Biomedical subjects

Rebecca J Dearman

Publications and source records attributed to Rebecca J Dearman.

At least 19 recordsLinked to original sources

PubMed2007

Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals.

The local lymph node assay (LLNA) is a murine model developed to evaluate the skin sensitization potential of chemicals. The LLNA is an alternative approach to traditional guinea pig methods and in comparison provides important animal welfare benefits. The assay relies on measurement of events induced during the induction phase of skin sensitization, specifically lymphocyte proliferation in the draining lymph nodes which is a hallmark of a skin sensitization response. Since its introduction the LLNA has been the subject of extensive evaluation on a national and international scale, and has been successfully validated and incorporated worldwide into regulatory guidelines. Experience gained in recent years has demonstrated that adherence to published procedures and guidelines for the LLNA (e.g., with respect to dose and vehicle selection) is critical for the successful conduct and eventual interpretation of the data. In addition to providing a robust method for skin sensitization hazard identification, the LLNA has proven very useful in assessing the skin sensitizing potency of test chemicals, and this has provided invaluable information to risk assessors. The primary method to make comparisons of the relative potency of chemical sensitizers is to use linear interpolation to estimate the concentration of chemical required to induce a stimulation index of three relative to concurrent vehicle-treated controls (EC3). In certain situations where there are available less than optimal dose response data a log-linear extrapolation method can be used to estimate an EC3 value which can reduce significantly the need for repeat testing of chemicals. The LLNA, when conducted according to published guidelines, provides a robust method for skin sensitization testing that not only provides reliable hazard identification information but also data necessary for effective risk assessment and risk management.

Animals↗

PubMed2007

A mouse model for food allergy using intraperitoneal sensitization.

Food allergy is an important health issue. With the increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches for the characterization of the allergenic potential of proteins. Although most foreign proteins are immunogenic (able to induce IgG antibody responses), relatively few are important food allergens with the capacity to provoke IgE antibody production. There is currently no validated animal model for the determination of allergenic potential of food proteins. One approach that appears to show some promise is outlined in the current chapter. BALB/c strain mice are immunized by intraperitoneal injection and the potential to cause allergenicity assessed as a function of the induction of specific IgE antibody, measured by homologous passive cutaneous anaphylaxis. Progress to date with this method is summarized, and comparisons are made with other experimental models, including considerations of route of exposure, use of adjuvants and selection of appropriate end points.

Adjuvants, Immunologic↗

PubMed2006

The IgE-facilitated allergen binding (FAB) assay: validation of a novel flow-cytometric based method for the detection of inhibitory antibody responses.

The IgE-facilitated allergen binding (IgE-FAB) assay represents an in vitro model of facilitated allergen presentation. Allergen-IgE complexes are incubated with an EBV-transformed B cell line and complexes bound to CD23 on the surface of cells are detected by flow cytometry. The addition of serum from patients who have received allergen-specific immunotherapy has been shown previously to inhibit allergen-IgE complex binding to CD23 on B cells. In this study, we describe the characterisation and analytical validation of the grass pollen-specific IgE-FAB assay according to guidelines from the International Conference on Harmonisation. We established the intra- and inter-assay variability of IgE-FAB and have defined the detection limits of this assay. We have also demonstrated assay linearity and robustness. Using the results from a randomised double-blind placebo-controlled trial of grass pollen immunotherapy (n=33), we have defined the clinical sensitivity and specificity of the IgE-FAB assay using ROC curve analysis. In conclusion, the IgE-FAB assay is reproducible, robust, sensitive and a specific method suitable as a tool for monitoring inhibitory antibody function from patients receiving allergen immunotherapy.

Allergens↗

PubMed2006

Identification of gene expression changes induced by chemical allergens in dendritic cells: opportunities for skin sensitization testing.

Cellular changes within resident skin dendritic cells (DCs) after allergen uptake and processing are critical events in the acquisition of skin sensitization. Here we describe the development of a set of selection criteria to derive a list of potential target genes from previous microarray analyses of human peripheral blood-derived (peripheral blood mononuclear cells (PBMCs)-DCs) treated with dinitrobenzene sulfonic acid for predicting skin-sensitizing chemicals. Based on those criteria, a probing evaluation of the target genes has been conducted using an extended chemical data set, comprising five skin irritants and 11 contact allergens. PBMCs-DCs were treated for 24 hours with various concentrations of chemicals and in each instance the expression of up to 60 genes was examined by real-time PCR analysis. Consistent allergen-induced changes in the expression of many genes were observed and further prioritization of the targets was conducted by analysis of the same genes in DCs treated with non-sensitizing chemicals to determine their specificity for skin sensitization. Real-time PCR analyses of multiple chemical allergens, irritants, and non-sensitizers have identified 10 genes that demonstrate reproducibly high levels of selectivity, specificity, and dynamic range consistent with providing the basis for robust and sensitive alternative approaches for the identification of skin-sensitizing chemicals.

Allergens↗

PubMed2006

Impaired Langerhans cell migration in psoriasis.

We have examined whether psoriasis is associated with systemic effects on epidermal Langerhans cell (LC) function and, specifically, the migration of LCs from the skin. Compared with normal skin, the frequency and morphology of epidermal LCs in uninvolved skin from patients with psoriasis was normal. However, mobilization of these cells in response to stimuli that normally induce migration (chemical allergen, tumor necrosis factor alpha [TNF-alpha], and interleukin-1beta [IL-1beta]) was largely absent, despite the fact that treatment with TNF-alpha and IL-1beta was associated with comparable inflammatory reactions in patients and controls. The failure of LC migration from uninvolved skin was not attributable to altered expression of receptors for IL-1beta or TNF-alpha that are required for mobilization, nor was there an association with induced cutaneous cytokine expression. Although a role for altered dynamics of LC migration/turnover has not been formally excluded, these data reveal a very consistent decrement of LC function in psoriasis that may play a decisive role in disease pathogenesis.

Adolescent↗

PubMed2006

The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?

The local lymph node assay (LLNA), an alternative approach to skin-sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut-down assay comprising a single high-dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.

Animal Testing Alternatives↗

PubMed2006

Skin sensitization potency of methyl methacrylate in the local lymph node assay: comparisons with guinea-pig data and human experience.

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.

Allergens↗

PubMed2005

Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment.

The identification of potential skin sensitizing chemicals is a key step in the overall skin safety risk assessment process. Traditionally, predictive testing has been conducted in guinea pigs. More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential. However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop non-animal test methods for skin sensitization. Mechanistic understanding of allergic contact dermatitis has increased substantially in recent years. For example, a number of changes are known to occur in epidermal Langerhans cells, the principal antigen-presenting dendritic cell in the skin, as a result of exposure to chemical allergens, including the internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis, the induction of tyrosine phosphorylation, the modulation of cell surface markers, and cytokine expression. The application of this knowledge to the design of predictive in vitro alternative tests provides both unique opportunities and challenges. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells and the potential use of that information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.

Allergens↗

PubMed2005

Impact of cutaneous IL-10 on resident epidermal Langerhans' cells and the development of polarized immune responses.

Prolonged topical exposure of BALB/c mice to chemical contact and respiratory allergens stimulates, respectively, preferential Th1- and Th2-type responses with respect to serum Ab isotype and cytokine secretion phenotypes displayed by draining lymph node cells. We now report that differential cytokine secretion patterns are induced rapidly in the skin following first exposure to the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory sensitizer trimellitic anhydride (TMA). TMA induced early expression of IL-10, a cytokine implicated in the negative regulation of Langerhans cell (LC) migration, whereas exposure to DNCB resulted in production of the proinflammatory cytokine IL-1beta. Associated with this, TMA provoked LC migration with delayed kinetics compared with DNCB, and local neutralization of IL-10 caused enhanced LC mobilization in response to TMA with concomitant up-regulation of cutaneous IL-1beta. We hypothesize that these differential epidermal cytokine profiles contribute to the polarization of immune responses to chemical allergens via effects on the phenotype of activated dendritic cells arriving in the draining lymph node. Thus, TMA-exposed dendritic cells that have been conditioned in vivo with IL-10 (a potent inhibitor of the type 1-polarizing cytokine IL-12) are effective APCs for the development of a Th2-type response.

Administration, Topical↗

PubMed2005

Exposure of mice to the nitroso metabolite of sulfamethoxazole stimulates interleukin 5 production by CD4+ T-cells.

Sulfamethoxazole hypersensitivity may be caused by production of the protein-reactive metabolite nitroso sulfamethoxazole (SMX-NO) and interaction of SMX-NO with T-cells. We have characterised the nature of the immune response induced by administration of sulfamethoxazole, sulfamethoxazole metabolites and nitrosobenzene to BALB/c mice. Drugs were administered over a 13-day period to induce polarised cytokine secretion profiles. Proliferation was measured by [(3)H] thymidine incorporation. Cytokine secretion was monitored by ELISA. Results were compared with those provoked by exposure to type 1 and type 2 chemical allergens, 2,4-dinitrochlorobenzene (DNCB) and trimellitic anhydride (TMA). CD4(+) or CD8(+) T-cells were depleted ex vivo to identify the primary source of cytokines. Lymph node activation was observed following treatment with DNCB, TMA, nitrosobenzene and SMX-NO, but not with sulfamethoxazole or sulfamethoxazole hydroxylamine (SMX-NHOH). DNCB and TMA induced type 1 and type 2 cytokine profiles, respectively. SMX-NO treatment stimulated the production of high levels of IL-5, variable amounts of IFN-gamma, and relatively low levels of IL-10 and IL-4. Nitrosobenzene-activated lymph node cells secreted only low levels of IFN-gamma and IL-5. Depletion of CD4(+) or CD8(+) T-cells from SMX-NO stimulated lymph node cells revealed that CD4(+) T-cells were the major source of IL-5. In conclusion, the data presented indicates that subcutaneous administration to mice of SMX-NO, but not the parent drug, stimulated the secretion of high levels of IL-5 from activated CD4(+) T-cells, which is consistent with the clinical profile of the drug.

Animals↗

PubMed2005

Selective haptenation of cellular or extracellular protein by chemical allergens: association with cytokine polarization.

Sensitizing chemicals can cause different forms of allergy, allergic contact dermatitis, or sensitization of the respiratory tract. These discrete types of chemicals induce in mice qualitatively divergent immune responses; contact allergens provoke preferential type 1 responses, whereas respiratory allergens stimulate selective type 2 responses. We have questioned whether the ability of chemicals to initiate polarized immune responses is in part a function of the nature of their association with protein. Cytokine secretion profiles provoked following topical exposure of BALB/c mice to dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), fluorescein isothiocyanate (FITC), trimellitic anhydride (TMA), and dinitrobenzenesulfonyl chloride (DNBSCl) were compared with the distribution of covalent binding to U937 cells and/or to serum proteins in vitro. DNCB and DNFB each provoked a type 1 cytokine secretion profile, with high levels of IFN-gamma, but relatively low levels of type 2 cytokines IL-4, -5, and -10. The converse selective type 2 phenotype was seen following equivalent exposure to TMA, FITC, or DNBSCl. Each chemical bound covalently to U937 cells and to serum proteins, when incubated with cells or serum alone. When incubated with cells and serum together, DNCB and DNFB bound selectively to cellular protein, whereas TMA, FITC, and DNBSCl bound selectively to serum. These investigations show that the distribution of antigen formation of chemical allergens in an in vitro model system segregates with the type of cytokines secreted from activated lymph node cells in an in vivo mouse model. Chemical allergens that stimulate type 1 cytokine secretion profiles bind selectively to cellular proteins, whereas others that provoke type 2 cytokine profiles bind preferentially to serum proteins.

Allergens↗

PubMed2005

Characterisation of immune responses to food allergens in mice.

There is considerable interest in the development and evaluation of approaches for the safety assessment of novel foods, and in particular in methods for characterisation of allergenic potential. One strategy that has found favour is a tiered approach in which the potential of novel proteins to induce allergic sensitisation is assessed based on considerations of stability of the protein in a simulated gastric juice and homology with, or structural similarity to, known allergens. Linked to such an approach may be evaluation of serological identity with proteins known to cause allergic disease. With the aim of supplementing such approaches with a more direct measurement of potential allergenic activity, attempts have been made to characterise the quality of immune responses elicited in BALB/c strain mice. Such evaluations comprise measurement of IgG and IgE antibody production and (to a lesser extent) of induced cytokine expression patterns. Investigations to date suggest that in mice proteins provoke variable immune responses, those with the potential to cause allergic sensitisation stimulating IgE (and IgG) antibody production. In contrast, non-allergenic, but nevertheless immunogenic, proteins are associated with IgG antibody responses in the absence of marked IgE production. Consistent with the selective activation of selective type 2 T lymphocyte responses, exposure of mice to allergenic protein is associated with preferential expression of IL-4, -5, -10 and -13. Collectively these data suggest that characterisation of the nature of immune response induced in mice by proteins may provide a useful adjunct or alternative to current strategies for the assessment of allergenic potential.

Allergens↗

PubMed2005

What makes a chemical a respiratory sensitizer?

PURPOSE OF REVIEW: In this article we consider the characteristics that are associated with chemical respiratory allergens, and that may be essential for effective sensitization of the respiratory tract. RECENT FINDINGS: Chemical respiratory allergens share some characteristics with other chemical allergens, specifically chemical allergens that cause skin sensitization and allergic contact dermatitis. The unique and defining characteristic of chemical respiratory allergens, which in most instances distinguishes them from contact allergens, is the ability to provoke the preferential development of T helper 2-type immune responses. There are, in addition, other characteristics, such as the ability to increase matrix metalloproteinase expression or to cause perturbation of redox homeostasis, that may in some instances facilitate the induction or expression of respiratory allergy, but it is not yet clear if these attributes are common or essential properties of all chemical respiratory sensitizers. SUMMARY: Predicting which chemical allergens may selectively induce respiratory sensitization is an important objective, but remains a significant challenge because our understanding of the relevant physicochemical characteristics and biological properties that confer on chemicals respiratory allergenic potential is incomplete.

Allergens↗

PubMed2005

Potency and risk assessment of a skin-sensitizing disperse dye using the local lymph node assay.

Disperse Blue 106 is an acknowledged skin-sensitization hazard. However, information about the relative sensitization potency of this chemical is lacking, and to provide this information was the purpose of the investigations described here. The approach taken was to measure dose-response relationships for C.I. Disperse Blue in the local lymph node assay, a method for the assessment of skin-sensitization potential in which activity is measured as a function of lymphocyte proliferative responses induced in draining lymph nodes. From these data, it was possible to derive EC3 values (such being the estimated concentration of chemical required to elicit a 3-fold increase in proliferation) that have been shown previously to reflect the relative sensitizing potency of contact allergens. These analyses revealed that Disperse Blue 106 had a relatively low EC3 value (0.01%), comparable to that measured concurrently for 2,4-dinitrochlorobenzene, a potent contact allergen. Collectively, these data reveal that Disperse Blue 106 represents a significant skin-sensitization hazard, and, in combination with information on dye migration and percutaneous penetration from various types of fabric and use conditions provide a basis for the development of effective and accurate risk assessments.

Allergens↗

PubMed2005

Predictive identification of human skin sensitization thresholds.

For years, methods have been available for the predictive identification of chemicals that possess the intrinsic potential to cause skin sensitization. However, many have proven less suitable for the determination of relative sensitizing potency. In this respect, the local lymph node assay (LLNA) has been shown to have a number of important advantages. Through interpolation of LLNA dose-response data, the concentration of a chemical required to produce a threshold positive response (a 3-fold increase in activity compared with concurrent vehicle controls, the EC3 value) can be measured. The robustness of this parameter has been demonstrated rigorously in terms of inter- and intralaboratory reproducibility. Additionally, the relationship between potency estimates from the LLNA and an appreciation of human potency based on clinical experience has been reported previously. In the present investigations, we have sought to consolidate further our understanding of the association between EC3 values and human skin-sensitization potency by undertaking a thorough and extensive analysis of existing human predictive assays, particularly where dose-response information is available, from historical human repeated insult patch tests (HRIPTs). From these human data, information on the approximate threshold for the induction of skin sensitization in the HRIPT was determined for 26 skin-sensitizing chemicals. These data were then compared with LLNA-derived EC3 values. The results from each assay, expressed as dose per unit area (microg/cm(2)), revealed a clear linear relationship between the 2 values, thereby substantiating further the utility of LLNA EC3 values for prediction of the relative human sensitizing potency of newly identified skin sensitizers.

Allergens↗

PubMed2005

Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods.

BACKGROUND: Within the toxicology community, considerable effort is directed toward the development of alternative methods for skin sensitization testing. The availability of high-quality, relevant, and reliable in vivo data regarding skin sensitization is essential for the effective evaluation of alternative methodologies. Ideally, data derived from humans would be the most appropriate source because the test methods are attempting to predict a toxicologic effect in humans. Unfortunately, insufficient human data of the necessary quality are available, so it is necessary to rely on the best available animal data. In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurately identifying skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency, information that is pivotal to the successful management of human health risks. OBJECTIVE: To provide a database of robust in vivo data to calibrate, evaluate, and eventually validate new approaches for skin sensitization testing. METHODS: LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources. RESULTS: We provide a database that comprises LLNA data on 211 individual chemicals. This extensive chemical data set encompasses both the chemical and biologic diversity of known chemical allergens. To cover the range of relative allergenic potencies, the data set includes data on 13 extreme, 21 strong, 69 moderate, and 66 weak contact allergens, classified according to each allergen's mathematically estimated concentration of chemical required to induce a threefold stimulation index. In addition, there are also 42 chemicals that are considered to be nonsensitizers. In terms of chemical diversity, the database contains data pertaining to the chemical classes represented by aldehydes, ketones, aromatic amines, quinones, and acrylates, as well as compounds that have different reactivity mechanisms. In addition to two-dimensional chemical structures, the physicochemical parameters included are log Kp, log K(o/w), and molecular weight. CONCLUSIONS: The list of chemicals contained in the data set represents both the chemical and biologic diversity that is known to exist for chemical allergens and non-allergens. It is anticipated that this database will help accelerate the development, evaluation, and eventual validation of new approaches to skin sensitization assessment.

Animals↗

PubMed2004

Gene expression changes in peripheral blood-derived dendritic cells following exposure to a contact allergen.

A critical step in the induction of allergic contact allergy is the activation and subsequent migration of Langerhans cells (LC), an important antigen presenting dendritic cell (DC) of the skin. As the Langerhans cells migrate, they undergo a maturation process. It has been proposed that contact allergen exposure can induce DC maturation. While changes in DC gene expression profiles induced by various maturation stimuli have been explored, there are no published reports describing genomic-scale analysis of the changes induced by chemical allergen exposure. Therefore, to explore the concept of chemical allergen-induced DC maturation and to identify genes that are regulated by exposure to allergens we examined, at the transcriptional level, the effects of exposure to a contact allergen on DC. Peripheral blood-derived DC were exposed for 24 h to either 1mM or 5 mM dinitrobenzenesulfonic acid (DNBS). Changes in gene expression were analyzed using Affymetrix U95Av2 GeneChip. Comparison of mean signal values from replicate cultures revealed 173 genes that were significantly different (P < or = 0.001) between 1 mM DNBS treated and untreated control DC and 1249 significant gene changes between 5 mM DNBS treated and control DC. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the observed transcript changes for selected genes in DC derived from a second donor. Comparison of the fold-changes in transcript levels between the two platforms and donors revealed a good correlation in both direction and magnitude. RT-PCR analysis was also used to assess the allergen specificity of a selected number of genes in DC derived from a third donor. Many of the gene expression changes were found to be induced only by exposure to the allergen, DNBS, and not by exposure to a structurally similar non-allergen, benzenesulfonic acid. A number of gene expression changes induced by allergen exposure were found to be consistent with what is known of the DC maturation process, and thus provide support for the theory of contact allergen-induced DC maturation. Additionally, it is hoped that some of the transcript changes identified through this approach will be shown to be suitable for use in the development of an in vitro predictive assay for contact sensitization.

Allergens↗

PubMed2004

Intradermal exposure of BALB/c strain mice to peanut protein elicits a type 2 cytokine response.

There is a growing need for the development of methods to characterize the allergenic properties of novel proteins, particularly those expressed by transgenic crop plants. Hence, there is considerable interest in the development of suitable animal models for this purpose. The production of specific IgE antibody has been reported following sensitization with food allergen via oral or systemic (intraperitoneal) routes of exposure. We have characterized cytokine profiles induced by intradermal treatment of BALB/c strain mice with a purified peanut allergen, Arachis hypogea lectin. Mice were exposed to peanut lectin by intradermal administration and the cytokine responses in the lymph node draining the site of exposure analyzed at the secreted protein level by enyzme-linked immunosorbent assay (ELISA) and cytokine mRNA level by ribonuclease protection assay (RPA). Exposure to peanut lectin, under conditions that induced robust IgE antibody titers, was found to be associated with a T helper 2 (Th2)-type cytokine expression profile at both the mRNA and secreted protein levels. Culture of naïve lymph node cells with peanut lectin failed to stimulate marked proliferation or cytokine production, confirming this protein is not mitogenic for mouse lymphocytes. Furthermore, the expression of Th2 cytokines was associated with the effector/memory CD62L- cell population. Similar treatment with a non-allergenic protein, potato acid phosphatase, failed to induce Th2 cytokine expression. These data demonstrate that exposure of mice to peanut allergen results in the selective stimulation of a Th2-type response.

Animals↗