Assessment of the inherent allergenic potential of proteins in mice.

There is considerable interest in the design of approaches that will permit the accurate identification and characterization of proteins that have the inherent potential to induce sensitization and cause food allergy. Among the methods used currently as part of such assessments are consideration of structural similarity to, or amino acid sequence homology with, known human allergens; whether there exists immunologic cross-reactivity with known allergens; and measurement of resistance to proteolytic digestion in a simulated gastric fluid. Although such approaches provide information that will contribute to a safety assessment, they do not--either individually or collectively--provide a direct evaluation of the ability of a novel protein to cause allergic sensitization. For this reason, work is in progress to design and evaluate suitable animal models that will provide a more holistic assessment of allergenic potential. In this laboratory, the approach we have taken has been to examine the characteristics of immune responses induced in mice following parenteral (intraperitoneal) exposure to test proteins. The basis of this method is to determine simultaneously the overall immunogenic potential of proteins [measured as a function of immunoglobulin (Ig) G antibody responses] and to compare this with their ability to provoke IgE antibody production, IgE being the antibody that effects allergic sensitization. Although this approach has not yet been evaluated fully, the results available to date suggest that it will be possible to distinguish proteins that have the inherent potential to induce allergic sensitization from those that do not. In this article we summarize progress to date in the context of the scientific background against which such methods are being developed

Mechanisms of allergic contact dermatitis

Allergenicity depends on several factors determined by the very physicochemical nature of the molecules themselves, i.e., their capacity to penetrate the horny layer, lipophilicity, and chemical reactivity. The sensitizing property of the majority of contact allergens could be predicted from these characteristics (Patlewicz et al., Contact Dermatitis 50:91-97, 2004; Gerberick et al., Altern Lab Anim 36(2):215-242, 2008). Two other factors, however, further contribute to the allergenicity of chemicals, namely, their pro-inflammatory activity and capacity to induce maturation of LC. These issues will be dealt with in more detail in the following sections. Along with their migration and settling within the draining lymph nodes, haptenized LC further mature, as characterized by their increased expression of costimulatory and antigen-presentation molecules (Cumberbatch et al., Arch Dermatol Res 289:277-284, 1997; Heufler et al. J Exp Med 167:700-705, 1988). In addition, they adopt a strongly veiled, interdigitating appearance, thus maximizing the chances of productive encounters with naive T lymphocytes and recognition of altered self (Steinman et al., J Invest Dermatol 105:2S-7S, 1995; Furue et al., Br J Dermatol 135:194-198, 1996; Schuler and Steinman, J Exp Med 161:526-546, 1985). The intricate structure of lymph node paracortical areas, the differential expression of chemokines and their receptors, the characteristic membrane ruffling of IDC, and the predominant circulation of naive T lymphocytes through these lymph node areas provide optimal conditions for T-cell-receptor binding, i.e., the first signal for induction of T-cell activation (Banchereau and Steinman, Nature 392:245-252, 1998). Intimate DC-T-cell contacts are further strengthened by secondary signals, provided by sets of cellular adhesion molecules, and growth-promoting cytokines (reviewed in Hommel, Immunol Cell Biol 82:62-66, 2004; Cella et al., Curr Opin Immunol 9:10-16, 1997). In healthy individuals, primary skin contacts with contact allergens lead to differentiation and expansion of allergen-specific effector T cells displaying Th1, Th2, and/or Th17 cytokine profiles. The same allergens, if encountered along mucosal surfaces, favor the development of allergen-specific Th2 and Th17 effector cells, and/or Th3 and Tr1 allergen-specific regulatory T cells. Whereas the first two subsets may assist or replace Th1 cells in pro-inflammatory effector functions, the latter two subsets are mainly known for downregulating immune responsiveness. For most, if not all allergens, along with prolonged allergenic contacts, the role of Th2 cells as effector cells gradually increases given reduced longevity of Th1 responses. The respective contributions of similar subsets of allergen-specific CD8 + T cells are still unknown, but distinct effector roles of allergen-specific Tc1 and Tc2 have been postulated. Priming via the skin results in CLA positive T cells, which upon inflammatory stimuli preferentially enter the skin; on the other hand, gut homing T cells have been primed and generated along mucosal surfaces. Upon priming, T cells loose much of their capacity to recirculate via the lymph nodes, but gain the capacity to enter the tissues. In particular recently activated T cells will enter skin inflammatory sites. ACD reactions are primarily infiltrated by CD4 and/or CD8 pro-inflammatory cells, later reactions may be dominated by Th2 cells and regulatory T cells. Skin infiltation by T cells is fine-tuned by sets of adhesion molecules and chemokine receptors, whose expression is not rigid, but can be modulated by micro-environmental factors. After antigenic activation the progeny of primed T cells is released via the efferent lymphatics into the bloodstream. Circulating allergen-specific cells can be challenged in vitro to provide diagnostic parameters for contact hypersensitivity. At least for water-soluble allergens, like metal salts, the degree of allergen-specific proliferation and cytokine production, in particular type-2 cytokines, correlate with clinical allergy. For routine application of a broad spectrum of allergens, culture conditions still need to be improved. For mechanistic in vitro studies in ACD, however, with selected sets of relatively nontoxic allergens, peripheral blood provides an excellent source of lymphocytes and antigen-presenting cells. ACD reactions can be mediated by classical effector cells, i.e., allergen-specific CD4+ type-1 T cells which, upon triggering by allergen-presenting cells, produce IFN-? to activate nonspecific inflammatory cells like macrophages. However, CD8 + T cells, and other cytokines, including IL-4, IL-17, and IL-22 can also play major roles in ACD. The conspicuous difference with DTH reactions induced by intradermal administration of protein antigens, i.e., the epidermal infiltrate, can largely be attributed to hapten-induced chemokine release by keratinocytes