Library

Notes: Allergic immune responses are initiated and maintained by T cells that recognize peptidic fragments of allergens in the context of major histocompatibility complex (MHC) class II molecules. An anomaly of this model exists in the T-cell response to haptens. Haptens are nonpeptide antigens that alone are too small to provoke an immune response. Nevertheless. T-cell responses to haptenic allergens clearly occur and are critically involved in allergic immune responses to drugs such as penicillin. Although the mechanisms that generate T-cell epitopes from protein antigens are well understood, haptens create T-cell epitopes by alternative mechanisms. These may include binding of haptens directly to preformed MHC-peptide complexes on the cell surface, or indirect association with MHC molecules after conjugation with self cell surface or serum proteins that are then processed and presented as haptenated peptide antigens. Which of these unorthodox mechanisms of epitope generation is dominant in allergy to penicillin is unknown. This study aims to determine the nature of the epitopes recognized by amoxicillin-specific T cells from allergic donors, and to clarify whether T-cell responses to penicillin antibiotics are MHC restricted and require haptenated self proteins to be processed before recognition. Human T-cell lines specific for amoxicillin were raised and used in assays with processing-disabled and MHC-class Il-typed antigenpresenting cells to determine the MHC restriction and processing requirements of T cells recognizing amoxicillin. Fixation of antigenpresenting cells with paraformaldehyde. before or after pulsing with amoxicillin. established that T cells can recognize amoxicillin-containing epitopes with a similar ezfficiency irrespective of whether the antigenic conjugate has been internalized and processed. These results suggest that amoxicillin can bind directly to preformed MHC-peptide complexes and need not necessarily involve the processing of haptenated self carrier proteins before recognition of the conjugate by amoxicillinpecific T cells.

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 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: Induced heteroduplex genotyping (IHG) is one of many methods that can be used to determine single nucleotide polymorphisms (SNPs). It is relatively new in comparison to other polymerase chain reaction (PCR)-based techniques. The aim of this study was to compare the results of genotyping using IHG with the results of genotyping using either polymerase chain reaction–sequence-specific primers (PCR-SSP) or polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) for SNPs in the tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 genes. Ninety patients who consented to participate in the study had their genotypes determined by IHG and either PCR-SSP (TNF-α−308 and IL-10 −1082/−819/−592) or PCR-RFLP (IL-1β+3953 and IL-6 −174). Results for each locus were compared between techniques by calculating the Kappa statistic as a measure of agreement. The IHG and more traditional genotyping methods produced very similar results at all loci. The Kappa statistics for each locus were as follows: TNF-α−308, K = 0.727; IL-1β+3953, K = 0.886; IL-6 −174, K = 0.909; IL-10 −1082, K = 0.876; IL-10 −592, K = 0.920. IHG is a valid method for the determination of genotypes at the loci examined in this study and produces comparable results to those of more traditional methods of genotyping.