Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.

BACKGROUND: Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens. OBJECTIVE: To analyse the allergenic composition of the basic peanut protein (BPP) fraction. METHODS: A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE. RESULTS: Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive. CONCLUSION: The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.

Molecular Allergen-Specific IgE Assays as a Complement to Allergen Extract-Based Sensitization Assessment.

Molecular allergen-based component-resolved diagnostic IgE antibody tests have emerged in the form of singleplex assays and multiplex arrays. They use both native and recombinant allergen molecules, sometimes in combination with each other, to supplement allergen extract-based IgE antibody analyses. The total number of available allergenic molecules has reached a diagnostically useful level; however, more molecules are needed to cover all the clinically important allergen specificities. Thus, for the foreseeable future, molecular allergen-specific IgE analyses will remain a supplement for initial allergen extract-based IgE antibody analyses in the diagnostic workup of the allergic patient. As a spin-off, it will enable manufacturers to improve the quality of extracts for in vitro testing. The 2 most exciting diagnostic developments linked to component-resolved diagnostic tests are the possibility to increase diagnostic sensitivity by the inclusion of allergens that are underrepresented in the current extracts and in vitro assays and to increase the diagnostic specificity by taking the information on allergen cross-reactivity into account. Particularly the latter application is still under development. This requires additional studies on the clinical relevance of serological cross-reactivity.

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis.

Heat shock protein 60 (hsp60) is a highly conserved stress protein and target of self-reactive T cells in various inflammatory diseases. Not much is known about a possible role in atopic disease. As atopic diseases are considered to be the result of a disturbance in the balance between T helper cells type 2 and regulatory T cells, it is of interest to know whether hsp60 acts as a bystander antigen in atopic disease. Our aim was to investigate whether hsp60 is involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expression of hsp60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood mononuclear cells (PBMC) of children with AD were cultured with hsp60 and proliferative responses, cytokine secretion, surface markers, and functional assays were compared to responses of PBMC of healthy controls (HC). Hsp60 was detected more in lesional skin of AD patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated more strongly in response to hsp60 compared to HC. hsp60-reactive T cells of atopic children produced high levels of IFNγ and low levels of IL-10. In vitro activation with hsp60 leads to the induction of CD4(+)CD25(bright) T cells expressing FOXP3 in both HC as well as in atopic children. However, despite their regulatory phenotype, hsp60-induced CD4(+)CD25(bright)CD127(-)FOXP3(+) T cells of AD patients were incapable of suppressing effector T cells in vitro. hsp60 is recognized by proinflammatory (IFNγ high, IL-10 low) T cells in atopic patients and is more present in lesional AD skin. This suggests that hsp60-specific T cell responses contribute to local inflammation in AD.

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut.

BACKGROUND: One of the IL-17 family members, IL-25, has been implicated with the initiation and amplification of Th2 responses in animal models and has been associated with airway hyper-reactivity. The involvement of IL-25 and also IL-17 in food allergic disease remains to be investigated. FINDINGS: In this study thirty children suspected of peanut allergic disease underwent a double-blind placebo controlled food challenge (DBPCFC) and IL-25 and IL-17 plasma levels were determined before and after challenge. IL-25 was highly elevated only in subgroup of children with a positive DBPCFC outcome. Plasma IL-25 was absent in children with a negative DBPCFC outcome and in healthy controls. CONCLUSIONS: This study shows that IL-25, an IL-17 family member, is highly elevated only in children with a clinical response to peanut. This suggests a role for IL-25 in the pathogenesis of peanut allergy and elevated plasma IL-25 may be a sign of a severe atopic phenotype.

HSP: Bystander Antigen in Atopic Diseases?

Over the last years insight in the complex interactions between innate and adaptive immunity in the regulation of an inflammatory response has increased enormously. This has revived the interest in stress proteins; proteins that are expressed during cell stress. As these proteins can attract and trigger an immunological response they can act as important mediators in this interaction. In this respect, of special interest are proteins that may act as modulators of both innate and adaptive immunity. Heat shock proteins (HSPs) are stress proteins that have these, and more, characteristics. More than two decades of studies on HSPs has revealed that they are part of intrinsic, "natural" mechanisms that steer inflammation. This has provoked comprehensive explorations of the role of HSPs in various human inflammatory diseases. Most studies have focused on classical autoimmune diseases. This has led to the development of clinical studies with HSPs that have shown promise in Phase II/III clinical trials. Remarkably, only very little is yet known of the role of HSPs in atopic diseases. In allergic disease a number of studies have investigated the possibility that allergen-specific regulatory T cell (Treg) function is defective in individuals with allergic diseases. This raises the question whether methods can be identified to improve the Treg repertoire. Studies from other inflammatory diseases have suggested HSPs may have such a beneficial effect on the T cell repertoire. Based on the immune mechanisms of atopic diseases, in this review we will argue that, as in other human inflammatory conditions, understanding immunity to HSPs is likely also relevant for atopic diseases. Specifically, we will discuss why certain HSPs such as HSP60 connect the immune response to environmental antigens with regulation of the inflammatory response. Thus they provide a molecular link that may eventually even help to better understand the immune pathological basis of the hygiene hypothesis.

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60).

BACKGROUND: To prevent harmful autoimmunity most immune responses to self proteins are controlled by central and peripheral tolerance. T cells specific for a limited set of self-proteins such as human heat shock protein 60 (HSP60) may contribute to peripheral tolerance. It is not known whether HSP60-specific T cells are present at birth and thus may play a role in neonatal tolerance. We studied whether self-HSP60 reactive T cells are present in cord blood, and if so, what phenotype these cells have. METHODOLOGY/PRINCIPAL FINDINGS: Cord blood mononuclear cells (CBMC) of healthy, full term neonates (n = 21), were cultured with HSP60 and Tetanus Toxoid (TT) to study antigen specific proliferation, cytokine secretion and up-regulation of surface markers. The functional capacity of HSP60-induced T cells was determined with in vitro suppression assays. Stimulation of CBMC with HSP60 led to CD4(+) T cell proliferation and the production of various cytokines, most notably IL-10, Interferon-gamma, and IL-6. HSP60-induced T cells expressed FOXP3 and suppressed effector T cell responses in vitro. CONCLUSION: Self-reactive HSP60 specific T cells are already present at birth. Upon stimulation with self-HSP60 these cells proliferate, produce cytokines and express FOXP3. These cells function as suppressor cells in vitro and thus they may be involved in the regulation of neonatal immune responses.