An in vitro method for detecting chemical sensitization using human reconstructed skin models and its applicability to cosmetic, pharmaceutical, and medical device safety testing.

Chemical sensitization is a serious condition caused by small reactive molecules and is characterized by a delayed type hypersensitivity known as allergic contact dermatitis (ACD). Contact with these molecules via dermal exposure represent a significant concern for chemical manufacturers. Recent legislation in the EU has created the need to develop non-animal alternative methods for many routine safety studies including sensitization. Although most of the alternative research has focused on pure chemicals that possess reasonable solubility properties, it is important for any successful in vitro method to have the ability to test compounds with low aqueous solubility. This is especially true for the medical device industry where device extracts must be prepared in both polar and non-polar vehicles in order to evaluate chemical sensitization. The aim of this research was to demonstrate the functionality and applicability of the human reconstituted skin models (MatTek Epiderm(®) and SkinEthic RHE) as a test system for the evaluation of chemical sensitization and its potential use for medical device testing. In addition, the development of the human 3D skin model should allow the in vitro sensitization assay to be used for finished product testing in the personal care, cosmetics, and pharmaceutical industries. This approach combines solubility, chemical reactivity, cytotoxicity, and activation of the Nrf2/ARE expression pathway to identify and categorize chemical sensitizers. Known chemical sensitizers representing extreme/strong-, moderate-, weak-, and non-sensitizing potency categories were first evaluated in the skin models at six exposure concentrations ranging from 0.1 to 2500 µM for 24 h. The expression of eight Nrf2/ARE, one AhR/XRE and two Nrf1/MRE controlled gene were measured by qRT-PCR. The fold-induction at each exposure concentration was combined with reactivity and cytotoxicity data to determine the sensitization potential. The results demonstrated that both the MatTek and SkinEthic models performed in a manner consistent with data previously reported with the human keratinocyte (HaCaT) cell line. The system was tested further by evaluating chemicals known to be associated with the manufacture of medical devices. In all cases, the human skin models performed as well or better than the HaCaT cell model previously evaluated. In addition, this study identifies a clear unifying trigger that controls both the Nrf2/ARE pathway and essential biochemical events required for the development of ACD. Finally, this study has demonstrated that by utilizing human reconstructed skin models, it is possible to evaluate non-polar extracts from medical devices and low solubility finished products.

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitization potency prediction. The results of the first phase ­ systematic evaluation of 16 test methods ­ are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data,potential for throughput, transferability and accessibility in cooperation with the test method developers.A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy ­ combined with bioavailability and skin metabolism data and exposure consideration ­ is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA-/-) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1). developed a novel methodology to detect pericellular proteolytic function, (2). demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3). demonstrated its usefulness in real-time studies of cell migration, and (4). showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.