Effect of Latex Purification and Accelerator Types on Rubber Allergens Prevalent in Sulphur Prevulcanized Natural Rubber Latex: Potential Application for Allergy-Free Natural Rubber Gloves.

Natural rubber (NR) gloves manufactured from NR latex are widely utilized in various applications as a personal protective device due to their exceptional barrier characteristics in infection control. However, the use of NR gloves was associated with concerns on NR protein allergy. With comprehensive leaching procedures now a common practice in NR latex glove factories to eliminate latent rubber proteins and chemical allergens, occurrences and complaints of protein allergy from medical glove users have decreased drastically over the past two decades. The present work aims to eliminate further the residual rubber allergens in NR latex through effective purification of the NR latex and compounding the thus purified latex with an established formulation for allergy-free NR for glove applications. NR latex was purified by deproteinization and saponification, respectively. Several analytical techniques were used to verify rubber allergens eliminated in the purified latexes. Saponified NR (SPNR) latex was the purified NR latex of choice since it is devoid of allergenic proteins and poses the lowest risk of Type I allergy. The purified NR latex was compounded with zinc diethyldithiocarbamate (ZDEC), zinc dibutyldithiocarbamate (ZDBC), and zinc 2-mercaptobenzothiazole (ZMBT), respectively, for glove dipping. Among the investigated accelerators, only ZDBC was not detected in the artificial sweat that came into contact with the dipped articles. Thus, it is deduced that ZDBC poses the lowest risk of Type IV allergy to consumers. Additionally, the morphological and physical properties of dipped articles were assessed. It was revealed that the dipped film from the SPNR latex compounded with ZDBC provided thinner and less yellow products with a more uniform internal structure and a tensile strength comparable to those of commercial NR gloves.

Application of Defined Approaches for Skin Sensitization to Agrochemical Products.

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the "2 out of 3 (2o3)" DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56-57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

Prior Exposure to Ortho-Phthalaldehyde Augments IgE-Mediated Immune Responses to Didecyldimethylammonium Chloride: Potential for 2 Commonly Used Antimicrobials to Synergistically Enhance Allergic Disease.

Health-care workers have an increased incidence of allergic disease compared with the general public and are exposed to a variety of high-level disinfectants. Although exposure to these agents has been associated with allergic disease, findings between epidemiology and animal studies often conflict respecting immunological mechanisms. Therefore, we hypothesized that previous exposure to a representative IgE-mediated sensitizer (ortho-phthalaldehyde [OPA]) alters immune responses to a representative T-cell-mediated sensitizer (didecyldimethlyammonium chloride [DDAC]). Here, BALB/c mice were topically exposed to OPA (0.5%) for 3 days, rested, then topically exposed to DDAC (0.0625%, 0.125%, and 0.25%) for 14 days. Coexposure resulted in phenotypic changes in draining lymph node (dLN) cells, including a decreased frequency of CD8+ T cells and increased frequency and number of B cells compared with DDAC-only treated mice. The coexposed mice also had enhanced Th2 responses, including significant alterations in: dLN Il4 (increased), B-cell activation (increased), CD8+ T-cell activation (decreased), and local and systemic IgE production (increased). These changes were not observed if mice were exposed to DDAC prior to OPA. Exposure to OPA alone shows Th2 skewing, indicated by increased activation of skin type 2 innate lymphoid cells, increased frequency and activation of draining lymph node B cells, and increased levels of type 2 cytokines. These findings suggest that the OPA-induced immune environment may alter the response to DDAC, resulting in increased IgE-mediated immune responses. This data may partially explain the discordance between epidemiological and laboratory studies regarding disinfectants and provide insight into the potential immunological implications of mixed chemical exposures.

Chemical or Drug Hypersensitivity: Is the Immune System Clearing the Danger?

Hypersensitivity reaction or allergy, initially perceived as a secondary disorder, is now believed to be a major public health concern. For instance, contact dermatitis is a type IV delayed-type hypersensitivity reaction to skin allergens. An estimated 15%-20% of the general population has acquired contact allergy. To maintain immune tolerance, our immune system needs to differentiate between harmful non-self and innocuous non-self. Living organisms have developed during their evolution a set of biological mechanisms to protect them from the "outside": physical barrier, inflammation, cell-based mechanisms, metabolism and elimination, and biochemical pathways such as glutathione. The innate and adaptive immune systems are also involved in the protection from the "outside" by mounting specific or non-specific responses leading to the clearance of dangerous triggers such as viruses, bacteria, or parasites. In the case of chemical or drug allergy, is our immune system clearing the danger resulting from environmental exposure to these harmful chemicals with a true immune response? Is the immune system then triggered specifically for its role: clearing a specific problem? Alternatively, should we perceive chemical or drug hypersensitivity as an accident with uncontrolled consequences leading to immunopathology?

A Role for Regulatory T Cells in a Murine Model of Epicutaneous Toluene Diisocyanate Sensitization.

Toluene diisocyanate (TDI) is a leading cause of chemical-induced occupational asthma which impacts workers in a variety of industries worldwide. Recently, the robust regulatory potential of regulatory T cells (Tregs) has become apparent, including their functional role in the regulation of allergic disease; however, their function in TDI-induced sensitization has not been explored. To elucidate the kinetics, phenotype, and function of Tregs during TDI sensitization, BALB/c mice were dermally exposed (on each ear) to a single application of TDI (0.5-4% v/v) or acetone vehicle and endpoints were evaluated via RT-PCR and flow cytometry. The draining lymph node (dLN) Treg population expanded significantly 4, 7, and 9 days after single 4% TDI exposure. This population was identified using a variety of surface and intracellular markers and was found to be phenotypically heterogeneous based on increased expression of markers including CD103, CCR6, CTLA4, ICOS, and Neuropilin-1 during TDI sensitization. Tregs isolated from TDI-sensitized mice were significantly more suppressive compared with their control counterparts, further supporting a functional role for Tregs during TDI sensitization. Last, Tregs were depleted prior to TDI sensitization and an intensified sensitization response was observed. Collectively, these data indicate that Tregs exhibit a functional role during TDI sensitization. Because the role of Tregs in TDI sensitization has not been previously elucidated, these data contribute to the understanding of the immunologic mechanisms of chemical induced allergic disease.

Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens.

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.

The lymphocyte transformation test in allergic contact dermatitis: New opportunities.

Allergic contact dermatitis (ACD) is driven by the activation and proliferation of allergen-specific memory T-lymphocytes and is currently diagnosed by patch testing with a selected panel of chemical allergens. The lymphocyte transformation test (LTT) can be used to monitor ex vivo T-lymphocyte responses to antigens, including contact allergens. The LTT is not viewed as being an alternative to patch testing, but it does seek to reflect experimentally skin sensitization to specific chemicals. The LTT is based on stimulation in vitro of antigen-driven T-lymphocyte proliferation. That is, exposure in culture of primed memory T-lymphocytes to the relevant antigen delivered in an appropriate configuration will provoke a secondary response that reflects the acquisition of skin sensitization. The technical aspects of this test and the utility of the approach for investigation of immune responses to contact allergens in humans are reviewed here, with particular emphasis on further development and refinement of the protocol. An important potential application is that it may provide a basis for characterizing those aspects of T-lymphocyte responses to contact allergens that have the greatest influence on skin sensitizing potency and this will be considered in some detail.

Evaluation of in silico models for the identification of respiratory sensitizers.

Low molecular weight (LMW) respiratory sensitizers can cause occupational asthma but due to a lack of adequate test methods, prospective identification of respiratory sensitizers is currently not possible. This article presents the evaluation of structure-activity relationship (SAR) models as potential methods to prospectively conclude on the sensitization potential of LMW chemicals. The predictive performance of the SARs calculated from their training sets was compared to their performance on a dataset of newly identified respiratory sensitizers and nonsensitizers, derived from literature. The predictivity of the available SARs for new substances was markedly lower than their published predictive performance. For that reason, no single SAR model can be considered sufficiently reliable to conclude on potential LMW respiratory sensitization properties of a substance. The individual applicability domains (ADs) of the models were analyzed for adequacies and deficiencies. Based on these findings, a tiered prediction approach is subsequently proposed. This approach combines the two SARs with the highest positive and negative predictivity taking into account model specific chemical AD issues. The tiered approach provided reliable predictions for one-third of the respiratory sensitizers and nonsensitizers of the external validation set compiled by us. For these chemicals, a positive predictive value of 96% and a negative predictive value of 89% were obtained. The tiered approach was not able to predict the other two-thirds of the chemicals, meaning that additional information is required and that there is an urgent need for other test methods, e.g., in chemico or in vitro, to reach a reliable conclusion.

Chemical allergen induced perturbations of the mouse lymph node DNA methylome.

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses and may determine the vigor, quality, or longevity of such responses. Chemical allergens can be divided into two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice, these are characterized by different T helper cell responses. To explore the regulation and maintenance of these divergent responses, mice were exposed to 2,4-dinitrochlorobenzene (DNCB, a contact allergen) or trimellitic anhydride (TMA, a respiratory allergen). DNA from draining lymph nodes was processed for methylated DNA immunoprecipitation followed by hybridization to a whole-genome DNA promoter array. 6319 differently methylated regions (DMRs) were identified following DNCB treatment, whereas 2178 DMRs were measured following TMA treatment, with approximately half of the TMA DMRs common to DNCB. When limited to promoter region-associated DMRs, 637 genes were uniquely associated with DNCB-induced DMRs but only 164 genes were unique to TMA DMRs. Promoter-associated DMRs unique to either DNCB or TMA were generally hypomethylated whereas DMRs common to both allergens tended to be hypermethylated. Pathway analyses highlighted a number of immune-related pathways, including chemokine and cytokine signaling. These data demonstrate that chemical allergen exposure results in characteristic patterns of DNA methylation indicative of epigenetic regulation of the allergic response.

Chemical allergy in humans: fresh perspectives.

There is considerable interest in the immunobiological processes through which the development of allergic sensitization to chemicals is initiated and orchestrated. One of the most intriguing issues is the basis for the elicitation by chemical sensitizers of different forms of allergic reaction; that is, allergic contact dermatitis or sensitization of the respiratory tract associated with occupational asthma. Studies in rodents have revealed that differential forms of allergic sensitization to chemicals are, in large part at least, a function of the selective development of discrete functional sub-populations of CD4(+) and CD8(+) T-lymphocytes. Evidence for a similar association of chemical allergy in humans with discrete T-lymphocyte populations is, however, limited. It is of some interest, therefore, that two recent articles from different teams of investigators have shed new light on the role of polarized T-lymphocyte responses in the development of allergic contact dermatitis and occupational asthma in humans. The implications for understanding of chemical allergy in humans are explored in this Commentary.