Thresholds in chemical respiratory sensitisation.

There is a continuing interest in determining whether it is possible to identify thresholds for chemical allergy. Here allergic sensitisation of the respiratory tract by chemicals is considered in this context. This is an important occupational health problem, being associated with rhinitis and asthma, and in addition provides toxicologists and risk assessors with a number of challenges. In common with all forms of allergic disease chemical respiratory allergy develops in two phases. In the first (induction) phase exposure to a chemical allergen (by an appropriate route of exposure) causes immunological priming and sensitisation of the respiratory tract. The second (elicitation) phase is triggered if a sensitised subject is exposed subsequently to the same chemical allergen via inhalation. A secondary immune response will be provoked in the respiratory tract resulting in inflammation and the signs and symptoms of a respiratory hypersensitivity reaction. In this article attention has focused on the identification of threshold values during the acquisition of sensitisation. Current mechanistic understanding of allergy is such that it can be assumed that the development of sensitisation (and also the elicitation of an allergic reaction) is a threshold phenomenon; there will be levels of exposure below which sensitisation will not be acquired. That is, all immune responses, including allergic sensitisation, have threshold requirement for the availability of antigen/allergen, below which a response will fail to develop. The issue addressed here is whether there are methods available or clinical/epidemiological data that permit the identification of such thresholds. This document reviews briefly relevant human studies of occupational asthma, and experimental models that have been developed (or are being developed) for the identification and characterisation of chemical respiratory allergens. The main conclusion drawn is that although there is evidence that the acquisition of sensitisation to chemical respiratory allergens is a dose-related phenomenon, and that thresholds exist, it is frequently difficult to define accurate numerical values for threshold exposure levels. Nevertheless, based on occupational exposure data it may sometimes be possible to derive levels of exposure in the workplace, which are safe. An additional observation is the lack currently of suitable experimental methods for both routine hazard characterisation and the measurement of thresholds, and that such methods are still some way off. Given the current trajectory of toxicology, and the move towards the use of non-animal in vitro and/or in silico) methods, there is a need to consider the development of alternative approaches for the identification and characterisation of respiratory sensitisation hazards, and for risk assessment.

In vitro pepsin resistance of proteins: Effect of non-reduced SDS-PAGE analysis on fragment observation.

The introduction of novel proteins to food products carries with it the need to assess the potential allergenicity of such materials. Resistance to in vitro pepsin digestion is one parameter considered in such a risk assessment using a weight of evidence approach; however, the methodology used to investigate this has not been fully standardised. In vitro pepsin resistance assays typically involve SDS-PAGE performed under reducing conditions, with limited published data available on the assay using non-reducing conditions despite the need to consider non-reducing analysis techniques having been highlighted by regulatory bodies such as the European Food Safety Authority (EFSA). The purpose of the work reported here was to investigate the applicability of (and additional insight provided by) non-reducing analyses, by digesting a set of proteins using a ring-trial validated method, with analysis by SDS-PAGE under both reducing and non-reducing conditions. In silico prediction of digest fragments was also investigated. Significant differences were observed between results under reduced and non-reduced conditions for proteins in which disulphide bonds have a major role in protein structure, such as ribulose 1,5-diphosphate carboxylase (RUBISCO) and bovine serum albumin. For proteins with no or few disulphide bonds, no significant differences were seen in the results. Structural information such as disulphide bond numbers and positions should be considered during experimental design, as a non-reduced approach may be appropriate for some proteins. The in silico approach was a useful tool to suggest potential digest fragments, however the predictions were not always confirmed in vitro and should be considered a guide only.

Human gut-specific homeostatic dendritic cells are generated from blood precursors by the gut microenvironment.

BACKGROUND: Dendritic cells (DC) dictate not only the type of T-cell immunity, but also homing patterns of T cells in mice. In humans, we characterized normal human gut DC and tested whether gut-specific homeostatic DC could be generated from blood precursors by factors in the gut microenvironment. METHODS: We characterized the phenotype and function of healthy human gut DC compared with blood and skin DC, and studied whether conditioning of blood DC in the presence of colonic biopsy supernatants (Bx-SN) induced gut-like phenotype and functions. RESULTS: Blood DC mostly expressed both gut and skin homing markers, indicating potential to migrate to both major immune surface organs, and induced multi-homing T cells. However, DC within gut or skin did not demonstrate this multi-homing phenotype, were tissue-specific, and induced tissue-specific T cells. Human gut DC were less stimulatory for allogeneic T cells than their dermal and blood counterparts. Human blood DC cultured in vitro lost homing marker expression. Conditioning of human enriched blood DC with colonic Bx-SN from healthy controls induced a gut-homing phenotype and a homeostatic profile. Moreover, Bx-SN-conditioned DC demonstrated a restricted T-cell stimulatory capacity and preferentially induced gut-specific T cells. Retinoic acid and transforming growth factor beta (TGF-ß) mediated the acquisition of the gut-homing and homeostatic properties, respectively, induced by colonic Bx-SN on blood enriched DC. CONCLUSIONS: Tissue-specific factors manipulate immunity via modulating characteristics of DC and may provide tools to generate tissue-specific immunotherapy.

Quantification and partial characterization of the residual protein in fully and partially refined commercial soybean oils.

A method has been developed to determine residual protein in refined oils, a potential trigger of allergic reactions. High-pH bicarbonate or borate buffers were found to be the most effective extractants, residual oil protein comprising a mixture of proteins of M(r) 6000-100000. Extracted protein could be quantified with superior precision using 3-(4-carboxybenzoyl)quinolone-2-carboxaldehyde (CBQCA). Residual protein content determined in a set of oils using the borate extraction-CBQCA assay was positively correlated with contents determined using a bicarbonate-total amino acid analysis method. Oil refining substantially reduced the oil protein content determined by the borate-CBQCA assay with neutralized/refined, bleached, and deodorized (fully refined) oils containing 62-265 ng/g oil, whereas crude un-degummed oils contained 86000-87900 ng/g of protein. These analyses and published data on cumulative threshold doses for soybean suggest that even the most sensitive individuals would need to consume at least 50 g of highly refined oil to experience subjective symptoms.

The effect of digestion and pH on the allergenicity of kiwifruit proteins.

It is suggested that patients with oral allergy syndrome (OAS) respond to pepsin-sensitive allergens, and systemic reactors identify pepsin-resistant allergens. We sought to assess the digestibility of kiwifruit proteins in simulated gastric fluid (SGF), and to compare the immunogenicity of the digests in patients with isolated oral and systemic reactions to kiwifruit. In addition, the effect of pH on digestibility of kiwifruit proteins was investigated. The in vitro resistance of kiwifruit proteins to digestion was determined using SGF. G-immunoglobulin (IgE) binding to digested proteins was investigated by Western blotting using sera from children and adults (aged 5-72 yr) with systemic reactions and patients with isolated oral symptoms. To determine whether pH conditions influence digestion of kiwifruit extracts, digestion at pHs 1.5-7 were compared by SDS-PAGE. Patients with systemic reactions showed IgE binding to digestion-resistant allergens, but patients with oral symptoms reacted only to digestion-labile allergens. An increase in pH from 1.5 to 2.5 significantly reduced pepsin breakdown of kiwifruit allergens. Immunoreactive digested protein fragments were detectable by immunoblot but not Coomassie stain. This study confirms a difference in the lability of food allergens recognized by patients with systemic reactions and those with OAS. Pepsin digestion of kiwifruit proteins was impaired by hypoacidic conditions suggesting that patients with hypoacidic gastric conditions are at increased risk of systemic absorption of allergens. The data indicate that commonly used methods for predicting allergenicity of novel proteins using Coomassie stains may be flawed.