Pityrosporum and Candida specific and non-specific humoral, cellular and cytokine responses in atopic dermatitis patients

BACKGROUND: Recent cytokine (RT-PCR, ELISA) analyses of inflammation in atopic dermatitis (AD) have suggested a role for IL-4, IL-5 and IFNgamma. Pityrosporum ovale and Candida albicans are important allergens in some patients with AD of the seborrhoic head, neck and shoulder region. In AD patients, the saprophytic yeasts induce IgE responses while they usually induce TH1 type responses. The cytokine responses induced by yeasts in AD are sparsely investigated. OBJECTIVE: To characterize the P. ovale- and C. albicans-specific and non-specific humoral, lymphoproliferative and cytokine (IL-2, 4, 5 and IFNgamma) responses in AD. METHODS: Fifteen AD patients and seven healthy controls (HC) were included. Ficoll-isolated PBMC were stimulated by PHA and laboratory-generated extracts of P. ovale and C. albicans. Lymphocyte proliferation was measured by 3H-thymidine incorporation and cytokine production by sandwich-ELISAs. The antigen-specific IgG and IgE antibodies were analysed by ELISA and nitrocellulose RAST. RESULTS: Pityrosporum ovale- and C. albicans-specific IgE (both P < 0.001) and P. ovale-induced PBMC proliferation (P < 0.02) were elevated in AD. In general, the IL-4/IFNgamma ratio induced by P. ovale was higher than that induced by C. albicans (P < 0.01). The PHA-induced IL-2 (P < 0.05) and IL-4 responses (P < 0.005), and the C. albicans-induced IL-5 response (P < 0.02) and IFNgamma response (P < 0.01), were elevated in AD. A network of correlations was seen between serum total and the yeast-specific IgE, P. ovale-specific lymphoproliferation, PHA-induced IL-2, IL-4 and IL-5, and C. albicans-induced IL-5. CONCLUSION: The cytokine profiles found in this study support the role of TH0 or TH1 cells by the side of TH2 cells in the pathogenesis of atopic dermatitis. Pityrosporum ovale appears to be associated more with IL-4 responses and C. albicans with IFNgamma responses.     

Interleukin-13: Targeting an underestimated cytokine in atopic dermatitis

Atopic dermatitis (AD) is a common inflammatory skin condition that has traditionally been considered a paradigmatic type 2 immunity (T2)-driven disease. Interleukin (IL)-4 and IL-13 are both pivotal cytokines involved in the generation of allergic diseases. Currently, besides dupilumab, which blocks the binding of both cytokines to their receptors, a number of new pharmacologic entities have been designed to target both T2 cytokines and/or their receptors and/or receptor-associated signal transduction machinery such as Janus kinases. Recently, IL-13 has been suggested to be the key T2 cytokine driving inflammation in the periphery, while IL-4 may merely have a central effect. There is increasing evidence that this concept holds true for the inflammatory reaction underlying AD, where IL-13 is overexpressed locally and has a significant impact on skin biology, including the recruitment of inflammatory cells, the alteration of the skin microbiome, and the decrease in the epidermal barrier function. This review provides an update on the role of IL-13 in AD and discusses the different strategies aimed at interfering with its biologic activity as well as their potential in a precision medicine approach in the management of AD.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.     

PPARgamma expression profile and its cytokine driven regulation in atopic dermatitis

BACKGROUND: Recent studies point to the pathophysiological role of the peroxisome proliferators-activated receptor gamma (PPARgamma) in the inflammatory immune response. We have showed that activation of PPARgamma by specific ligands attenuates the allergic immune response via monocytes and lymphocytes. The objective of this study was to analyse the PPARgamma expression and its regulation via inflammatory cytokines. METHODS: We examined the PPARgamma expression in the lesional and nonlesional skin of atopic patients by immunohistochemistry. The expression patterns of PPARgamma mRNA and its isoforms were investigated in peripheral mononuclear blood cells of atopic and nonatopic donors and in cytokine-stimulated populations by quantitative real-time RT-PCR. RESULTS: Our data show an increased PPARgamma expression in lesional skin from atopic dermatitis patients. The analysis of PPARgamma mRNA reveals a significantly up-regulated expression of PPARgamma1 but not of PPARgamma2 in monocytes and CD4(+) T-cells from atopic dermatitis patients. Furthermore, we demonstrate that Th-cytokines, like IL-4, IL-13 and IFNgamma, which regulate the biphasic atopic immune response, directly regulate the expression of PPARgamma1. CONCLUSION: Taken together, these data demonstrate that PPARgamma isoforms are differently expressed and regulated by the local cytokine-milieu. Whether the increased expression of the PPARgamma1 receptor may be beneficial or not for a PPARgamma ligand-based treatment of atopic dermatitis, is currently under investigation.     

Inflammatory cell numbers and cytokine expression in atopic dermatitis after topical pimecrolimus treatment

BACKGROUND: In several clinical trials the topical application of pimecrolimus was shown to be effective in the treatment of atopic dermatitis (AD). By targeting calcineurin-dependent signaling pathways, pimecrolimus controls cytokine gene expression. The purpose of this study was to investigate the effect of pimecrolimus on the inflammatory infiltrate and cytokine expression pattern in AD upon topical therapy. METHODS: From 10 patients with acute AD, skin biopsies as well as immunophenotype and cytokine production of peripheral blood mononuclear cells (PBMC) were examined before and 3 weeks after therapy. RESULTS: The clinical improvement was associated with a marked regression of histopathological features. In particular, the density of the inflammatory infiltrate mostly containing lymphocytes and eosinophils declined. By double immunofluorescent staining, a reduced expression of the T helper (Th) 2 cytokines interleukin (IL)-5, IL-10, and IL-13 in both CD4+ and CD8+ T cells was demonstrated after therapy. Pimecrolimus therapy was also associated with a reduced expression of the Th1 cytokine interferon (IFN)-gamma. Interestingly, the numbers of epidermal CD1a+ dendritic cells increased following treatment. In the peripheral blood, a decrease of lymphocytes and eosinophils was noticed, but the distribution of lymphocyte subpopulations and their capacity of cytokine production did not change. CONCLUSIONS: Topical pimecrolimus exhibits anti-inflammatory effects in AD by reducing the inflammatory cell infiltrate and cytokine expression in the dermis.     

Immediate hypersensitivity to Malassezia furfur and Candida albicans mannans in vivo and in vitro

BACKGROUND: Elevated and correlative Malassezia furfur (M. furfur) and Candida albicans (C. albicans) mannan-specific IgE have been demonstrated in atopic eczema dermatitis syndrome (AEDS) of the head, neck and shoulder (HNS) region of the skin. The significance of these antibodies in vivo has not been demonstrated. METHODS: Sixty-five AEDS patients with HNS distribution were included. Serum total IgE (S-IgE) and yeast antigen-specific (Cetavlon-purified mannan and whole extract antigens of M. furfur and C. albicans) IgE were measured and skin prick tests (SPT) were performed with the yeast antigens. RESULTS: Mannan-specific IgE and SPT were positive in 51 and 48% of patients with M. furfur and in 42 and 22% with C. albicans, respectively. Whole extract-specific IgE and SPT were positive in 85 and 95% of patients with M. furfur and in 91 and 57% with C. albicans, respectively. The highest correlation between specific IgE and SPT was seen with M. furfur mannan (r = 0.60; P < 0.0001). Both M. furfur mannan-specific IgE (r = 0.76; P < 0.0001) and SPT (r = 0.44; P = 0.0005) correlated with S-IgE. CONCLUSIONS: Mannan-induced immediate hypersensitivity in vivo was demonstrated in SPT. The significant correlation between M. furfur mannan-specific IgE and SPT suggests that mannan is an important allergen in yeast hypersensitive AEDS in vivo.     

IgE antibodies to protein and mannan antigens of pityrosporum ovale in atopic dermatitis patients

Background Pityrosporum ovale is a common saprophyte on the skin capable of inducing IgE antibody production in atopic dermatitis (AD) patients. Allergens ofP. ovale have been examined in several studies, but consensus on them is lacking. Objective This study was carried out to obtain more information about the IgE antibody response against P. ovale. including niunnun. Methods Sera from 64 AD patients and 10 healthy controls were analysed with immuno-blotting and the nitrocellulose radio allergosorbent test (RAST) method specifically developed to detect antimannan P. ovale IgE antibodies. Results In immunoblotting a total of 39 different IgE stained protein bands were seen. A high molecular weight staining was also seen especially in patients who displayed elevated mannan P. ovale RAST values. The most commonly stained protein bands in immunoblotting were 9 and 96 kD bands with antibodies in 73 and 65% of AD patients who had been positive in commercial P. orbiculare RAST with total serum IgE less than 4000 kU/I. Mannan RAST appeared positive in 77% of them. Positive immunoblotting to either of these bands was seen in 90% and, if added with staining with ihe 20 kD band, in 100% of these AD patients. A comhination of 9 kD IgE staining and mannan P. ovale RAST was positive in 92% of the patients and % kD and mannan P. ovale RAST in 85% of the patients. Conclusion It is evident that P. ovale has several allergens, the 9. 96 and 20 kD regions being the most important. According to our results mannan is also an important allegen of P. ovale     

Impaired TLR-2 expression and TLR-2-mediated cytokine secretion in macrophages from patients with atopic dermatitis

Background:  In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus . The pattern recognition receptor toll-like receptor (TLR)-2 recognizes components of S. aureus , for example, lipoteichoic acid (LTA) and peptidoglycan (PGN) and, therefore, might be crucial in the pathogenesis and flare-ups of AD.
Objective:  To investigate TLR-2 expression and cytokine secretion in macrophages from patients with AD compared to healthy controls upon TLR-2 stimulation with PGN, LTA and Pam3Cys.
Methods:  Macrophages were cultivated from highly purified peripheral blood monocytes of AD patients and nonatopic healthy controls and stimulated with PGN, LTA and Pam3Cys in a time and dose–dependent manner. Afterwards, TLR-2 expression and cytokine secretion were measured on protein and mRNA level. TLR-1 and TLR-6 expression were investigated on the mRNA level. Immunohistochemical stainings from punch biopsies were performed to investigate TLR-2 expression in skin macrophages .
Results:  We could clearly show that macrophages from patients with AD expressed significantly less TLR-2, whereas the expression pattern of TLR-1 and TLR-6 were not altered. Macrophages had a reduced capacity to produce pro-inflammatory cytokines such as IL-6, IL-8 and IL-1β after stimulation with TLR-2 ligands.
Conclusion:  Our findings clearly show an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls which might contribute to the enhanced susceptibility to skin infections with S. aureus in AD.     

The effects of Mycobacteria vaccae derivative on allergen-specific responses in children with atopic dermatitis

The capacity of microbial products to inhibit allergic inflammation make them logical candidates for novel therapies in allergic diseases such as atopic dermatitis. To assess the effects of intradermal Mycobacterium vaccae derivative on allergen‐specific immune responses in children with moderate to severe atopic dermatitis. Peripheral blood mononuclear cells were isolated from children aged 5–16 years who received intradermal injections of M. vaccae derivative AVAC^TM (n = 26) or placebo (n = 34) three times at 2‐weekly intervals, weeks 0, 2 and 4. Cytokine [interleukin (IL)‐13, interferon (IFN)‐γ and IL‐10] responses to allergen [house dust mite (HDM)], mitogen [phytohaemagglutinin (PHA)], Staphylococcal enterotoxin B (SEB) and Toll‐like receptor (TLR) ligands were assessed. At week 8 (1 month after all injections given) children in the AVAC group showed a significant increase in IL‐10 (P = 0·009), T helper type 1 (Th1) IFN‐γ (P = 0·017) and Th2 IL‐13 (P = 0·004) responses to HDM compared with baseline (week 0). There were no significant changes in any cytokine production in the placebo. HDM‐specific IL‐10 responses remained significantly higher (P = 0·014) than at baseline in the AVAC group by week 12; however, the HDM‐specific IL‐13 and IFN‐γ responses were no longer significantly different from baseline. IL‐13 (r = 0·46, P < 0·001) and IL‐10 (r = 0·27, P = 0·044) responses to HDM were correlated with total immunoglobulin E but not with disease severity. There were no effects of AVAC on mitogen, SEB, TLR‐2‐ or TLR‐4‐mediated responses. This M. vaccae derivative appeared to modulate responses to HDM selectively, suggesting the capacity for in vivo effects on allergen‐specific immune responses.     

Increased plasma LIGHT levels in patients with atopic dermatitis

LIGHT [the name of which is derived from 'homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for herpes simplex virus entry mediator (HVEM), and expressed by T lymphocytes'], is a member of the tumour necrosis factor superfamily that is involved in various inflammatory diseases. We aimed to estimate the relevance of plasma LIGHT levels as a biomarker for atopic dermatitis (AD). In order to understand the putative role of LIGHT in AD pathogenesis, we also investigate the effects of LIGHT on a monocytic cell line, human acute monocytic leukaemia cell line (THP-1). We examined plasma LIGHT levels, total serum IgE, serum value of CCL17 and peripheral blood eosinophil counts in patients with AD and healthy subjects. The effects of LIGHT on activation and apoptosis in THP-1 cells were also investigated. The plasma concentrations of LIGHT in AD patients were significantly higher than those in healthy individuals and the concentrations decreased as the symptoms were improved by treatment. The LIGHT plasma concentrations correlated with IgE levels and the Severity Scoring of AD (SCORAD) index. In addition, LIGHT stimulation increased expression of CD86 and induced production of interleukin-1β in THP-1 cells. Apoptosis was inhibited, the Bcl-2 level increased and the caspase-3 level decreased in THP-1 cells stimulated with LIGHT, compared to unstimulated control cells. These results suggest that plasma LIGHT levels may be one of the promising biomarkers for AD.     

Allergens of Pityrosporum ovale and Candida albicans

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-F. ovafe-negative sera showed a very weak reaction with C. albicans . The correlation was due to a marked cross-reactivity, as shown by inhibition ELISA. Eluid-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 fiuid-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 .     

Interleukin 2 therapy in severe atopic dermatitis

Interleukin 2 (IL-2) at a dose of 10,000 to 20,000 U/kg/q 8 hr was given for 9–12 days to six patients with cases of severe atopic dermatitis (AD) which were refractory to conventional therapy. After IL-2 therapy, the clinical symptoms and signs of eczema including pruritus, scratching, papulovesicles, and lichenification were much improved, but all of them recurred 2–6 weeks after stopping treatment. Adverse reactions were similar to those reported previously, but all of them subsided after discontinuation of therapy. Laboratory findings showed decreased T-cell subsets, especially CD4+ cells, and increased IL-2R+ (CD25) cells, but there was no significant change in serum IL-2, serum IgE, orin vitro IgE production. Immunopathological studies of the skin biopsies showed decreased mononuclear-cell infiltration, depletion of CD4+ cells, and enhanced expression of CD25 and HLA-DR antigens. As lymphokine-activated killer (LAK)-cell activity against cultured fibroblasts was similar in patients with AD and in normals and CD1+ Langerhans cells were not decreased after IL-2 therapy, we speculate that the depletion of helper/inducer CD4+ cells and hence abrogation of the exaggerated antigen processing and cellular activation in diseased skin are the explanation for the transient efficacy of IL-2 in the treatment of atopic dermatitis.     

Differential in vivo cytokine mRNA expression in lesional skin of intrinsic vs. extrinsic atopic dermatitis patients using semiquantitative RT-PCR

BACKGROUND: A small subgroup of atopic dermatitis (AD) patients with normal serum IgE levels and without specific IgE sensitization has been termed 'intrinsic type of AD' (ADi) as a counterpart to the term 'extrinsic type of AD' (ADe). However, there are neither molecular markers nor clinically diagnostic tools for distinguishing between ADi and ADe. OBJECTIVE: The present studies were undertaken to clarify the pathogenesis and in vivo cytokine micromilieu of ADi patients in comparison with ADe patients. METHODS: We used semiquantitative RT-PCR to investigate the expression of various cytokines and assessed the tissue eosinophil counts in skin biopsies from both types of AD patients. RESULTS: Although there was no significant difference of cellular infiltrates in the lesional skin between ADe and ADi patients, ADe had significantly increased tissue eosinophilia than ADi. Based on our RT-PCR, the expression patterns of cytokines could be categorized into four groups. The first group includes IL-5, IL-13, and IL-1beta, whose levels of mRNA expression were higher in both types of AD patients than non-atopic (NA) subjects, while ADe patients had even higher levels than ADi patients. The second group includes interferon-gamma (IFN-gamma), IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and IL-10, whose levels of mRNA expression were elevated in both types of AD patients without differences between ADe and ADi patients. The third group includes tumour necrosis factor-alpha (TNF-alpha), whose mRNA expression was more decreased in both types of AD patients than NA, and the fourth group includes IL-6 and transforming growth factor-beta (TGF-beta), which did not show any differences among the three groups. CONCLUSION: These current data demonstrate that the expressions of cytokines IL-5, IL-13, and IL-1beta mRNA and the number of dermal infiltrating eosinophils are increased in ADe patients compared with ADi patients.     

Bronchial and cutaneous responses in atopic dermatitis patients after allergen inhalation challenge

Background Atopic dermatitis (AD) is often associated with allergic asthma (AA). Inhalation of allergens influences the activity of AA but the effect on the skin in AD is unclear. Objectives We evaluated the degree of bronchial hyperresponsiveness to methacholine in eight AD patients with AA (AD⁺) and eight AD patients without AA (AD^–) and studied bronchial and cutaneous responses after allergen inhalation challenge. Methods All patients were treated in hospital for their eczema with tar ointment (pix liquida) and orally administered antihistamines (mean hospital stay 37 days). After clearing of the skin lesions allergen inhalation challenge was performed. Cutaneous responses were studied by measuring the‘Costa’ score before and 24 h after allergen inhalation challenge. Results The median value of the provocative concentration of methacholine causing a 20% fall (PC₂₀ Mch) in forced expiratory volume in 1 second (FEV₁) was significantly higher in the AD^– group compared to the AD⁺ group with median values of 10.70 and 0.60mg/mL, respectively. These values did not change significantly in both groups during hospital stay. After challenge all AD⁺ patients showed early and late asthmatic responses whereas only four AD^– patients showed early asthmatic responses (mean values of the maximal fall in FEV_l during the EAR 37%/16% and in PEF during the LAR 27%/4% for AD⁺ and AD^–patients, respectively). The‘Costa’ score increased in both groups (mean score before 19.1/ 24.4 and after challenge 26.8/26.9 for AD⁺ and AD⁺ patients, respectively). The increase in the AD^– group was significantly higher compared with the AD^– group (P= 0.016). Conclusion We conclude that allergen inhalation challenge causes a flare up of the skin lesions in atopic dermatitis patients. This was more prominent in atopic dermatitis patients who already suffered from an IgE-mediated allergic inflammation in the lung.     

Cross-reacting IgE and IgG antibodies to Pityrosporum ovale mannan and other yeasts in atopic dermatitis.

Atopic dermatitis (AD) patients often demonstrate positive skin prick test results and serum IgE antibodies to a range of different yeasts. This has been thought to be due to cross-reactivity. In this study, the cross-reactivity of IgE and IgG antibodies between mannan and crude antigens of Pityrosporum ovale, Candida albicans, and Saccharomyces cerevisiae and crude antigens of Cryptococcus albidus and Rhodotorula rubra was examined by RAST and ELISA inhibition with two serum pools of AD patients. We found cross-reacting IgE and IgG antibodies. In the IgE response, the main cross-reacting pattern was the mannan region, although inhibition could be achieved also with crude antigens of C. albicans, S. cerevisiae, and, to some extent, C. albidus. P. ovale was the most potent inhibitor of IgE-binding components, and against it the highest IgE antibody levels were detected in AD serum pools. In contrast, C. albicans was found to be the most important inducer of IgG antibodies, since the IgG level against P. ovale mannan in both AD serum pools was very low. Cross-reacting antibodies were also seen in ELISA inhibition with both crude and mannan antigens, but since the IgG antibody level of P. ovale mannan in AD serum pools was low, further studies are needed to confirm the IgG results.     

Clinical effects of probiotics are associated with increased interferon-γ responses in very young children with atopic dermatitis

BACKGROUND: We recently demonstrated that administration of probiotics resulted in significant clinical improvement in very young children with moderate-to-severe atopic dermatitis (AD). The purpose of this study was to determine the underlying immunological effects that are associated with these apparent clinical benefits. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from children (n = 53) at baseline and at the end of an 8-week supplementation period during which they received a probiotic (Lactobacillus fermentum PCCtrade mark) (n = 26) or a placebo (n = 27). A further sample was collected at 16 weeks (8 weeks after ceasing the supplement). Cytokine (IL-5, IL-6, IL-10, IL-13, IFN-gamma and TNF-alpha) responses to allergens (egg ovalbumin (OVA), beta lactoglobulin (BLG), house dust mite (HDM)), vaccines (tetanus toxoid (TT)), diphtheria toxoid (DT)), intestinal flora (heat-killed Lactobacillus (HKLB)), heat-killed Staphylococcus aureus (HKSA), Staphylococcus aureus enterotoxin B (SEB) and mitogen (phytohaemaglutinin (PHA)) were compared. RESULTS: The administration of probiotics was associated with a significant increase in T-helper type 1(Th1-type) cytokine IFN-gamma responses to PHA and SEB at the end of the supplementation period (week 8: P = 0.004 and 0.046) as well as 8 weeks after ceasing supplementation (week 16: P = 0.005 and 0.021) relative to baseline levels of response. No significant changes were seen in the placebo group. The increase in IFN-gamma responses to SEB was directly proportional to the decrease in the severity of AD (r = -0.445, P = 0.026) over the intervention period. At the end of the supplementation period (week 8) children receiving probiotics showed significantly higher TNF-alpha responses to HKLB (P = 0.018) and HKSA (P = 0.011) but this was no longer evident when supplementation ceased (week 16). Although IL-13 responses to OVA were significantly reduced in children receiving probiotics after 8 weeks (P = 0.008), there were no other effects on allergen-specific responses, and this effect was not sustained after ceasing supplementation (week 16). There were no effects on vaccine-specific responses, or on responses to any of the stimuli assessed. CONCLUSION: The improvement in AD severity with probiotic treatment was associated with significant increases in the capacity for Th1 IFN-gamma responses and altered responses to skin and enteric flora. This effect was still evident 2 months after the supplementation was ceased. The lack of consistent effects on allergen-specific responses suggests that the effects of probiotics may be mediated through other independent pathways, which need to be explored further.