Tolerogenic phenotype of dendritic cells is induced after hapten sensitization followed by attenuated contact hypersensitivity response in atopic dermatitis model NC/Nga mice.

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are common inflammatory diseases. We previously reported attenuated contact hypersensitivity (CHS) responses in AD model mice using 2,4-dinitrofluorobenzene, reflecting clinical experiments. However, previous studies have not addressed the commonality of findings across haptens and mechanisms focused on dendritic cells (DCs). Thus, this study evaluated CHS responses to fluorescein isothiocyanate (FITC) and DC migration and maturation in the sensitization phase of CHS in AD. CHS responses to FITC were compared between NC/Nga mice without and with AD induction (non-AD and AD mice, respectively). T-cell responses and DC migration and maturation after FITC-induced sensitization were examined in the draining lymph nodes of non-AD and AD mice. AD mice demonstrated reduced CHS responses to FITC under decreased T-cell proliferation following sensitization and interferon-γ production by hapten-specific T cells compared with non-AD mice. In addition, the number of FITC+CD11c+MHC class IIhigh migratory DCs 24 h after FITC sensitization was comparable between non-AD and AD mice. However, FITC+CD11c+MHC class IIhigh migratory DCs in AD mice exhibited lower expression levels of CD80 and CD86 and higher expression levels of PD-L1 and mRNA of transforming growth factor beta than non-AD mice. These findings suggest that attenuated CHS responses may be hapten-independent and the induction of the tolerogenic phenotype of hapten-bearing DCs can contribute to reduced T-cell proliferation after sensitization and CHS responses in AD.

CTLA-4 suppresses hapten-induced contact hypersensitivity in atopic dermatitis model mice.

Atopic dermatitis (AD) patients with skin barrier dysfunction are considered to be at a higher risk of allergic contact dermatitis (ACD), although previous studies showed that attenuated ACD responses to strong sensitizers in AD patients compared to healthy controls. However, the mechanisms of ACD response attenuation in AD patients are unclear. Therefore, using the contact hypersensitivity (CHS) mouse model, this study explored the differences in CHS responses to hapten sensitization between NC/Nga mice with or without AD induction (i.e., non-AD and AD mice, respectively). In this study, ear swelling and hapten-specific T cell proliferation were significantly lower in AD than in non-AD mice. Moreover, we examined the T cells expressing cytotoxic T lymphocyte antigen-4 (CTLA-4), which is known to suppress T cell activation, and found a higher frequency of CTLA-4+ regulatory T cells in draining lymph node cells in AD than in non-AD mice. Furthermore, the blockade of CTLA-4 using a monoclonal antibody eliminated the difference in ear swelling between non-AD and AD mice. These findings suggested that CTLA-4+T cells may contribute to suppressing the CHS responses in AD mice.

Implementation of a dermal sensitization threshold (DST) concept for risk assessment: structure-based DST and in vitro data-based DST.

Skin sensitization resulting in allergic contact dermatitis represents an important toxicological endpoint as part of safety assessments. When available substance-specific sensitization data are inadequate, the dermal sensitization threshold (DST) concept has been proposed to set a skin exposure threshold to provide no appreciable risk of skin sensitization. Structure-based DSTs, which include non-reactive, reactive, and high potency category (HPC) DSTs, can be applied to substances with an identified chemical structures. An in vitro data-based "mixture DST" can be applied to mixtures based on data from in vitro test methods, such as KeratinoSens™ and the human Cell Line Activation Test. The purpose of this review article is to discuss the practical use of DSTs for conducting sound sensitization risk assessments to assure the safety of consumer products. To this end, several improvements are discussed in this review. For application of structure-based DSTs, an overall structural classification workflow was developed to exclude the possibility that "HPC but non-reactive" chemicals are misclassified as "non-reactive", because such chemicals should be classified as HPC chemicals considering that HPC rules have been based on the chemical structure of high potency sensitizers. Besides that, an extended application of the mixture DST principle to mixtures that either is cytotoxic or evaluated as positive was proposed. On a final note, we also developed workflows that integrate structure-based and in vitro-based mixture DST. The proposed workflows enable the application of the appropriate DST, which serves as a point of departure in the quantitative sensitization risk assessment.

Hapten sensitization to vaginal mucosa induces less recruitment of dendritic cells accompanying TGF-ß-expressing CD206+ cells compared with skin.

INTRODUCTION: Contact hypersensitivity (CHS), a type of delayed-type hypersensitivity, is induced by hapten exposure to the skin and mucosa. We previously reported that, in a murine model of CHS, the vaginal mucosa (VM) sensitization showed lower T-cell responses as compared with the abdominal skin sensitization. To investigate mechanisms of impaired CHS by the VM sensitization, we compared migration of hapten-captured dendritic cells (DCs) in the draining lymph nodes (dLNs) and recruitment of DCs at the sensitized local sites. METHODS: Fluorescein isothiocyanate (FITC) or 2,4-dinitrofluorobenzene (DNFB) was used as hapten, and migration of FITC+ DCs in the dLNs and local recruitment of MHC class II+ and CD11c+ cells were compared between abdominal skin and VM sensitization by flow cytometric analyses and immunohistochemistry. Expression of tumor growth factor (TGF)-ß at mRNA and protein levels, and local recruitment of CD206+ cells were examined after VM sensitization. RESULTS: VM sensitization showed less numbers of FITC+ MHC class IIhigh CD11c+ migratory DCs in the dLNs at 6 and 24 h, as compared with skin sensitization. Both skin and VM sensitization induced the recruitment of dermal/submucosal DCs at 6 h, but the number of submucosal DCs in the VM was significantly decreased at 24 h. VM showed persistently higher mRNA levels of TGF-ß2/ß3 expression than those of the skin before and after sensitization. In the VM sensitization, increment of CD206+ MHC class II+ cells was observed especially at the deep lamina propria at 24 h. Most of CD206+ cells were also positive for the binding to Fc chimeric TGF-ß receptor that interacts with all TGF-ß isoforms, suggesting TGF-ß expression. CONCLUSION: DC migration to dLNs and localization of DCs at the sensitized sites are limited in the VM sensitization. Our results suggest that the existence of TGF-ß-expressing CD206+ cells may contribute less sensitization ability and CHS responses in the VM.

Differential susceptibility between skin and vaginal mucosa in sensitization phase of allergic contact dermatitis in mice.

INTRODUCTION: Mechanisms underlying skin sensitization in allergic contact dermatitis have been actively studied using the murine contact hypersensitivity (CHS) model. However, much less is known about sensitization at the vaginal mucosa (VM). METHODS: We developed a CHS model with VM sensitization and epicutaneous elicitation at the ear. We then examined the proliferation activity of lymphocytes, the frequencies of T cells and the differentiation of hapten-specific T cells in draining lymph nodes (dLNs) after sensitization. RESULTS: Hapten-specific CHS responses to 2,4-dinitrofluorobenzene (DNFB), 2,4,6-trinitrochrolobenzene, and oxazolone assessed by ear swelling suggested that the VM would be an inductive site of CHS to haptens. In the comparisons of CHS responses to each of the three haptens examined, the lower responses in VM-sensitized mice were observed than skin-sensitized mice (e.g., DNFB-induced responses, -56%; p < .001, at 48 h after challenge). Consistent with the CHS responses, the DNFB-induced proliferation of cells in dLNs examined by 5-bromo-2'-deoxyuridine assay was lower (-62%; p < .001) in VM-sensitized mice than skin-sensitized mice. On the other hand, between skin and VM sensitization, no significant differences were observed in the frequencies of interferon-γ-producing CD4+ and CD8+ effector, and regulatory T cells in dLNs after sensitization. We also observed no significant differences with respect to differentiation of hapten-specific T cells based on the examination of cytokine production from dLN cells stimulated in vitro with 2,4-dinitrobenzene sulfonate. CONCLUSION: These findings suggested that the lower T cell proliferation after VM sensitization is important for the lower CHS responses with VM sensitization than skin sensitization.

Application of the dermal sensitization threshold concept to chemicals classified as high potency category for skin sensitization assessment of ingredients for consumer products.

Skin sensitization evaluation is a key part of the safety assessment of ingredients in consumer products, which may have skin sensitizing potential. The dermal sensitization threshold (DST) concept, which is based on the concept of the thresholds of toxicological concern, has been proposed for the risk assessment of chemicals to which skin exposure is very low level. There is negligible risk of skin sensitization if a skin exposure level for the substance of interest was below the reactive DST which would protect against 95% of protein-reactive chemicals. For the remaining 5%, the substance with the defined knowledge of chemical structure (i.e., High Potency Category (HPC) rules) needs to be excluded from the application. However, the DST value for HPC chemicals has not yet been proposed. In this study, we calculated the 95th percentile probabilities estimate from distributions of skin sensitization potency data and derived a novel DST for HPC chemicals (HPC DST) of 1.5 µg/cm2. This value presents a useful default approach for unidentified substances in ingredients considering, as a worst-case scenario, that the unidentified compound may be a potent skin sensitizer. Finally, we developed a novel risk assessment workflow incorporating the HPC DST along with the previously published DSTs.

Adjustment of a no expected sensitization induction level derived from Bayesian network integrated testing strategy for skin sensitization risk assessment.

Skin sensitization is a key adverse effect to be addressed during hazard identification and risk assessment of chemicals, because it is the first step in the development of allergic contact dermatitis. Multiple non-animal testing strategies incorporating in vitro tests and in silico tools have achieved good predictivities when compared with murine local lymph node assay (LLNA). The binary test battery of KeratinoSensTM and h-CLAT could be used to classify non-sensitizers as the first part of bottom-up approach. However, the quantitative risk assessment for sensitizing chemicals requires a No Expected Sensitization Induction Level (NESIL), the dose not expected to induce skin sensitization in humans. We used Bayesian network integrated testing strategy (BN ITS-3) for chemical potency classification. BN ITS-3 predictions were performed without a pre-processing step (selecting data from their physic-chemical applicability domains) or post-processing step (Michael acceptor chemistry correction), neither of which necessarily improve prediction accuracy. For chemicals within newly defined applicability domain, all under-predictions fell within one potency class when compared with LLNA results, indicating no chemicals that were incorrectly classified by more than one class. Considering the potential under-prediction by one class, a worst case value to each class from BN ITS-3 was used to derive a NESIL. When in vivo and human data from suitable analogs cannot be used to estimate the uncertainty, adjusting the NESIL derived from BN ITS-3 may help perform skin sensitization risk assessment. The overall workflow for risk assessment was demonstrated by incorporating the binary test battery of KeratinoSensTM and h-CLAT.

Sensitivity of KeratinoSensTM and h-CLAT for detecting minute amounts of sensitizers to evaluate botanical extract.

Cosmetic ingredients are often complex mixtures from natural sources such as botanical extracts that might contain minute amounts of constituents with sensitizing potential. The sensitivity of in vitro skin sensitization test methods such as KeratinoSensTM and h-CLAT for the detection of minute amounts of sensitizer in mixtures remains unclear. In this study, we assessed the detection sensitivity of the binary test battery comprising KeratinoSensTM and h-CLAT for minute amounts of sensitizers by comparing the LLNA EC3 (estimated concentration of a substance expected to produce a stimulation index of 3) values to the minimum detection concentrations (MDCs) exceeding the positive criteria for each of the two in vitro test methods. 146 sensitizers with both sets of in vitro data and LLNA data were used. MDC values for KeratinoSensTM and h-CLAT were calculated from exposure concentrations exceeding positive criteria for each in vitro test method (EC1.5 and minimum induction thresholds, respectively). The dilution rate used to expose culture medium was also considered. For 86% of analyzed sensitizers, the in vitro test methods showed MDC values lower than LLNA EC3 values, suggesting that the binary test battery with KeratinoSensTM and h-CLAT have greater sensitivity for detection of minute amounts of sensitizer than LLNA. These results suggest the high applicability of KeratinoSensTM and h-CLAT for detecting skin sensitizing constituents present in botanical extract.

The dermal sensitization threshold (DST) approach for mixtures evaluated as negative in in vitro test methods; mixture DST.

Cosmetic ingredients often comprise complex mixtures, such as botanical extracts, which may contain skin sensitizing constituents. In our previous study for the sensitivity of the evaluations of skin sensitizing constituents in mixtures using the binary in vitro test battery with KeratinoSensTM and h-CLAT, some sensitizers showed higher detection limits in in vitro test methods than in murine local lymph node assays (LLNA). Thus, to minimize the uncertainty associated with decreased sensitivity for these sensitizers, a risk assessment strategy was developed for mixtures with negative results from the binary test battery. Assuming that the no expected sensitization induction level of mixtures (mixture NESIL) can be derived for mixtures with negative in vitro test results, we assessed 146 sensitizers with in vitro and LLNA data according to the assumption of indeterminate constituents in mixtures. Finally, we calculated 95th percentile probabilities of mixture NESILs and derived dermal sensitization thresholds for mixtures (mixture DST) with negative in vitro test results of 6010 µg/cm2. Feasibility studies indicated that this approach was practical for risk assessments of products in the cosmetic industry. This approach would be a novel risk assessment strategy for incorporating the DST approach and information from in vitro test methods.

Binary test battery with KeratinoSens™ and h-CLAT as part of a bottom-up approach for skin sensitization hazard prediction.

Skin sensitization is one of the key safety endpoints for chemicals applied directly to the skin. Several integrated testing strategies (ITS) using multiple non-animal test methods have been developed to accurately evaluate the sensitizing potential of chemicals, but there is no regulatory-accepted ITS to classify a chemical as a non-sensitizer. In this study, the predictive performance of a binary test battery with KeratinoSens™ and h-CLAT compared to the local lymph node assay (LLNA) and human data was examined using comprehensive dataset of 203 chemicals. When two negative results indicate a non-sensitizer, the binary test battery provided sensitivity of 93.4% or 94.4% compared with the LLNA or human data. Taking into account the predictive limitations (i.e. high log Kow, pre-/pro-haptens and acyl transfer agents (or amine-reactive)), the binary test battery had extremely high sensitivity comparable to that of the 3 out of 3 ITS where three negative results of the DPRA, KeratinoSens™ and h-CLAT indicate a non-sensitizer. Therefore, the data from KeratinoSens™ or h-CLAT may provide partly redundant information on the molecular initiating event derived from DPRA. Taken together, the binary test battery of KeratinoSens™ and h-CLAT could be used as part of a bottom-up approach for skin sensitization hazard prediction.

Densified electrochemical sensors based on local redox cycling between vertically separated electrodes in substrate generation/chip collection and extended feedback modes.

A new local redox cycling-based electrochemical (LRC-EC) device integrated with many electrochemical sensors has been developed into a small chip device. The LRC-EC chip device was successfully applied for detection of alkaline phosphatase and horseradish peroxidase activity in substrate generation/chip collection (SG/CC) and extended feedback modes, respectively. The new imaging approach with extended feedback mode was particularly effective for sharpening of the image, because this mode uses feedback signals and minimizes the undesired influence of diffusion. The LRC-EC chip device is considered to be a useful tool for bioanalysis.

Noninvasive measurement of alkaline phosphatase activity in embryoid bodies and coculture spheroids with scanning electrochemical microscopy.

Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.

Noninvasive measurement of respiratory activity of mouse embryoid bodies and its correlation with mRNA levels of undifferentiation/differentiation markers.

Mouse embryoid bodies (mEBs) were evaluated in detail on the basis of respiratory activity and high-throughput quantitative reverse transcription-PCR (RT-qPCR) analysis. The hanging drop culture method was applied to prepare various sizes of mEBs ranging from 100 to 250 µm in radius by causing the aggregation of embryonic cells. The respiratory activity of individual mEBs was noninvasively measured using scanning electrochemical microscopy in a cone-shaped microwell. For gene expression analysis, we used 48.48 Dynamic Array chips (Fluidigm) integrating microfluidic circuits, which allowed high-throughput qPCR analysis in parallel. The respiratory activity of the mEBs that were cultured for 1 to 6 days could predict the mRNA levels of undifferentiation and differentiation markers. However, the sizes of the mEBs could also predict the gene expression of the undifferentiation/differentiation markers because the radii of the mEBs increased by more than 2-fold after incubation in hanging drop culture for 6 days. Next, mEBs with identical sample sizes were evaluated for respiratory activity and gene expression. For mEBs cultured at 1500 cells per droplet for 3 days, the respiratory activity was negatively correlated with the mRNA levels of pluripotent markers such as Nanog and Sox2. Many differentiation markers were positively correlated with the respiratory activity. However, there was no significant difference in respiration activity between the beating and nonbeating samples on day 3. Finally, principal component analysis (PCA) confirmed the relationship between respiratory activity and the mRNA levels of undifferentiation/differentiation markers.

LSI-based amperometric sensor for real-time monitoring of embryoid bodies.

A large scale integration (LSI)-based amperometric sensor is used for electrochemical evaluation and real-time monitoring of the alkaline phosphatase (ALP) activity of mouse embryoid bodies (EBs). EBs were prepared by the hanging drop culture of embryonic stem (ES) cells. The ALP activity of EBs with various sizes was electrochemically detected at 400 measurement points on a Bio-LSI chip. The electrochemical measurements revealed that the relative ALP activity was low for large EBs and decreased with progress of the differentiation level of the ES cells. The ALP activity of the EBs was successfully monitored in real time for 3.5h, and their ALP activity in a glucose-free buffer decreased after 2h. To the best of our knowledge, this is the first report on the application of an LSI-based amperometric sensor for real-time cell monitoring over 3h. The chip is expected to be useful for the evaluation of cell activities.

Electrochemical detection for dynamic analyses of a redox component in droplets using a local redox cycling-based electrochemical (LRC-EC) chip device.

This report describes the electrochemical detection of a redox component in droplets using a local redox cycling-based electrochemical (LRC-EC) chip device consisting of 256 sensors. The time-course analyses showed that the redox compound in the droplet was dynamically changed during droplet evaporation or mass transfer through a water/oil interface.