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Notes: Background Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross-reactive allergens remains unknown.Objective The aim of this study was to investigate the presence of allergenic cross-reactivity between peanut and tree nuts.Methods Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between peanut and tree nuts.Results Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut- and tree nut-allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre-incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross-reactivity. Pre-incubation of sera with cashew nut extract did not cause any inhibition.Conclusion These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross-reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross-reactivity.

Notes: Background Peanut and tree nuts are a major cause of food-induced anaphylaxis with an appreciable mortality. Co-sensitization to peanuts and tree nuts is a common clinical observation and may be because of peanut-specific serum IgE antibodies that cross-react with tree nut allergens. It is, however, unclear whether these cross-reactive IgE antibodies are involved in effector-cell activation.Objective To determine if cross-reactivity of peanut-specific IgE antibodies with tree nuts can cause effector cell activation using an in vitro basophil activation assay.Methods Two peanut allergic subjects with positive specific IgE for peanut and tree nuts (as measured by CAP-FEIA) were studied. Basophil activation to peanut and tree nuts, as indicated by CD63 expression, was assessed by flow cytometry to confirm co-sensitization to peanut and tree nuts. Inhibition ELISA using sera from the subjects was performed to detect peanut-specific IgE antibodies that cross-reacted with tree nut proteins. To determine whether cross-reactive tree nut allergens can induce effector-cell activation, peanut-specific antibodies were affinity purified from the subject sera and used to resensitize non-peanut/tree nut allergic donor basophils stripped of surface IgE. Basophil activation was then measured following stimulation with peanut and tree nut extracts.Results The two peanut allergic subjects in this study showed positive basophil activation to the peanut and tree nut extracts. Inhibition ELISA demonstrated that pre-incubation of the peanut allergic subject sera with almond, Brazil nut and hazelnut extracts inhibited IgE binding to peanut extract. IgE-stripped basophils from non-peanut/tree nut allergic subjects resensitized with affinity-purified peanut-specific antibodies from the peanut allergic subject sera became activated following stimulation with peanut, almond and Brazil nut extracts, demonstrating biological activity of cross-reactive IgE antibodies.Conclusion Peanut-specific IgE antibodies that cross-react with tree nut allergens can cause effector-cell activation and may contribute to the manifestation of tree nut allergy in peanut allergic subjects.

Notes: Background Allergen and fungal exposures are important risk factors for asthma. We conducted a longitudinal analysis of allergen levels in Melbourne homes between 1996 and 1998 to examine the effects of changing residential characteristics on allergen and fungal levels. We also examined the changes in levels of indoor allergens.Methods The subjects were participants in the European Community Respiratory Health Survey (ECRHS) in Melbourne. In 1996, 485 subjects participated in a follow-up study, which involved both home and laboratory visits. Dust and air samples were collected from participants' bedrooms and a validated residential questionnaire was administered. In 1998, 360 participants underwent further follow-up. House dust mite (Der p 1) and cat allergens (Fel d 1) and ergosterol were measured in dust.Results We observed moderate within home correlations between 1996 and 1998 in floor Der p 1 (intraclass correlation ICC=0.48), bed Der p 1 (ICC=0.61), Fel d 1 (κ=0.53) and ergosterol (ICC=0.28) levels. We found that the floor Der p 1 levels decreased from 1996 to 1998 in the homes of participants who moved to an attached home, moved their bedrooms to the first floor, removed fitted carpet or central heating. Replacing or vacuuming the mattress more than twice per year reduced levels of Der p 1 in the bed. Ergosterol levels were reduced by removing visible mould and fitted carpet.Conclusions These findings provide evidence to support current advice with regard to allergen avoidance in patients with dust mite and fungal allergies.

Notes: Background The influence of current levels of indoor fungi on asthma is a controversial issue that needs to be resolved in order to advise patients appropriately.Objective To assess the seasonal variation in indoor fungal levels and the impact of these levels on asthma among mould-sensitized individuals.Methods Thirty-five young adults with current asthma and sensitization to fungi were visited four times over 1 year. At each home visit a questionnaire was administered and samples of dust and air were collected. Participants also recorded information on symptoms, peak expiratory flows (PEF) and medication use. Dust samples were analysed for house dust mite allergen (Der p 1) and total fungal biomass (ergosterol). Total and genus-specific fungal propagules were identified in air samples. Seasonal variation in allergen levels and significant independent effects of fungal levels on peak flow variability (PFV) were identified by repeated measures analysis of variance.Results Significant seasonal variations were observed in viable airborne fungi, ergosterol levels in the floor dust and PFV. PFV correlated significantly with symptom scores and the dose of reliever medication. PFV was also significantly associated with smoking and visible mould. The association between visible mould and PFV was independent of season, smoking and the dose of reliever medication. However, there was no association between total fungi, specific fungi or ergosterol and PFV. Der p 1 levels had no significant influence on asthma, even in HDM-sensitized individuals.Conclusions Mouldy homes adversely influence asthma in asthmatics sensitized to fungi.

Notes: Background  Hev b 5 is a major latex allergen recognized predominantly by latex-allergic health care workers (HCWs). Recombinant Hev b 5 (rHev b 5) was previously expressed as a fusion protein with maltose binding protein (MBP), itself an immunogenic molecule; therefore non-fusion rHev b 5 is desirable. Moreover, standardized immunological assays for the detection of Hev b 5 are currently lacking and may have important implications for both allergen avoidance and diagnosis in latex allergy.Objectives  To generate and use Hev b 5-specific mAbs to determine the relative abundance of Hev b 5 in different latex extracts, correlating this with the IgE reactivity of latex-allergic HCWs and to produce non-fusion rHev b 5.Methods  For the production of mAbs, mice were immunized with rHev b 5/MBP fusion protein and mAbs selected with rHev b 5/MBP but not MBP reactivity. The mAb reactivity was compared with polyclonal IgE from latex-allergic HCWs using direct and inhibition ELISA and immunoblot assays. Recombinant Hev b 5 was expressed and purified in the pPROEX-HTa bacterial expression system.Results  Four Hev b 5-specific mAbs were produced. Immunoblotting and ELISA using the mAbs indicate abundant Hev b 5 in high protein powdered latex glove extracts as compared with crude latex sap extracts. High quality surgical gloves with no detectable protein have no detectable Hev b 5. Inhibition ELISAs using serum IgE from latex-allergic HCWs and Hev b 5-specific mAbs gave strong correlation. Non-fusion recombinant Hev b 5 was successfully expressed and purified, showing reactivity with both the Hev b 5-specific mAbs and serum IgE of latex-allergic HCWs.Conclusion  Hev b 5-specific mAbs and human IgE from latex-allergic HCWs demonstrate the greater content of Hev b 5 in high protein powdered glove extracts. This may explain the observed higher frequency of sensitization to this allergen in HCWs.

Notes: Background Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4+ T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy.Objective To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users.Methods Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4+ T cell intracellular cytokines, IL-4 and IFN-γ were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2.Results All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10–29) and Hev b 6.01 p(19–38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-γ was evident from intracellular cytokine staining.Conclusion Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.

Notes: Background Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs.Objective This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT.Methods A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-γ and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation.Results At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P〈0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P〈0.05) compared with baseline. CD4+ and CD8+ T cell IFN-γ production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L−, CD4+CD49dhi, CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P〈0.05). IL-10 staining co-localized with CD4+CD25+ T cells.Conclusion Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.