T helper 1 (Th1) and Th2 polarizing conditions were stimulated with CD3 and/or CD28 in the presence or absence of TGF-beta. Proliferation, cytokine production and mRNA expression of transcription factors was measured. TGF-beta enhanced the IL-10 production in Th1 and naive cells only, and suppressed the T(H)1 phenotype as demonstrated in cytokine levels and T-box expression in T cells (T-bet) expression. Interestingly, forkhead box p3 (Foxp3) expression tended to increase in both Th1 and Th2 cells. These data indicate that TGF-beta can induce a regulatory phenotype in both naive and Th1-polarized cells derived from cord blood. The induction of IL-10 was not observed in Th2-polarized phenotype, indicating that TGF-beta might be especially of interest for immunomodulation in Th1 cells.

Differences in antigen-specific T-cell responses between infants with atopic dermatitis with and without cow's milk allergy: relevance of TH2 cytokines.

BACKGROUND: Cow's milk is the most important food antigen in infancy and may lead to acute cutaneous symptoms and atopic dermatitis (AD). The role of circulating allergen-specific T cells in the pathogenesis of food-allergic skin symptoms is still under investigation. OBJECTIVE: This study was designed to analyze the cow's milk protein (CMP)-specific T-cell response at the clonal level in infants with AD and cow's milk allergy (CMA) in comparison with infants with AD without CMA. METHODS: We used an antigen-specific culturing system with autologous B cells as antigen-presenting cells to establish CMP-specific T-cell clones derived from PBMCs in infants with AD. T-cell reactivity, measured by using a lymphocyte stimulation test, and cytokine production, measured by using ELISA, was compared between infants with AD with and without CMA. RESULTS: Both infants with and without allergy to cow's milk had a CMP-specific T helper cell response directed against the major proteins in milk. Analysis of antigen-specific cytokine production showed that this response was T(H)2 skewed in infants with CMA, with production of high levels of IL-4, IL-5, and IL-13. In contrast, infants without CMA had a T(H)1-skewed response, with high levels of IFN-gamma and low levels of IL-4, IL-5, and IL-13. CONCLUSION: These data confirm for the first time at the clonal level that food allergy in infants with AD is associated with production of T(H)2 cytokines by circulating antigen-specific CD4(+) T cells, whereas tolerance to food antigens is associated with low levels of these cytokines. This suggests a key role for the T helper cell-derived T(H)2 cytokines in food allergy-related skin symptoms.

Clinical and immunologic variables in skin of patients with atopic eczema and either positive or negative atopy patch test reactions.

BACKGROUND: Epicutaneous application of aeroallergens induces a positive atopy patch test (APT) response in about 50% of patients with atopic eczema (AE) and sensitization for these allergens. OBJECTIVE: To elucidate the mechanisms determining the outcome of the APT, the following questions were addressed. Are there differences in clinical features between patients with AE who have positive versus negative APT responses? Is a macroscopically negative APT response also histologically negative, and if so, are there differences in clinically noninvolved skin between the two groups regarding (1) the sensitivity toward an irritant, (2) the composition of cellular infiltrate, (3) the presence of aeroallergen-specific T cells, and (4) the number of IgE(+) cells? METHODS: Punch biopsy specimens from both house dust mite patch tested and the clinically noninvolved skin of patients with AE who have positive APT responses (n = 10) and negative APT responses (n = 10) and those from the normal skin of atopic individuals without AE (n = 10) and nonatopic volunteers (n = 10) were analyzed by using immunohistochemistry with mAbs against eosinophil cationic protein, IgE, the high-affinity receptor for IgE, and CD3 and CD25 mAbs. Furthermore, T-cell lines were propagated from noninvolved skin of all patient and control groups. The T-cell lines were tested for house dust mite specificity. RESULTS: Negative APT sites were immunohistochemically similar to clinically noninvolved AE skin. There were no significant differences between patients with AE who had positive and negative APT results regarding either clinical features, the composition of cellular infiltrate, or the presence of allergen-specific T cells in clinically noninvolved skin. However, differences were observed regarding the presence of IgE on epidermal CD1a(+) cells. CONCLUSION: Our results indicate that a positive APT reaction requires the presence of epidermal IgE(+) CD1a(+) cells in clinically noninvolved skin, but that also other, as yet unknown, discriminatory factors are involved.

Influence of culture period on the allergenic composition of Pityrosporum orbiculare extracts.

BACKGROUND: Previous characterization studies of Pityrosporum orbiculare allergens have led to contradictory results. In immunoblotting studies a range of IgE-binding proteins of 10-100 kDa have been identified. In another study, however, the IgE-binding structures were claimed to be associated with high-molecular-weight polysaccharides or glycoproteins, presumably mannans or mannoproteins. OBJECTIVE: In the present study the reasons for these discrepancies were investigated. METHODS: P. orbiculare preparations were compared in IgE ELISA and IgE-inhibition ELISA, as well as in immunoblotting with sera from atopic dermatitis patients. RESULTS: It was inferred that variations in the period of in vitro culture of P. orbiculare constituted the most important factor determining the different compositions of the resulting yeast cell extracts. After 2 days of culture a wide range of allergenic proteins was present but upon more prolonged culture (> 4 days) most proteins of 10-100 kDa were lost. Accordingly, the protein concentration of the extracts gradually declined from 40% to 25% between days 4 and 15 of culture. On the other hand, the carbohydrate content remained fairly constant (approximately 30%). Using inhibition ELISA it was demonstrated that the high-molecular-weight glycoproteins or polysaccharides presumably involved in most of the IgE-binding capacity in extracts from old cultures, were also present in comparable concentrations in all extracts tested, even after culture for only 2 or 4 days. CONCLUSION: Preparations obtained from the exponential phase of yeast cultures (2-4 days old), should preferably be used in studies of the IgE response to P. orbiculare.

Different growth factor requirements for human Th2 cells may reflect in vivo induced anergy.

We previously reported the isolation of allergen-specific Th2 lines and clones from atopy patch test (APT) sites of atopic dermatitis (AD) patients. Upon stimulation with allergen or anti-CD3+ phorbol myristate acetate (PMA) IL-4 was released with or without IL-5, while no (or extremely low concentrations of) IL-2 and interferon-gamma (IFN-gamma) were detectable. A high IL-4/IFN-gamma ratio facilitates production of allergen-specific IgE, of which high levels are observed in AD patients. Here we show that the above mentioned Th2 cells are notably different from murine Th2 cells. Not IL-4, which is the autocrine acting growth factor for murine Th2 cells, but IL-2 was needed for proliferation of these human APT-derived Th2 lines and clones. Of significance, unless exogenous IL-2 was added, no proliferative response to allergen, presented by Epstein-Barr virus-transformed B (EBV-B) cells, non-T cells or IgE-bearing Langerhans cells (LC), occurred. Lack of proliferation and IL-2 production after full T cell receptor (TCR) triggering is a characteristic first described for in vitro anergized T cells. However, like the clones we describe in this study, anergic T cells may retain production of cytokines other than IL-2. A further resemblance between anergic T cells and the human Th2 clones reported here is that IL-4 can enhance IL-2-driven proliferation, but is not capable of inducing T cell growth by itself. The absence of IL-4-driven proliferation differentiates human Th2 cells from murine Th2 cells. Both produce IL-4 when stimulated in a cognate fashion, but only murine Th2 cells will proliferate. We conclude that the presently reported human Th2 cells are different from murine Th2 cells, in that they need other T cells to produce IL-2 required for their expansion. Moreover, the Th2 cells phenotypically resemble anergic T cells. As yet, however, we have no clue as to whether these features account for the current Th2 cells only or for human Th2 cells in general. We hypothesize that the Th2 phenotype of AD skin-derived, allergen-specific T cells may be induced in vivo by LC, which lack CD80, and therefore do not provide secondary signals through CD28-CD80 interaction.

Allergens of Pityrosporum ovale and Candida albicans. I. Cross-reactivity of IgE-binding components.

In atopic dermatitis (AD), a high prevalence has been reported of type I reactions and specific IgE to extracts of the commensal lipophilic skin yeast Pityrosporum ovale. In the present study, a highly significant correlation (r = 0.77) was found between levels of anti-P. ovale IgE and of IgE reacting with extracts of Candida albicans, both measured by a sensitive ELISA method. In a series of 128 AD sera, 34 sera reacted positively with both yeast extracts, 38 reacted with P. ovale but not with C. albicans, and only one of the 56 anti-P. ovale-negative sera showed a very weak reaction with C. albicans. The correlation was due to a marked cross-reactivity, as shown by inhibition ELISA. Fluid-phase preincubation of double-positive sera with either of the two yeast extracts resulted in a dose-dependent, and at high concentrations complete, inhibition of the IgE reactions with both coated P. ovale and C. albicans allergens. Mutual inhibition of IgE-binding could also be achieved with pools of glycoproteins and/or polysaccharides isolated from the crude extracts by Con A affinity chromatography. P. ovale allergens were, however, more potent fluid-phase inhibitors than the corresponding C. albicans components. The apparently higher avidity for P. ovale allergens suggests that these antiyeast IgE antibodies in AD result from sensitization to P. ovale and cross-react with C. albicans.

Allergens of Pityrosporum ovale and Candida albicans. II. Physicochemical characterization.

Pityrosporum ovale has recently been recognized as a source of allergens to which many patients with atopic dermatitis (AD) show type I skin reactions and specific IgE antibodies. In this study the IgE-binding components and/or epitopes in P. ovale extract were shown to be partially sensitive to pronase or trypsin treatment, whereas periodate oxidation resulted in a complete loss of IgE-binding capacity, thus suggesting the involvement of carbohydrate structures. In Con A affinity chromatography most of the IgE-binding capacity of crude P. ovale extract bound to the column, and could be eluted with mannoside. Gel filtration on Sephacryl S-400 revealed a marked heterogeneity with respect to molecular mass, with most of the IgE-binding activity associated with high-mol.-mass fractions (from 5 x 10(4) up to 2 x 10(6) Da). A similar heterogeneity was found after chromatofocusing, with IgE-binding in the whole pI-range from 7.0 to 4.0. Essentially identical results were obtained with extracts of Candida albicans, in agreement with the previously shown cross-reactivity of IgE-binding components in the two yeast extracts. In inhibition ELISA, gel filtration and chromatofocusing fractions containing components with widely different mol. mass or pI showed complete reciprocal cross-inhibition, and were all capable of inhibiting the binding of IgE to unfractionated extracts. We therefore conclude that the cross-reacting anti-P. ovale/anti-C. albicans IgE antibodies in the sera of AD patients are mainly directed at a restricted number of carbohydrate epitopes that are expressed on a heterodisperse range of high-mol.-mass components, probably mannans or mannoproteins.