Library

Notes: 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 PCC™) (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-γ and TNF-α) 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-γ 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-γ 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-α 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-γ 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.

Notes: Background Breast milk contains many immunomodulatory factors (soluble CD14 (sCD14), IgA and cytokines) with the potential to influence infant immune development.Objective To determine if changes in breast milk ω-3 polyunsaturated fatty acid (n-3 PUFA) composition as a result of maternal dietary fish oil supplementation during pregnancy can modify levels of these immunological parameters in breast milk.Method In a randomized controlled trial, 83 atopic women received either 4 g fish oil capsules (containing 3.7 g n-3 PUFA) (n=40) or 4 g olive oil capsules (n=43) from 20 weeks gestation until delivery. Breast milk was collected 3 days post-partum and fatty acids were analysed by gas liquid chromatography and IgA, sCD14 and cytokines (IL-5, IL-6, IL-10, TNF-α and IFN-γ) were quantitated by ELISA or time resolved fluorescence (TRF).Results ω-3 docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels were significantly higher (P〈0.001) in breast milk from women supplemented with fish oil (n=33, DHA mean 1.15%, SD 0.47% and EPA mean 0.16%, SD 0.07%) than in samples from the control group (n=40, DHA mean 0.50%, SD 0.17% and EPA mean 0.05%, SD 0.02%). Breast milk arachidonic acid (AA; 20:4n-6) levels were significantly lower (P=0.045) in the fish oil group (mean 0.55%, SD 0.12%) compared with the control group (mean 0.61%, SD 0.14%). Breast milk IgA was positively correlated with DHA (P=0.046) and 22:5n-3 (P=0.003), but inversely correlated with linoleic acid (LA; 18:2n-6) (P=0.034). Levels of sCD14 were also positively correlated with 22:5n-3 (P=0.009). Cytokines involved in IgA synthesis (IL-10 and IL-6) were also significantly correlated with both IgA and n-3 PUFA levels, although there were no differences in the levels of breast milk IgA, sCD14 or cytokines between study groups.Conclusion Supplementation with fish oil during pregnancy significantly alters early post-partum breast milk fatty acid composition. ω-3 PUFA levels were positively associated with IgA and sCD14 levels, suggesting a relationship between fatty acid status and mucosal immune function.

Notes: Background There is increasing evidence that the T-cell reactivity to environmental allergens underlying expression of allergic disease in adulthood, develops initially during childhood. However, there is little information available on the kinetics of these early responses, or on the patterns of cytokine production during this period.Objective The purpose of this study was twofold: to obtain further information on the reported differences between responses to food versus inhalant allergens during early childhood, and to ascertain the age-range over which T-cell responses to inhalant allergens become polarized towards the TH2 cytokine profile, in potentially atopic children.Methods In vitro cytokine responses to house dust mite (HDM) and egg (OVA) were assessed by semiquantitative RT-PCR in panels of 2- and 5-year-old children and adults; lymphoproliferative responses to OVA were subjected to epitope analysis.Results At age 2 years IL-4/IL-5 responses to HDM grouped with positive atopic family history, and by age 5 years cytokine responses correlated strongly with individual SPT reactivity to HDM. In contrast, OVA responses were restricted to weak and transient IL-5 signals in the 2-year-old family history positive group. Lymphoproliferation assays performed in parallel indicate a log-scale greater postnatal expansion of T-cell reactivity to the inhalant allergen; preliminary epitope analysis of OVA responses indicate that the number of OVA epitopes recognised decrease during early childhood.Conclusions Inhalant allergen-specific in vitro cytokine production associated with positive skin-prick test (SPT) reactions, one of the hallmarks of adult atopy, manifests in children at or before 5 years of age; additionally, cytokine responses in SPT negative 5 year-olds are restricted to IFNγ, as per normal adults. In contrast, T-cell responses to a typical food allergen appear to be deleted during early childhood.

Notes: Background and objectives A reduced capacity of antigen presenting cells (APC) to provide pro-T helper 1 (Th1) signals, such as IL-12, to T cells during early life may be implicated in the development of T helper 2 (Th2)-mediated allergic disease. In this study we examined the relationships between the capacity for IL-12 responses in the neonatal period and atopic risk (family allergy), in vitro T cell responses to allergens, and the subsequent development of allergic disease at 6 yearsMethods The capacity of circulating neonatal (and maternal) APC to produce IL-12 p70 in response to LPS (and IFN-γ) stimulation was assessed in a group of 60 children with previously well-characterized immune responses to allergens and atopic outcomes. The IL-12 responses were compared with allergen-induced lymphoproliferation (to house dust mite (HDM) ovalbumin (OVA), cat and β-lactoglobulin (BLG)) and IL-13 and IFN-γ cytokine responses (to OVA, HDM and phytohaemaglutinin (PHA)) in the neonatal and postnatal periods. IL-12 responses were also compared according to atopic risk and atopic outcomes (doctor-diagnosed asthma, eczema, food allergies and sensitization as evidenced by skin prick testing) at 6 years clinical follow-up.Results Maternal peripheral blood mononuclear cells (PBMC) synthesized significantly greater amounts of IL-12 than neonatal PBMC, though within maternal-infant pairs IL-12 responses were significantly correlated (r = 0.4, P = 0.019). Moreover, neonatal IL-12 responses were positively correlated with neonatal allergen proliferation for HDM (r = 0.6, P 〈 0.0001), OVA (r = 0.55, P 〈 0.0001), cat (r = 0.5, P = 0.003) and BLG (r = 0.55, P = 0.001), but negatively correlated with neonatal IL-13 responses to both allergens tested (HDM: r = − 0.4, P = 0.03 and OVA: r = − 0.5, P = 0.001). Both neonatal and maternal IL-12 responses were positively correlated with postnatal IFN-γ responses to HDM at 12, 18 and 24 months of age (responses after age of 2 years were not assessed). There was no relationship between atopic risk and IL-12 capacity in the neonatal period, but there was a (non-significant) trend for neonatal IL-12 responses to be lower in the high-risk children who developed clinical allergy at 6 years (compared with the low risk group) although the number in this analysis was small.Conclusions Reduced APC IL-12 production in the perinatal period was associated with reduced T cell activation (lymphoproliferation), stronger neonatal Th2 responses, and weaker Th1 responses to allergen in the postnatal period. This supports the notion that variations in APC function in early life may contribute to altered allergen-specific cytokine responses associated with later allergy.

Notes: Background and objectives The epidemiological association between higher dietary n-3 polyunsaturated fatty acids (PUFA) and lower prevalence of asthma, has led to interest in the role of early dietary modification in allergic disease prevention. In this study we examined the effects of maternal n-3 (PUFA)-rich fish oil supplementation on cord blood (CB) IgE and cytokine levels in neonates at risk of developing allergic disease.Methods In a randomized double-blind, placebo-controlled trial, 83 atopic pregnant women received either fish oil capsules (n = 40) containing 3.7 g n-3 PUFA/day or placebo capsules (n = 43) from 20 weeks gestation until delivery. CB cytokine levels (IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-α and IFN-γ) and total IgE levels were measured and compared between the two groups. Fatty acid composition of red cell membranes was analysed by gas chromatography and the relationships among PUFA, cytokine and IgE levels were examined.Results Maternal fish oil supplementation resulted in a significant increase in n-3 PUFA levels (P 〈 0.001) in neonatal erythrocyte membranes. Neonates whose mothers had fish oil supplementation had significantly lower plasma IL-13 (P 〈 0.05) compared to the control group. There was also a significant inverse relationship between levels of n-3 PUFA in neonatal cell membranes and plasma IL-13. There was no difference in levels of IgE and the other cytokines measured.Conclusions This study provides preliminary evidence that increasing neonatal n-3 PUFA levels with maternal dietary supplementation can achieve subtle modification of neonatal cytokine levels. Further assessment of immune function and clinical follow-up of these infants will help determine if there are any significant effects on postnatal immune development and expression of allergic disease.

Notes: Background Maternal allergy confers stronger allergy risk (than paternal allergy) suggesting that maternal patterns of immune response can directly influence immune development in offspring. Women prone to allergic immune responses to allergens may also have altered immune responses to other antigens including fetal antigens.Objective This exploratory study examines relationships between maternal immune responses to fetal antigens and the subsequent risk of allergy.Methods Mononuclear cells (MNC) were collected from 36 mother–infant pairs to compare maternal (and fetal) cellular immune responses to alloantigens (fetal, maternal or unrelated donor [URD]), and allergens in allergic (18 pairs) and non-allergic (18 pairs) mothers. Thirty children had documented allergic outcomes at 6 years of age.Results In this population, allergic outcomes in the offspring were associated more strongly with materno-fetal immune interactions than with a maternal family history of allergy. Specifically, allergic disease at 6 years of age was associated with significantly higher maternal responses to fetal alloantigens (lymphoproliferation, P=0.008; IL-13, P=0.02 and IFN-γ, P=0.015), whereas associations with maternal allergy did not reach significance (P=0.07). Fetal IFN-γ alloantigen responses were significantly correlated with the degree of human lymphocyte antigen (HLA) mismatch (maternal HLA class II antibodies) (τ=0.3, P=0.03). The capacity of the fetus to produce IL-13 (τ=0.4, P=0.001) and IL-10 (τ=0.3, P=0.029) was directly related to the level of these cytokines produced by the mother in response to fetal antigens. Allergic mothers showed a non-significant trend for stronger lymphoproliferation to fetal alloantigens. The number of previous pregnancies (gravidity) was associated with stronger maternal responses to fetal alloantigens, as shown by lymphoproliferation (Kendall τ=0.3, P=0.04) and IFN-γ (τ=0.3, P=0.04) synthesis, but did not affect fetal responses to the various stimuli.Conclusions Maternal responses to fetal antigens were related to fetal immune responses and subsequent allergy. This novel observation suggests that events at the materno-fetal interface have an important influence on early immune development and should be investigated further.

Notes: Background A significant proportion of children with food allergy and more severe forms of atopic dermatis (AD) go on to develop persistent forms of allergic disease such asthma. Defining immune dysregulation in these children will be of great value in understanding disease pathogenesis.Objective In this study we characterized the immune responses of young infants (6–18 months of age) with moderate-to-severe AD (a modified SCORAD〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:09547894:CEA2348:ges" location="ges.gif"/〉25) and compared these (n=53) with responses of non-allergic children with no history of dermatitis or sensitization of the same age (n=20).Methods Mononuclear cell cytokine responses to allergens (egg ovalbumin (OVA), β-lactoglobulin (BLG), house dust mite (HDM)), vaccines (tetanus toxoid (TT), diphtheria toxoid (DT)), intestinal flora (heat-killed Lactobacillus species (HKLB)), heat-killed Staphylococcus aureus (HKSA), S. aureus enterotoxin B (SEB) and mitogen (phytohaemaglutinin (PHA)) were compared in children with AD with unaffected children.Results Children with AD had significantly lower spontaneous (unstimulated) production of regulatory cytokine IL-10 (P〈0.001), as well as IFN-γ (P〈0.001) and TNF-α (P〈0.001) compared with the unaffected children. After allowing for differences in baseline levels IL-10 responses to virtually all stimuli (food allergens (P=0.003), vaccines P=0.01, intestinal flora (heat-killed Lactobacillus species (HKLB), P=0.005) and skin flora (heat-killed Staphylococcus aureus (HKSA), P=0.003)) were also significantly attenuated in children with AD. The only exception was HDM, to which responses were stronger in children with AD [P=0.05]. Although there were no significant correlations between HDM IgE and HDM cytokine responses at this age, T-helper type 2 (Th2) IL-5 (P=0.014) and IL-13 (P=0.004) responses to HDM were significantly more frequent in the children with AD. However, while children with AD showed significantly attenuated Th1 IFN-γ responses to food allergens (OVA, P=0.007 and BLG, P〈0.001) and vaccines (DT, P=0.008 and TT, P〈0.001), these children showed no difference in Th1 IFN-γ responses to HDM or microbial agents (HKSA and HKLB).Conclusion A increase in propensity for Th2 responses to aeroallergens in children with AD is associated with early impaired production of IL-10 regulatory cytokine to a broad range of environmental stimuli including foods, intestinal flora, S. aureus, and vaccines.

Notes: Background The T-helper type 1 (Th1) trophic properties of bacterial cytosine–phosphate–guanine (CpG) motifs have made them logical adjuvants both for the suppression of Th2-mediated allergic disease in early life and for promoting vaccine responses in neonates who have relatively immature Th1 function. However, little is known about their effects on immature immune responses in this period.Objectives To compare the effects of CpG on adult and neonatal cellular immune responses to various stimuli.Methods The immune responses of mononuclear cells (MC) derived from neonates (n=25) and their mothers (n=25) were compared in vitro. These were stimulated with house dust mite (HDM), CpG B, CpG C, non-CpG oligodeoxynucleotides (ODN) or diphtheria toxoid (DT) in optimized conditions. In parallel cultures, CpGs were combined with HDM or DT antigens to assess the effect of the various ODN on these antigen-specific responses. Lymphoproliferation and cytokine responses IL-13, IFN-γ, IL-6, IL-10, TNF-α) were measured for all of the cultures described above.Results Although neonates showed attenuated lymphoproliferation to CpG, the production of antigen-presenting cell-derived cytokines such as IL-6 and IL-10 and the up-regulation of major histocompatibility complex class II (HLA-DR) were detected at adult levels. T cell-derived cytokines (IL-13 and IFN-γ) were not detectable in response to CpG alone. Most neonates also failed to produce detectable IFN-γ to HDM or DT (unlike adults), but readily produced IL-13 to these stimuli. The addition of CpG resulted in an increase in neonatal IFN-γ production in response to HDM (P=0.011) and a similar but non-significant trend with DT. However, rather than inhibiting Th2 IL-13 responses, the addition of CpGs was associated with a significant increase in the IL-13 responses to HDM (P=0.016) and DT (P=0.03), effects not seen in adults.Conclusions This study provides further evidence that neonatal MC responses to CpG are functionally different from adults, and do not show clear Th1 polarization. The CpG associated increase in Th2 responses may reflect a potentiation of the normal neonatal Th2 propensity, or non-specific activation of neonatal MC.