Rebecca J. Dearman

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 Methods: LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources.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 allergens 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 KO/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.

The local lymph node assay: developments and applications.

The murine local lymph node assay is a predictive test method for the identification of contact allergens in which sensitizing activity is measured as a function of induced proliferative responses in lymph nodes draining the site of application. In this article the development and validation of the assay are described and comparisons with guinea pig predictive test methods discussed. In addition we examine the advantages and limitations of the method and consider new opportunities and applications of the assay in the context of the toxicological evaluation of sensitizing potential.

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.

The local lymph node assay: past, present and future

The local lymph node assay (LLNA) was developed originally as a method for the identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. The assay is based on an understanding that the acquisition of contact sensitization is associated with, and dependent upon, the stimulation by chemical allergens of lymphocyte proliferative responses in skin‐draining lymph nodes. Those chemicals that provoke a defined level of lymph node cell (LNC) proliferation (a 3‐fold or greater increase compared with concurrent vehicle controls) are classified as skin sensitizers. Following its original inception and development, the LLNA was the subject of both national and international interlaboratory collaborative trials, and of very detailed comparisons with other test methods and with human skin sensitization data. The assay has now been validated fully as a stand‐alone test for the purposes of hazard identification. In recent years, there has been a growing interest also in the use of the LLNA to assess the potency of contact allergens and in risk assessment. There is reason to believe that the extent of skin sensitization achieved is associated with the vigour of LNC proliferation induced in draining nodes. Given this relationship, the relative potency of skin sensitizing chemicals is measured in the LLNA by derivation of an EC3 value, this being the concentration of chemical required to provoke a 3‐fold increase in the proliferation of LNC compared with controls. Experience to date indicates that relative potency as determined using this approach correlates closely with what is known of the activity of skin sensitizing chemicals in humans. In this article, we review the development, evaluation and validation of the LLNA for the purposes of hazard identification, and the more recent application of the method for evaluation of potency in the context of risk assessment. In addition, we consider what new applications and modifications are currently being investigated.

Interleukin (IL)-18 induces Langerhans cell migration by a tumour necrosis factor-alpha- and IL-1beta-dependent mechanism.

Following skin sensitization a proportion of epidermal Langerhans cells (LC) are stimulated to leave the skin and to migrate, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor‐α (TNF‐α), an inducible product of epidermal keratinocytes, and interleukin (IL)‐1β, produced exclusively by LC in murine epidermis, provide important signals for the initiation of this response. Recently, it has been demonstrated that IL‐18, a cytokine produced by both LC and keratinocytes within the epidermis, may also participate in immune responses induced following skin sensitization. In the present investigations, the ability of IL‐18 to contribute to the regulation of LC migration and the accumulation of DC in draining lymph nodes has been examined. It was found that, like IL‐1β, IL‐18 administered intradermally to mice resulted in a significant reduction in epidermal major histocompatibility complex (MHC) class II+ LC densities and a marked increase in lymph node DC numbers. Using neutralizing anti‐TNF‐α and blocking anti‐type I IL‐1 receptor (IL‐1RI) antibodies, it was shown also that the induction by IL‐18 of both LC mobilization and DC accumulation in regional lymph nodes was dependent upon availability of TNF‐α and the integrity of IL‐1RI signalling. Furthermore, using IL‐1β converting enzyme (caspase‐1) knockout mice, IL‐18‐induced LC migration was found to have a mandatory requirement for active IL‐1β. Importantly, not only was IL‐18 able to contribute to the regulation of LC migration, it was found to be essential for the manifestation of these processes in response to topical sensitization with the contact allergen oxazolone.

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögrens syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynauds disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log10 odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.

Immunogenicity of therapeutic proteins: Influence of aggregation

Abstract The elicitation of anti-drug antibodies (ADA) against biotherapeutics can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immunogenicity of biotherapeutics is, therefore, an important issue. There is evidence that protein aggregation can result in enhanced immunogenicity; however, the precise immunological and biochemical mechanisms responsible are poorly defined. In the context of biotherapeutic drug development and safety assessment, understanding the mechanisms underlying aggregate immunogenicity is of considerable interest. This review provides an overview of the phenomenon of protein aggregation, the production of unwanted aggregates during bioprocessing, and how the immune response to aggregated protein differs from that provoked by non-aggregated protein. Of particular interest is the nature of the interaction of aggregates with the immune system and how subsequent ADA responses are induced. Pathways considered here include ‘classical’ activation of the immune system involving antigen presenting cells and, alternatively, the breakdown of B-cell tolerance. Additionally, methods available to screen for aggregation and immunogenicity will be described. With an increased understanding of aggregation-enhanced immune responses, it may be possible to develop improved manufacturing and screening processes to avoid, or at least reduce, the problems associated with ADA.

Langerhans cell migration

Epidermal Langerhans cells (LC) play pivotal roles in the induction of cutaneous immune responses. Encounter with antigen in the skin, or other stimuli, cause the mobilization of LC and their migration, via afferent lymphatics, to draining lymph nodes where they localize within the paracortex. During their movement from the skin LC acquire the characteristics of immunostimulatory dendritic cells (DC) such that the antigen‐bearing cells which accumulate in lymph nodes are able to provoke specific T‐lymphocyte responses. Epidermal cytokines initiate and regulate LC migration (and maturation), of particular importance being interleukin‐1β and tumour necrosis factor‐α. Collectively, these cytokines, together with relevant chemokine receptor–ligand interactions, effect the liberation of LC from the epidermis and their directed movement to, and localization within, peripheral lymph nodes. Described here are the phenotypic changes induced during the activation of LC and the mechanisms through which their migration is regulated.

Differential regulation by thalidomide and dexamethasone of cytokine expression in human peripheral blood mononuclear cells

Immunosuppressive drugs are used routinely to reduce the inappropriate production of cytokines in an immune response. Recent attention has focused on drugs that selectively inhibit specific cytokines. Both thalidomide and dexamethasone have been reported to exhibit immunomodulatory effects on cytokines in vitro. We wished to examine the effects of thalidomide and dexamethasone on the production of cytokines by peripheral blood mononuclear cells (PBMC), following mitogenic stimulation, at the level of both secreted product and mRNA production. PBMC from healthy human volunteers were stimulated optimally with phytohaemagglutinin (PHA) in the presence of varying concentrations of thalidomide and dexamethasone using dimethyl sulphoxide (DMSO) as the solvent. Analysis of supernatants by enzyme-linked immunosorbent assay (ELISA) showed that thalidomide caused a dose-dependent inhibition of the pro-inflammatory cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha), maximally reducing production by 20 (P < 0.05) and 30% (P < 0.01), respectively, compared with controls. However, thalidomide did not affect either proliferation or the production of interleukin 2 (IL-2), interleukin 4 (IL-4) or interleukin 10 (IL-10). A slight bell shaped inhibition of interferon gamma (IFN-gamma) was seen which was statistically significant (P < 0.05). In contrast, dexamethasone inhibited markedly the expression of all cytokines tested (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) in dose-dependent fashion, reducing levels to near to background. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that thalidomide inhibited selectively the expression of TNF-alpha and IL-6 mRNA, whereas dexamethasone inhibited mRNA levels of all cytokines examined. The data indicate that dexamethasone is a broad range immunosuppressant inhibiting all cytokines tested in a dose-dependent manner at the level of both secreted product and mRNA. Conversely, thalidomide selectively inhibits the production of IL-6 and TNF-alpha. Due to their markedly different effects on cytokine production, and the fact that both drugs act at the level of transcription, we believe they influence separate pathways involved in cytokine gene regulation.