Biomedical subjects

R J Dearman

Publications and source records attributed to R J Dearman.

155 records · Page 9Linked to original sources

PubMedDate not supplied

A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses.

Effective risk assessment and management of allergic contact dermatitis require three key factors: adequate hazard identification, measurement of the relative potency of identified hazards and an understanding of the nature, extent and duration of exposure. Suitable methods for hazard identification, such as the murine local lymph node assay (LLNA) and the guinea-pig maximization test, are well established and conditions of human exposure normally can be well anticipated. Thus, the need is for a robust and quantitative method for the estimation of relative skin sensitizing potency. One possible approach is via the analysis of LLNA dose-response data. In the LLNA, contact allergens are defined currently as those chemicals that cause a threefold or greater increase in lymph node cell proliferative activity compared with concurrent vehicle-treated controls. It is possible to estimate the concentration of a sensitizer required to generate a threefold stimulation of proliferation in draining lymph nodes; such a concentration is known as the EC3 value. Using a variety of statistical approaches to derive EC3 values from LLNA dose-response data for 10 chemicals, it has been demonstrated that simple linear interpolation between the values either side of the threefold stimulation index provides a robust assessment of the EC3 value without the need for recourse to more sophisticated statistical techniques. Provided that the appropriate concentrations of test chemical have been selected, EC3 values obtained in this way are reproducible both within and between laboratories and form the basis for examination of the utility of this approach for the estimation of relative skin sensitizing potency.

Allergens↗

PubMedDate not supplied

Local lymph node assay: use in hazard and risk assessment.

The murine local lymph node assay (LLNA) was developed as an alternative method for the identification of chemicals that have the ability to cause skin sensitization and allergic contact dermatitis. The assay now has been evaluated extensively in the context of both national and international inter-laboratory collaborative trials and has been the subject of detailed comparisons with guinea pig test methods and human skin sensitization data. On the basis of these evaluations the LLNA has been endorsed recently by the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) as a stand-alone method for skin sensitization testing. The assay offers a number of important benefits compared with conventional guinea pig test methods, among these being provision of an objective and quantitative endpoint. Moreover, the LLNA provides advantages in the context of animal welfare; compared with guinea pig tests, fewer animals are required and these animals are subject to less trauma. It is important now that the validation status of the LLNA is recognized and the method applied widely so that these advantages may be realized. Hazard identification represents only the first step in the risk assessment process. A full toxicological evaluation of skin sensitization activity requires an understanding of relative potency. Guinea pig methods do not lend themselves readily to assessment of potency, and interest recently has focused on the utility of the LLNA for this purpose. Contained within this review article are brief descriptions of the history of the LLNA and the immunobiological basis for the method, together with detailed accounts of the conduct and interpretation of the assay. Procedural modifications to, and alternative endpoints for, the LLNA are considered also. Finally, the current regulatory status of the LLNA is summarized and the application of the method for the purposes of defining relative potency and developing risk assessments is reviewed.

Allergens↗

PubMedDate not supplied

Characterization in mice of the immunological properties of five allergenic acid anhydrides.

Occupational exposure to certain acid anhydrides, including trimellitic anhydride (TMA), maleic anhydride (MA), phthalic anhydride (PA), hexahydrophthalic anhydride (HHPA) and methyltetrahydrophthalic anhydride (MTHPA), has been associated with the development of respiratory allergy or asthma. There is considerable debate about the mechanisms through which such chemicals may cause respiratory sensitization, particularly concerning a universal requirement for specific IgE antibody. Despite the controversy regarding an obligatory role for IgE, there is a growing consensus that chemical respiratory hypersensitivity is associated with the selective development of T lymphocytes with a type 2 (Th2) phenotype. In the current investigations we have characterized in mice the nature of immune responses provoked by prolonged topical exposure to five acid anhydrides. Under application conditions where similar overall immunogenicity was achieved, we have compared cytokine responses induced by PA, MA, HHPA and MTHPA with those provoked by concurrent exposure to TMA or to the reference contact allergen 2, 4-dinitrochlorobenzene (DNCB). Lymph node cells (LNC) draining the site of topical exposure to DNCB invariably expressed high levels of the type 1 cytokines interferon-gamma (IFN-gamma) and interleukin-12 (IL-12), but only low levels of the type 2 cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). In each experiment, TMA-activated LNC displayed the converse, type 2, phenotype of cytokine production. The other acid anhydrides in each case provoked a type 2 cytokine secretion profile, with comparable IL-10 expression but somewhat less vigorous IL-4 production compared with that observed following exposure to the reference respiratory allergen TMA. In every experiment relatively low levels of IFN-gamma and IL-12 were elaborated by acid anhydride-activated LNC, with the exception of PA-stimulated LNC that displayed increased amounts of IL-12 in comparison with other acid anhydrides. Thus, prolonged topical exposure of mice to five different acid anhydrides in each case resulted in the development of a predominantly Th2-type cytokine secretion phenotype, consistent with the ability of these materials to provoke asthma and respiratory allergy through a type 2 (possibly IgE-mediated) mechanism. Taken together with the results of previous investigations with a wider range of chemical allergens, these data suggest that induced cytokine secretion patterns or 'fingerprints' allow discrimination between contact and respiratory allergens and consequently represent a suitable approach to prospective evaluation of respiratory sensitization hazard.

Allergens↗

PubMedDate not supplied

Induction of mast cell sensitization by chemical allergens: a comparative study.

In previous investigations we have shown that chemical allergens of different classes induce in mice qualitatively divergent immune responses. Respiratory allergens provoke substantial increases in total serum concentration of IgE. In contrast, contact allergens which are known or suspected not to cause respiratory sensitization or which at most have only a very limited potential to do so, have little or no effect on total serum IgE. Such differences, we propose, provide a novel approach for the prospective identification of chemicals with potential to cause respiratory allergy. In the absence of a robust method for the direct measurement of respiratory hypersensitivity reactions in mice we have sought in the present study to determine whether the IgE responses induced in mice by respiratory allergens are specific and of sufficient magnitude to cause the active sensitization of mast cells in vivo, a prerequisite for immediate hypersensitivity, including acute-onset respiratory hypersensitivity. Topical exposure of BALB/c mice to concentrations of > or = 10% of the human respiratory allergen trimellitic anhydride (TMA) caused the specific sensitization of peritoneal mast cells in situ as measured by the conjugate-induced release of [3H]-5-hydroxytryptamine in vitro. Experiments were performed also with 2,4-dinitrochlorobenzene (DNCB), a contact allergen that fails to induce respiratory hypersensitivity. Treatment of mice with concentrations of DNCB of comparable immunogenicity failed to cause mast cell sensitization. These data demonstrate that a known human chemical respiratory allergen induces in mice specific mast-cell-sensitizing IgE antibody and reinforce the value of the mouse as a model for the evaluation of respiratory sensitization potential.

Allergens↗

PubMedDate not supplied

The local lymph node assay: an interlaboratory evaluation of interleukin 6 (IL-6) production by draining lymph node cells.

The murine local lymph node assay is a predictive method for the identification of skin-sensitizing chemicals in which activity is measured as a function of proliferative activity induced in lymph nodes draining the site of exposure. In the present study, the induction by topically applied chemicals of draining lymph node cell (LNC) production of the cytokine interleukin 6 (IL-6) has been evaluated as an alternative endpoint for the local lymph node assay. In addition, results derived from studies of IL-6 production by LNC performed independently in two separate collaborating laboratories have been compared. Of the nine skin sensitizing chemicals examined, six provoked detectable levels (> 150 pg ml-1) of IL-6 production by draining LNCs (as measured by enzyme-linked immunosorbent assay) following exposure of mice to at least one test concentration of the material in both of the laboratories. Three other sensitizing chemicals failed to induce measurable IL-6 production at any test concentration in either one or both of the participating laboratories. Both of the non-sensitizing chemicals evaluated (sodium lauryl sulphate and methyl salicylate) also failed to result in detectable IL-6 synthesis. There was a high level of agreement between the two laboratories. The rank order of chemicals with respect to IL-6 production by LNCs was identical in both cases, as was the dose-response relationship observed with each test material. These data reveal that, although inducible IL-6 production by draining LNCs provides a robust approach to the measurement of strong sensitizing activity, as performed here the method is of insufficient sensitivity for the routine identification of skin allergens.

Allergens↗

PubMedDate not supplied

Allergen-induced changes in interleukin 1 beta (IL-1 beta) mRNA expression by human blood-derived dendritic cells: inter-individual differences and relevance for sensitization testing.

The development of in vitro methods for the identification of skin sensitizers based upon analysis of Langerhans cell (LC) function has been constrained by the fact that these cells represent only a minority population in the skin that, once isolated, alter their phenotype spontaneously and rapidly. Methods have been developed recently that allow the expansion in culture using appropriate cytokine conditions of LC-like dendritic cells (DCs) from certain tissues, including human peripheral blood. It has been demonstrated that culture of human blood-derived LC-like cells with selected potent contact allergens such as 2,4-dinitrofluorobenzene (DNFB) stimulates selective phenotypic changes, including the up-regulation of interleukin 1 beta (IL-1 beta) mRNA expression, under conditions where skin irritants are without effect. However, in our own previous investigations, we have observed that there appear to be differences between blood donors with respect to the responsiveness of DCs to DNFB-induced changes in IL-1 beta expression, differences that could compromise the utility of this approach as a screening method for contact allergens. We have therefore investigated donor variability in DC responsiveness to a panel of known human contact allergens (DNFB; paraphenylene diamine, PPD; methyl- chloroisothiazolinone/methylisothiazolinone, CMIT), to the skin irritant benzalkonium chloride and to the mitogen phorbol myristate acetate (PMA). Dendritic cells derived from all donors expressed IL-1 beta mRNA constitutively. Treatment of DCs isolated from donors with a responder phenotype to DNFB with PPD or CMIT resulted also in up-regulation of IL-1 beta mRNA expression, although such changes were always comparatively modest, generally resulting in a twofold induction compared with vehicle-treated controls. Dendritic cells derived from donors with a non-responder phenotype to DNFB failed also to respond to these additional contact allergens under conditions where the mitogen PMA caused similar increases in IL-1 beta expression to those observed for allergen-responsive donors. Benzalkonium chloride failed to provoke changes in the expression of this cytokine in any donor examined, irrespective of their responder phenotype. The temporal stability of the responder/non-responder DC phenotype was confirmed, with stable phenotypes with respect to DNFB-induced changes in IL-1 beta mRNA expression observed over a period of some 18 months. Fifty per cent (6/12) of donors tested over this period displayed a responder phenotype. These data demonstrate that chemical allergens do stimulate consistent changes in IL-1 beta mRNA expression in the proportion of donors who have a responsive phenotype, and that such responses are apparently selective for allergen using the relatively narrow range of materials assessed to date. However, the modest response to very strong contact allergens, coupled with the difficulties of responder/non-responder phenotypes, means that in its present form this approach does not lend itself to the routine assessment of skin sensitizing activity.

Adult↗

PubMedDate not supplied

Cytokine fingerprinting and hazard assessment of chemical respiratory allergy.

Allergic sensitization of the respiratory tract resulting in occupational asthma and other symptoms can be caused by a variety of chemicals and represents an important occupational health problem. Although there is a need to identify and characterize those chemicals that are able to cause respiratory allergy, there are currently no well validated or widely accepted predictive test methods. Some progress has been made with guinea pig assays, but our attention in this laboratory has focused instead on the development of novel approaches based on an understanding of the nature of immune responses induced in mice by chemical allergens. We have shown that whereas contact allergens provoke in mice selective type 1 immune responses, characterized by the secretion by draining lymph node cells (LNC) of high levels of the cytokine interferon gamma (IFN-gamma), chemical respiratory allergens stimulate instead preferential type 2 responses associated with comparatively high levels of interleukins 4 and 10 (IL-4 and IL-10). The divergent immune responses provoked by different classes of chemical allergens, and the phenotypes of selective cytokine secretion that characterize such responses, form the basis of a novel method-cytokine fingerprinting--that permits chemicals that have the potential to cause respiratory allergy to be identified and distinguished from those that are associated primarily with contact sensitization. In this article the immunobiological basis for cytokine fingerprinting is considered and the development, evaluation and practical application of the assay are reviewed.

Allergens↗

PubMedDate not supplied

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.

Administration, Topical↗

PubMedDate not supplied

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.

Administration, Topical↗

PubMedDate not supplied

Induction by tumour necrosis factor alpha of dose-related changes in Langerhans cell frequency in mice.

It has been demonstrated previously that tumour necrosis factor alpha (TNF-alpha) provides an important signal for the migration of epidermal Langerhans cells (LC) from the skin. Intradermal administration to mice of homologous recombinant TNF-alpha induces both a rapid reduction in the frequency of LC local to the site of exposure and, somewhat later, an accumulation of dendritic cells in draining lymph nodes. It has been proposed recently, however, that the influence of TNF-alpha on LC function may be dose-dependent in nature with lower concentrations inducing migration, but higher concentrations immobilizing LC in the epidermis. To investigate this proposal we examined the kinetics and dose-response relationships of TNF-alpha-induced LC migration in mice. At all concentrations tested (50, 150 or 300 ng/ear), intradermal exposure to TNF-alpha caused within 30 min a significant reduction in the frequency of MHC class II (Ia)+ LC within epidermal sheets. With the lower concentrations of TNF-alpha this effect was still apparent when LC were enumerated in the epidermis up to 4 h following cytokine treatment. In contrast, however, exposure of mice to 300 ng of TNF-alpha was consistently associated with a considerably less marked, and statistically insignificant, reduction in LC frequency by 4 h. These data indicate that at all concentrations of the cytokine examined here, TNF-alpha was able to stimulate a rapid (within 30 min) reduction in epidermal LC numbers, but that the rapidity with which the epidermis was repopulated following the initiation of LC migration was influenced by the concentration of TNF-alpha administered. It is suggested that TNF-alpha may influence not only the tempo of LC migration, but also the kinetics of epidermal repopulation.

Animals↗

PubMedDate not supplied

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.

Allergens↗