Off to a good start? Review of the predictivity of reactivity methods modelling the molecular initiating event of skin sensitization.

The assessment of skin sensitizing properties of chemicals has moved away from animal methods to new approach methodologies (NAM), guided by qualitative mechanistic understanding operationalized in an adverse outcome pathway (AOP). As with any AOP, the molecular initiating event (MIE) of covalent binding of a chemical to skin proteins is particularly important. This MIE has been modelled by several test methods by measuring the reaction of a test chemical with model peptides in chemico. To better understand the similarities and differences, a data repository with publicly available data for the direct peptide reactivity assay (DPRA), amino acid derivative reactivity assay (ADRA) and kinetic DPRA (kDPRA), as well as the peroxidase peptide reactivity assay (PPRA) was assembled. The repository comprises 260 chemicals with animal and human reference data, data on four relevant physicochemical properties, and between 161 to 242 test chemical results per test method. First, an overview of the experimental conditions of the four test methods was compiled allowing to readily compare them. Second, data analyses demonstrated that the test methods' predictivity was consistently reduced for poorly watersoluble chemicals and that the DPRA and ADRA can be used interchangeably. It also revealed new categorization thresholds for the DPRA and ADRA that are potentially relevant for strategic uses. In summary, a detailed assessment of reactivity test methods is provided, highlighting their potential and limitations. The results presented are intended to stimulate scientific discussion around test methods modelling the MIE of the skin sensitization AOP.

Amending the U-SENS™ skin sensitization test method for interfering auto-fluorescent chemicals.

Limitations of the applicability domain of new approach methodologies (NAM) present a major challenge for the testing of cosmetic ingredients in Europe, as the regulation does not allow to resort to in vivo test method. Therefore, research focused on overcoming such limitations of established in vitro test methods is frequently conducted. Here, we address a limitation of the U-SENS™, an in vitro skin sensitization test method that addresses the key event 3 on activation of dendritic cells of the adverse outcome pathway (AOP) for skin sensitization. The applicability domain of the U-SENS™ excludes autofluorescent substances that can interfere with the measurement of the expression of CD86, i.e., the primary readout. An evaluation of several fluorochromes identified APC as most suitable for testing auto-fluorescent chemicals. Acceptance criteria were reproducibly met when using the APC-labelled antibody. Equivalent performance in terms of reproducibility and skin sensitisation hazard assessment of the standard FITC-labelled antibodies and the APC-labelled antibodies was demonstrated by testing 40 substances. Finally, the value of the expanded technical applicability domain was highlighted with a case study using sulfuretin. In conclusion, we successfully demonstrated the expansion of the U-SENS™ applicability domain to interfering auto-fluorescent chemicals by using APC-labelled antibodies.

Coordination of the health policy dialogue process in Guinea: pre- and post-Ebola.

BACKGROUND: Policy dialogue can be defined as an iterative process that involves a broad range of stakeholders discussing a particular issue with a concrete purpose in mind. Policy dialogue in health is increasingly being recognised by health stakeholders in developing countries, as an important process or mechanism for improving collaboration and harmonization in health and for developing comprehensive and evidence-based health sector strategies and plans. It is with this perspective in mind that Guinea, in 2013, started a policy dialogue process, engaging a plethora of actors to revise the country's national health policy and develop a new national health development plan (2015-2024). This study examines the coordination of the policy dialogue process in developing these key strategic governance documents of the Guinean health sector from the actors' perspective. METHODS: A qualitative case study approach was undertaken, comprising of interviews with key stakeholders who participated in the policy dialogue process. A review of the literature informed the development of a conceptual framework and the data collection survey questionnaire. The results were analysed both inductively and deductively. RESULTS: A total of 22 out of 32 individuals were interviewed. The results suggest both areas of strengths and weaknesses in the coordination of the policy dialogue process in Guinea. The aspects of good coordination observed were the iterative nature of the dialogue and the availability of neutral and well-experienced facilitators. Weak coordination was perceived through the unavailability of supporting documentation, time and financial constraints experienced during the dialogue process. The onset of the Ebola epidemic in Guinea impacted on coordination dynamics by causing a slowdown of its activities and then its virtual halt. CONCLUSIONS: The findings herein highlight the need for policy dialogue coordination structures to have the necessary administrative and institutional support to facilitate their effective functioning. The findings also point to the need for further research on the practical and operational aspects of national dialogue coordination structures to determine how to best strengthen their capacities.

Guiding principles for the implementation of non-animal safety assessment approaches for cosmetics: skin sensitisation.

Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the 'gold standard' test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.

Metallic haptens induce differential phenotype of human dendritic cells through activation of mitogen-activated protein kinase and NF-kappaB pathways.

Dendritic cells (DCs) play a major role in the regulation of immune responses to a variety of antigens (Ag) and haptens which participate in the process of DC maturation. Indeed, metallic haptens are able to induce DC maturation in vitro but the mechanism of this maturation is not well understood. We and others have already shown that NiSO(4) activates p38 mitogen-activated protein kinases (p38MAPK), c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and the transcription factor NF-kappaB during the early events of DCs maturation. However, the effect of other metallic haptens on DC maturation is still poorly understood. In the present study, using dendritic cells derived from CD34(+) cord blood cells, we showed that both NiSO(4) and CoCl(2) induced the expression of CD86, CD83, HLA-DR and CD40 and the production of IL-6 in human DCs while K(2)Cr(2)O(7) induced only a slight upregulation of CD86. Interestingly, only NiSO(4) was able to induce the production of IL-12p40. NiSO(4) and CoCl(2) but not K(2)Cr(2)O(7) were able to activate the MAPK pathway and the transcription factor NF-kappaB. The role of MAPKs in metals-induced DC maturation was then evaluated using well-described pharmacological inhibitors. Our results suggest that p38MAPK activation regulates the expression of CD86 and CD83 induced by NiSO(4) while it only affects the expression of CD83 induced by CoCl(2). IL-6 production induced by NiSO(4) and CoCl(2) strongly depended on all MAPKs. IL-12p40 synthesis after NiSO(4) treatment was regulated by both p38MAPK and JNK pathways whereas ERK may play an inhibitory role. Our results show that both NiSO(4) and CoCl(2) activate similar signaling pathways that are playing different roles in DC maturation depending on the hapten used.

HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway.

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

NF-kappaB plays a major role in the maturation of human dendritic cells induced by NiSO(4) but not by DNCB.

Dendritic cell (DC) activation is a critical event for the induction of an immune response to haptens. Although signaling pathways such as mitogen-activated protein kinase (MAPK) family members have been reported to play a role in DC activation by haptens, little is known about the implication of the nuclear factor kappa B (NF-kappaB) pathway. In this work, we showed that NiSO(4) induced the expression of HLA-DR, CD83, CD86, and CD40 and the production of interleukin (IL)-8, IL-6, and IL-12p40 in human DCs, whereas DNCB induced mainly the expression of CD83 and CD86 and the production of IL-8. NiSO(4) but not DNCB was able to activate the degradation of IkappaB-alpha leading to the binding of the p65 subunit of NF-kappaB on specific DNA probes. Inhibition of the NF-kappaB pathway using BAY 11-7085 prevents both CD40 and HLA-DR expression and cytokine production induced by NiSO(4). However, BAY 11-7085 only partially inhibited CD86 and CD83 expression induced by NiSO(4). In addition, p38 MAPK and NF-kappaB were independently activated by NiSO(4) since SB203580 did not inhibit NF-kappaB activation by NiSO(4). Interestingly, we also showed that DNCB inhibited the degradation of IkappaB-alpha induced by tumor necrosis factor-alpha leading to alteration of CD40, HLA-DR, and CD83 expression but not of CD86 and CCR7. Extensive modifications of DC phenotype by NiSO(4) in comparison to DNCB are probably the consequence of NF-kappaB activation by NiSO(4) but not by DNCB.

Activation of U937 cells by contact sensitizers: CD86 expression is independent of apoptosis.

Among the different phenotypic changes induced by contact sensitizers in dendritic cells and myeloid cell lines, CD86 appears to be a consensus marker, since constantly described as systematically up-regulated. To evaluate the robustness of this marker, interference of cytotoxicity on CD86 expression was investigated in U937 myelomonocytic cell line. In this study, cytotoxicity observed at 48 hr (reading-time for CD86 expression) after treatment with DNCB, NiSO(4) and pPD was shown to result from apoptosis taking place at earlier time points. This allergen-induced apoptosis was at least partly caspase-dependent as demonstrated by caspase-3 activation in response to DNCB and NiSO(4) and inhibition of DNCB-induced apoptosis by Z-VAD-fmk. Inhibition of apoptosis did not modify the stimulation index of CD86 expression in DNCB-treated cells, indicating that apoptosis did not interfere with up-regulation of CD86 expression. In addition, similar CD86 expression level was found in DNCB-treated cells whether calculated from the whole non-necrotic cell population including apoptotic cells or from viable non-apoptotic cell population only. Altogether, these results brought evidence that the presence of cells engaged in death process are not a confusing factor for CD86 expression in response to contact sensitizers. They also pointed out apoptosis as another possible key marker of cellular response to contact sensitizers.