The lipophilic hapten parthenolide induces interferon-gamma and interleukin-13 production by peripheral blood-derived CD8+ T cells from contact allergic subjects in vitro.

BACKGROUND: Peripheral blood mononuclear cells (PBMC) from persons with contact allergy to nickel react in vitro predominantly with nickel-induced CD4+ T cell-mediated production of both T-cell type 1 and 2 cytokines. OBJECTIVES: The aim of the present study was to investigate if the contact allergen parthenolide, a sesquiterpene lactone of lipophilic character, elicits an immune response which differs from that induced by water-soluble nickel ions. PATIENTS AND METHODS: Ten allergic subjects with strong (n = 6), moderate (n = 2), or weak (n = 2) patch-test reactivity to parthenolide and five patch test-negative control subjects participated in the study. PBMC from the subjects were analysed for in vitro reactivity with parthenolide by an enzyme-linked immunospot (ELISpot) assay, measuring cytokine production at the single-cell level. RESULTS: The allergic group, but not the control group, responded to parthenolide with increased numbers of cells producing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5 (P < 0.05 for all) and IL-13 (P < 0.01). The responses manifested by T-cell type 1 (IFN-gamma and IL-2) and type 2 (IL-4, IL-5 and IL-13) cytokines were positively correlated between cytokines. Subjects with a strong or moderate, but not weak or negative, patch-test reaction displayed detectable in vitro responses. In contrast to the CD4+ T cell-mediated peripheral reactivity induced by nickel, cell depletion experiments identified the parthenolide-reactive IFN-gamma- and IL-13-producing cells as CD8+ T cells. CONCLUSIONS: The finding that the PBMC reactivity to parthenolide in humans involves a CD8+ T cell-mediated type 1 and 2 cytokine response warrants further studies on the relationship between the chemical nature of a hapten and the resulting immune response.

Dose-response studies of contact allergens using 3 guinea pigs models.

A multi-dose-response induction protocol for the guinea pig maximization test (GPMT), including a statistical computer program, has earlier been developed to improve the power of predictive tests for identification of contact allergens. This dose-response protocol, with 2 modifications (i.e., increased number of animals in each group and increased number of challenge concentrations) was evaluated in the GPMT, the cumulative contact enhancement test (CCET) and the Freund's complete adjuvant test (FCAT), using potassium dichromate and hydroxycitronellal as model contact allergens. Application of the dose-response protocol on the CCET and the FCAT resulted in either monotone or non-monotone curves with significant dose-response. However, application of the dose-response protocol on the GPMT gave curves with no significant dose-response. The protocol makes it possible to obtain an EC50 value, thus improving the possibility of ranking contact allergens, which is of substantial use for risk assessments. The dose-response protocol could benefit from a few adjustments: a wider span in the induction doses; change to simultaneous increase in intradermal and topical induction doses to obtain a proper dose-response for the GPMT; the addition of further challenge concentrations. In addition the computer program should allow calculation of threshold concentration for sensitization and EC50 value for a non-monotone curve.

Sensitizing potential in mice, guinea pig and man of the preservative Euxyl K 400 and its ingredient methyldibromo glutaronitrile.

The allergenicity of the preservative Euxyl K 400 and its principal allergen methyldibromo glutaronitrile (MDBGN) (1,2-dibromo-2,4-dicyanobutane) was investigated using 3 animal models; in mice, the local lymph node assay (LLNA) and in guinea pigs, the guinea pig maximization test (GPMT) and the cumulative contact enhancement test (CCET) with a dose-response protocol included. Previous attempts to define the sensitization capacity of these chemicals have given conflicting results. For comparison, the frequency and causes of positive patch test reactions to Euxyl K 400 and MDBGN were studied in patients referred to an occupational dermatology clinic. This investigation showed that Euxyl K 400 and MDBGN can give rise to contact allergy in man and that the relevant cases found mainly had similar exposure as non-occupational cases. A contact allergenic potential could be detected for MDBGN in 2 animal models, i.e., the CCET and the LLNA, and also for Euxyl K 400 in the LLNA. However, statistical analysis of the results from the GPMT with MDBGN failed to detect the sensitizing potential of this particular allergen. The results indicate that to be able to detect the allergenic potential of Euxyl K 400 and MDBGN, a predictive test method with multiple topical applications at induction is required. It is therefore important that an investigator is aware of the possibility of using various predictive test models for investigation of potential contact allergens.

Murine local lymph node assay for predictive testing of allergenicity: two irritants caused significant proliferation.

The murine local lymph node assay is a method for predictive testing of contact allergenicity, but its ability to discriminate between allergens and irritants has been questioned. To explain some of the conflicting results with irritants, the proliferation induced by methyl salicylate and nonanoic acid, both considered to be non-sensitisers, was further investigated. Both substances showed a dose--response relationship and clearly positive results when tested at higher concentrations (> or = 50%) and would thus be classified as potential sensitisers according to the present criteria for a positive assay result. In the case of methyl salicylate, the use of either dimethyl formamide or methyl ethyl ketone as vehicle did not significantly influence the results. The negative results obtained for methyl salicylate in some earlier reports were probably due to testing at too low concentrations. The proliferation induced by irritants such as methyl salicylate and nonanoic acid and inter alia sodium dodecyl sulfate, Triton X-100, oxalic acid, chloroform/methanol (2:1) must be better recognized and elucidated before the assay can be generally accepted as a predictive test method.

An evaluation of the suitability of benzocaine as a positive control skin sensitizer.

Although they are subject to some limitations, sensitization tests such as the guinea pig maximization test (GPMT) have for many years provided a valuable basis for the identification of skin sensitization potential. Thus they have been used widely by regulatory authorities, such as those in Europe, as a means to identify significant sensitization hazards associated with new chemicals. However, the standard of performance of guinea pig sensitization assays has been demonstrated to be widely variable. Consequently, the OECD sensitization test guideline (the de facto world standard) has been updated to incorporate recommendations for action whose aim is to achieve a minimum standard of test conduct. The principle is that a test laboratory should be able to demonstrate an acceptable level of response using a moderately sensitizing chemical. A list of 3 such chemicals is provided, hexyl cinnamic aldehyde, mercaptobenzothiazole and benzocaine. It is our experience that whilst good results can readily be obtained with the first 2 of these, benzocaine is much more difficult. Using both the GPMT and the local lymph node assay (LLNA), an OECD-recommended screening test, benzocaine has given highly variable results. A range of from 0% to 60% positive in the GPMT was found and, in most tests, benzocaine would not classify as a skin sensitizer according to EU criteria. In the LLNA, from a series of 12 tests conducted in 2 laboratories, only occasional positive results were obtained. Furthermore, these positive results were not reproducible. Reasons for this variability are discussed. However, the main conclusion must be that benzocaine does not represent a useful moderately sensitizing positive control.

Experience with the murine local lymph node assay: inability to discriminate between allergens and irritants.

The murine local lymph node assay is a new predictive test for identifying contact sensitizers. It measures the proliferative response in the lymph nodes during the sensitization phase. In the present study, moderate-to-extreme allergens (from human and guinea pig experience) gave clearly positive results in this assay. However, irritants tested, i.e. sodium dodecyl sulphate (SDS), chloroform/methanol, oxalic acid, triton X-100 and methylsalicylate, also gave positive results, not distinguishable from the results with low-grade/moderate allergens. Two allergens were also tested in the presence of 10% SDS. The effect on proliferation was additive in the first case and synergistic in the second. The local lymph node assay in its present design and with the criteria used for a positive response requires further validation studies and perhaps further development before it can be accepted as an alternative to guinea pig tests for allergenicity. Substances with exclusively irritating properties could falsely be classified as allergens by the method or, alternatively, the allergenicity of chemicals with both allergenic and irritating properties could be overestimated.