The direct Peptide reactivity assay: selectivity of chemical respiratory allergens

It is well known that some chemicals are capable of causing allergic diseases of the skin and respiratory tract. Commonly, though not exclusively, chemical allergens are associated with the selective development of skin or respiratory sensitization. The reason for this divergence is unclear, although it is hypothesized that the nature of interactions between the chemical hapten and proteins is influential. The direct peptide reactivity assay (DPRA) has been developed as a screen for the identification of skin-sensitizing chemicals, and here we describe the use of this method to explore whether differences exist between skin and respiratory allergens with respect to their peptide-binding properties. Known skin and respiratory sensitizers were reacted with synthetic peptides containing either lysine (Lys) or cysteine (Cys) for 24h. The samples were analyzed by HPLC/UV, and the loss of peptide from the reaction mixture was expressed as the percent depletion compared with the control. The potential for preferential reactivity was evaluated by comparing the ratio of Lys to Cys depletion (Lys:Cys ratio). The results demonstrate that the majority of respiratory allergens are reactive in the DPRA, and that in contrast to most skin-sensitizing chemicals, preferentially react with the Lys peptide. These data suggest that skin and respiratory chemical allergens can result in different protein conjugates, which may in turn influence the quality of induced immune responses. Overall, these investigations reveal that the DPRA has considerable potential to be incorporated into tiered testing approaches for the identification and characterization of chemical respiratory allergens.     

A murine model for assessing the respiratory hypersensitivity potential of chemical allergens

Using equimolar quantities of 2 chemical allergens, toluene diisocyanate (TDI), noted for its ability to cause respiratory hypersensitivity, and dinitrochlorobenzene (DNCB), noted for its dermal sensitizing activity, the mouse was evaluated as a possible model to indicate respiratory hypersensitivity. A previously published procedure (Garssen et al. (1989) Immunology 68, 51–58) was followed whereby chemicals were applied epicutaneously to the shaved flank of BALB/c mice. Eight days later, animals were challenged by intranasal application of the chemical. The lungs were evaluated at 48 h. Both TDI and DNCB elicited mild mononuclear inflammatory cuffing around pulmonary vasculature. No reaction was noted around pulmonary airways. Sera, drawn 48 h following the intranasal challenge with chemical allergen, were evaluated for total IgE, hapten-specific IgE and IgG, and for IL-2, IL-4, IL-5, IL-6, and interferon gamma. Animals exposed to TDI demonstrated decreased total IgE and the presence of TDI-specific IgG. Cytokine levels were unchanged in both groups. These results indicate that in this mouse model, total serum IgE and the production of hapten-specific IgG antibodies distinguished a respiratory from a contact sensitizing chemical. Further comparison of the serologic response of mice to these two classes of chemicals is required to determine if the murine model can be used to predict dermal versus respiratory sensitizing activity of chemical allergens.     

直接肽段结合方法(DPRA)在中药注射剂过敏反应预测中的应用

目的:采用直接肽段结合方法（DPRA）考察两种中药注射剂的过敏反应,评估该方法在中药注射剂过敏反应检测中应用的可能性。方法:运用HPLC法,通过测定两种中药注射剂对半胱氨酸多肽和赖氨酸多肽的消耗,来推测其产生过敏反应的可能性。结果:两种中药注射剂对半胱氨酸多肽和赖氨酸多肽均有不同程度的消耗,预示有潜在的致敏性。结论:该方法可以作为中药注射剂过敏反应的预测方法。     

Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation

Asthma resulting from sensitisation of the respiratory tract to chemicals is an important occupational health issue, presenting many toxicological challenges. Most importantly there are no recognised predictive methods for respiratory allergens. Nevertheless, it has been found that all known chemical respiratory allergens elicit positive responses in assays for skin sensitising chemicals. Thus, chemicals failing to induce a positive response in skin sensitisation assays such as the local lymph node assay (LLNA) lack not only skin sensitising activity, but also the potential to cause respiratory sensitisation. However, it is unclear whether it will be possible to regard chemicals that are negative in in vitro skin sensitisation tests also as lacking respiratory sensitising activity. To address this, the behaviour of chemical respiratory allergens in the LLNA and in recently validated non-animal tests for skin sensitisation have been examined. Most chemical respiratory allergens are positive in one or more newly validated non-animal test methods, although the situation varies between individual assays. The use of an integrated testing strategy could provide a basis for recognition of most respiratory sensitising chemicals. However, a more complete picture of the performance characteristics of such tests is required before specific recommendations can be made.     

Chemical reactivity measurements: Potential for characterization of respiratory chemical allergens

Allergic diseases of the skin and respiratory tract resulting from exposure to low molecular weight chemicals remain important issues for consumer product development and occupational/environmental health. Widespread opportunities for exposure to chemical allergens require that there are available effective methods for hazard identification and risk assessment. In the search for new tools for hazard identification/characterization there has been interest in developing alternative methods that will reduce, refine or replace the need for animals. One approach that shows promise is based on the measurement of the peptide reactivity of chemicals; the potential to form stable associations with protein/peptide being a key requirement for the induction of sensitization. Recent investigations using these systems have focused primarily on skin sensitizing chemicals. However, there is interest in the possibility of exploiting these same experimental approaches to distinguish between different forms of chemical allergens - as individual materials are primarily associated with one or the other form of sensitization in humans. These investigations may also provide insight into why chemical sensitizers can differ in the form of allergic disease they will preferentially induce. These opportunities are surveyed here against a background of the immunobiology of allergic sensitization and current state-of-the-art approaches to measurement of peptide/protein reactivity.     

胸腺肽及制剂多肽含量测定方法研究

目的：研究胸腺肽溶液及其制剂不同方法测定多肽含量结果的差异。方法：用高效液相色谱法及Folin法测定多肽含量。结果：高效液相色谱法方法准确、灵敏、专属性强；Folin法胸腺肽含量测定结果与高效液相色谱法无显著性差异，测定胸腺肽制剂多肽含量则受赋形剂干扰较大。     

Methods for the identification of chemical respiratory allergens in rodents: comparisons of cytokine profiling with induced changes in serum IgE

No validated or widely recognized test methods are currently available for the prospective identification of chemicals with the potential to cause respiratory allergy. The cellular and molecular mechanisms that result in the induction of chemical sensitization of the respiratory tract are unclear, although there is evidence for the selective development of T helper 2 (Th2)-type responses and, in some cases, the production of IgE antibody. We have therefore examined the utility of cytokine profiling using BALB/c mice, together with the measurement of induced increases in the total serum concentration of IgE in the Brown Norway (BN) rat, as markers for the prospective identification of chemical respiratory allergens. Responses provoked by the reference respiratory allergen trimellitic anhydride (TMA) have been compared with those stimulated by the respiratory sensitizing diisocyanates toluene diisocyanate (TDI) and hexamethylene diisocyanate (HDI) and by the acid anhydride hexahydrophthalic anhydride (HHPA). Topical exposure of BN rats to TMA, TDI and HHPA each provoked marked immune activation (increases in lymph node cellularity and proliferation). However, only treatment with TMA stimulated vigorous increases in the total serum concentration of IgE. In contrast, exposure to HHPA, TDI or HDI failed to provoke significant changes in serum IgE concentration or induced only transient and relatively weak increases in serum IgE levels. In parallel experiments using BALB/c strain mice, however, topical application of all four chemical respiratory allergens provoked a marked Th2-type cytokine secretion profile in draining lymph node cells. These data suggest that the measurement of induced changes in serum IgE is not sufficiently sensitive for the robust identification of chemical respiratory allergens. Furthermore, irrespective of the reasons for variations in TMA-induced IgE production among BN rats, doubts remain regarding the utility of these animals for the characterization of immune responses to chemical allergens. Cytokine profiling using the BALB/c strain mouse apparently provides a more robust method for the hazard assessment of chemical respiratory allergens.     

Induced changes in total serum IgE concentration in the Brown Norway rat: potential for identification of chemical respiratory allergens.

A variety of chemicals can cause sensitization of the respiratory tract and occupational asthma that may be associated with IgE antibody production. Topical exposure to chemical respiratory allergens such as trimellitic anhydride (TMA) has been shown previously to induce increases in the total serum concentration of IgE in BALB/c strain mice. Contact allergens such as 2,4-dinitrochlorobenzene (DNCB), which apparently lack respiratory sensitizing potential, fail to provoke similar changes. However, it became apparent with time that there was some inter-animal variation in constitutive and inducible IgE levels. We have now examined the influence of topical exposure to TMA and DNCB on serum IgE levels in the Brown Norway (BN) rat. Such animals can be bled serially and thus it is possible to perform longitudinal analyses of changes in serum IgE concentration. The kinetics of IgE responses therefore can be followed on an individual animal basis, allowing discrimination between transient and sustained increases in serum IgE concentration. Rats (n = 5) were exposed on shaved flanks to 50% TMA, to 1% DNCB (concentrations that elicit comparable immune activation with respect to draining lymph node cellularity and proliferation) or to vehicle alone. Total IgE was measured by enzyme-linked immunosorbent assay in serum samples taken prior to and 14-42 days following initial exposure. Those animals having high pre-existing IgE levels (>1.0 microg ml(-1)) were excluded from subsequent analyses. The levels of serum IgE in the majority of rats exposed to DNCB or vehicle alone remained relatively stable throughout the duration of all the experiments conducted, although some animals displayed transient increases in serum IgE. Only TMA treatment was associated with a significant and sustained increase in the level of serum IgE in the majority of experiments. The elevated concentrations of IgE induced by topical exposure to TMA are persistent, the results reported here demonstrating that induced changes in IgE are maximal or near maximal at approximately 35 days, with a significant increase in IgE demonstrable for at least 42 days following the initiation of exposure. Interestingly, although TMA and DNCB at the test concentrations used were found to be of comparable overall immunogenicity with regard to lymph node activation and the induction of lymph node cell proliferation, there were apparent differences in humoral immune responses. Thus, not only did exposure to TMA stimulate increases in total serum IgE concentration and the production of specific IgE antibody, but also a more vigorous IgG antibody response was provoked by TMA compared with DNCB. These data suggest that the measurement of induced changes in serum IgE concentration in the BN strain of rat is able to differentiate between different classes of chemical allergen. Given the inter-animal variation in IgE production, it would be prudent to incorporate a concurrent assessment of responses induced by treatment with TMA as a positive control against which to assess the activity of other test materials.     

Assessing the potency of respiratory allergens: Uncertainties and challenges

In contrast to skin sensitisation, there are no accepted tests for the identification of chemicals or proteins with the potential to cause sensitisation of the respiratory tract. Although progress has been made, the assessment of respiratory sensitisation potential remains associated with significant challenges and uncertainties. Nevertheless, there is interest in determining whether it is possible to assess the relative potency of respiratory sensitisers. The second Adaptation to Technical Progress (ATP) to the EU Classification, Labelling and Packaging (CLP) Regulation recently introduced changes to criteria for classification and labelling of chemicals and preparations, bringing it in line with the 3rd revision to the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Among other things, the second ATP introduces sub-categories for respiratory and skin sensitisers, discriminating between strong sensitisers and other sensitisers. Here we examine whether such categorisation of protein and/or chemical respiratory allergens is realistic and/or feasible. For this purpose comparisons have been drawn with skin sensitisation, where potency categorisation has now been widely accepted and successfully integrated into the regulatory process. The conclusion drawn is that, on the basis of the currently available information, potency categorisation for respiratory sensitisers is premature and could potentially be misleading.     

Assessing the potency of respiratory allergens: uncertainties and challenges

In contrast to skin sensitisation, there are no accepted tests for the identification of chemicals or proteins with the potential to cause sensitisation of the respiratory tract. Although progress has been made, the assessment of respiratory sensitisation potential remains associated with significant challenges and uncertainties. Nevertheless, there is interest in determining whether it is possible to assess the relative potency of respiratory sensitisers. The second Adaptation to Technical Progress (ATP) to the EU Classification, Labelling and Packaging (CLP) Regulation recently introduced changes to criteria for classification and labelling of chemicals and preparations, bringing it in line with the 3rd revision to the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Among other things, the second ATP introduces sub-categories for respiratory and skin sensitisers, discriminating between strong sensitisers and other sensitisers. Here we examine whether such categorisation of protein and/or chemical respiratory allergens is realistic and/or feasible. For this purpose comparisons have been drawn with skin sensitisation, where potency categorisation has now been widely accepted and successfully integrated into the regulatory process. The conclusion drawn is that, on the basis of the currently available information, potency categorisation for respiratory sensitisers is premature and could potentially be misleading.     

Alternative approaches to the identification and characterization of chemical allergens.

Chemical allergy can take a variety of forms, those of greatest importance in an occupational setting being skin sensitization resulting in allergic contact dermatitis and sensitization of the respiratory tract associated with asthma and other symptoms. In both cases there is a need for predictive test methods that allow the accurate identification of sensitizing chemicals. Well characterized methods are available for skin sensitization testing, and although to date no tests for respiratory sensitization have been formally validated, progress has been made in defining suitable animal models. In recent years there have been significant advances in our understanding of the cellular and molecular mechanisms through which allergic sensitization to chemicals is induced and regulated. Such progress provides us now with new opportunities to consider alternative approaches to sensitization testing, including the design of in vitro test methods. The greatest investment has been in exploring novel methods for the identification of contact sensitizers and it is upon this aspect of chemical allergy that this article is focused. Described here are some of the general requirements of in vitro test methods for skin sensitization, and progress that has been made in developing suitable approaches with particular emphasis on the utility of dendritic cell culture systems.     

Reactivity of Langerhans cells in human reconstructed epidermis to known allergens and UV radiation.

Epidermal Langerhans cells are the outmost guards of our immune defence system. These cells are directly involved in phenomena such as contact hypersensitivity and UV-induced immunosuppression. Some years ago we succeeded in introducing CD34(+)-derived Langerhans cells into a reconstructed human epidermis. Here we describe their reactivity after topical exposure of the reconstructed epidermis to known allergens, allergen-inducible cytokines, irritants and UV irradiation. Exposure to allergens for 24 h resulted in an activated appearance of the Langerhans cells and in some cases a decrease in their number. Concomitantly, IL-1beta and CD86 mRNA over-expressions were detected in the reconstructed epidermis. A topical treatment with TNF-alpha or IL-1beta revealed that both cytokines induced an activated appearance of the Langerhans cells as early as 4 h following application. Irritants had no effect on the integrated Langerhans cells. Exposure of the reconstructed epidermis to Solar Simulated Radiation caused a dramatic decrease in the number of Langerhans cells and a loss of dendricity in the remaining cells 24 h after irradiation. The topical application of a large spectrum UVA/B filter before irradiation prevented these UV-induced alterations. In our hands, this model provides a promising tool to evaluate the sensitization potential of new compounds and to validate the efficacy of sunscreens to prevent UV-induced immunosuppression.     

Categorisation of protein respiratory allergens: The case of Subtilisin

Characterisation of the relative sensitizing potency of protein and chemical allergens remains challenging, particularly for materials causing allergic sensitization of the respiratory tract. There nevertheless remains an appetite, for priority setting and risk management, to develop paradigms that distinguish between individual respiratory allergens according to perceptions of the hazards and risks posed to human health. One manifestation thereof is recent listing of certain respiratory allergens as Substances of Very High Concern (SVHC) under the provisions of REACH (Registration, Evaluation, Authorisation and restriction of Chemicals). Although priority setting is a laudable ambition, it is important the process is predicated on evidence-based criteria that are transparent, understood and owned. The danger is that in the absence of rigorous criteria unwanted precedents can be created, and confidence in the process is compromised. A default categorisation of sensitisers as SVHC requiring assessment under the authorisation process is not desirable. We therefore consider here the value and limitations of selective assignment of certain respiratory allergens as being SVHC. The difficulties of sustaining such designations in a sound and equitable way is discussed in the context of the challenges that exist with respect to assessment of potency, and information available regarding the effectiveness of exposure-based risk management.     

Variable effects of chemical allergens on serum IgE concentration in mice. Preliminary evaluation of a novel approach to the identification of respiratory sensitizers.

A wide variety of chemicals may induce allergic contact dermatitis (contact sensitivity). Some chemical allergens may, in addition, cause respiratory sensitization. Topical exposure of mice to contact and respiratory chemical sensitizers results in the initiation of divergent immune responses characteristic of preferential activation of different functional subpopulations of T helper (TH) cells. In the present study we have sought to make use of these differences, particularly differences in the ability of contact and respiratory allergens to provoke IgE responses, and to question whether opportunities exist for the identification of chemicals with the potential for respiratory sensitization. We have examined alterations in the serum concentration of IgE following topical exposure of mice to seven chemical allergens; trimellitic anhydride (TMA), phthalic anhydride, diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), oxazolone and 2,4-dinitrochlorobenzene (DNCB). Three of these--TMA, phthalic anhydride and MDI--are known human respiratory sensitizers. The other four--HMDI, IPDI, oxazolone and DNCB--appear not to cause respiratory allergy, or at least have a very limited potential to do so. At the concentrations tested, exposure to all chemicals caused a lymphocyte proliferative response in lymph nodes draining the site of application. However, exposure only to TMA, phthalic anhydride and MDI resulted in a substantial increase in the concentration of serum IgE. Treatment with HMDI and IPDI failed to induce any change in serum IgE concentration. DNCB and oxazolone caused only small and transient elevations of IgE that were considerably less marked than those observed with respiratory sensitizers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inter‐relationships between different classes of chemical allergens

Although allergic sensitization of the respiratory tract induced by chemicals is not as common as skin sensitization, it is nevertheless an important occupational health issue. Respiratory allergy to chemicals, characterized typically by rhinitis and asthma, is associated with considerable morbidity and with related socioeconomic costs. Several experimental approaches have been proposed for the prospective identification of chemical respiratory allergens, but none of these has yet been validated formally. In the absence of a widely accepted method for respiratory allergen identification, it is appropriate and relevant to explore their relationship with skin‐sensitizing chemicals. A series of chemicals known to cause immune‐mediated respiratory allergy in humans has been examined. The majority of the respiratory allergens tested were found to elicit positive responses in one or more standard tests used for the identification of skin‐sensitizing potential (guinea pig maximization test, the Buehler test and/or the local lymph node assay). We suggest that this observation might form the basis of a potentially useful paradigm for initial characterization of the respiratory‐sensitizing potential of chemicals. Specifically, chemicals that fail to elicit positive responses in accepted skin‐sensitization test methods might also be regarded as lacking the inherent potential to cause allergic sensitization of the respiratory tract. Copyright © 2012 John Wiley & Sons, Ltd.     

Peptide Stability in Drug Development. II. Effect of Single Amino Acid Substitution and Glycosylation on Peptide Reactivity in Human Serum

The determination of peptide stability in human serum (HS) or plasma constitutes a powerful screening assay for eliminating unstable peptides from further development. Herein we report on the stability in HS of several major histocompatibility complex (MHC)-binding peptides. Some of these peptides are in development for the novel treatment of selected autoimmune disorders such as rheumatoid arthritis and insulin-dependent diabetes. For most of the 1-amino acid peptides studied, the predominant degradation mechanism is exopeptidase-catalyzed cleavage. Peptides that were protected by d-amino acids at both termini were found to be more stable than predicted, based on additivity of single substitutions. In addition, N-acetylglucosamine glycopeptides were significantly stabilized, even when the glycosylation site was several amino acids from the predominant site(s) of cleavage. This indicates that long-range stabilization is possible, and likely due to altered peptide conformation. Finally, the effect of single amino acid substitutions on peptide stability in HS was determined using a model set of poly-Ala peptides which were protected from exopeptidase cleavage, allowing the study of endopeptidase cleavage pathways.     

注射用肌氨肽苷中多肽的含量测定方法研究

目的：建立注射用肌氨肽苷中多肽的含量测定方法。方法：用福林-酚法测定多肽含量。结果：三批样品多肽含量均在85．0％-115．0％之间。结论：用国家药品标准肌氨肽苷注射液规定的福林酚法检测多肽,可用于本品质量标准的研究。     

Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses

The cellular and molecular mechanisms that result in the induction of chemical respiratory sensitization are unclear, although there is evidence for the development of T helper (Th) 2 type responses and, in some cases, the production of IgE. We have compared cytokine secretion patterns stimulated by topical exposure of BALB/c strain mice or Brown Norway (BN) strain rats to the reference respiratory allergen trimellitic anhydride (TMA), or to the reference contact allergen 2,4-dinitrochlorobenzene (DNCB). Under conditions where TMA and DNCB provoke similar levels of immune activation [increases in lymph node cell (LNC) cellularity and proliferation] divergent cytokine expression patterns are elicited. TMA-activated LNC isolated from BALB/c mice or BN rats elaborated high levels of the Th2-type cytokines interleukin (IL)-10 and IL-13, but relatively little of the Th1-type cytokines IL-12 or interferon γ. For LNC derived from both species there was a requirement for restimulation in vitro with the mitogen concanavalin A for IL-4 production. Generally, DNCB-stimulated LNC displayed the converse type 1 cytokine phenotype. The cytokine secretion profiles of LNC isolated from BN rats were considerably more variable than those observed for LNC from BALB/c mice. Statistically significant differences (P<0.01) between DNCB- and TMA-activated LNC were recorded for all cytokines in BALB/c strain mice. For the BN rat, differences reached statistical significance (P<0.01) only for the expression of IL-4 and IL-13. These data demonstrate that the intrinsic ability of DNCB and TMA to promote preferential Th1- and Th2-type responses, respectively, is species-independent and provide further evidence that chemical respiratory allergens are associated with polarized Th2-type responses. For the prospective assessment of chemical respiratory allergens as a function of induced cytokine secretion profiles, however, these data suggest that the use of the BALB/c strain mouse will provide the more robust method.