Maja Aleksic

Reactivity Profiling: Covalent Modification of Single Nucleophile Peptides for Skin Sensitization Risk Assessment

The molecular basis of chemical allergy is rooted in the ability of an allergen (hapten) to modify endogenous proteins. This mechanistic understanding aided development of screening assays which generate reproducible quantitative and qualitative reactivity data. Such assays use model peptides with a limited number and type of protein nucleophiles, and the data does not reflect the specificity, variety, and complexity of hapten interactions with multiple nucleophiles. Building on these developments, we extended the standardized approach to maximize the type and the amount of information that can be derived from an in chemico assay. We used a panel of six single nucleophile peptides and individually optimized the incubation conditions to favor chemical modification. Employing liquid chromatography tandem mass spectrometry (LC-MS/MS) technique, we simultaneously obtained multiple quantitative and qualitative measurements (% peptide depletion, adducts formation, and peptide dimerization for Cys-containing peptide). Using these methods, we obtained reactivity data for 36 chemicals of known skin sensitizing potency. By optimizing incubation conditions, we ensured detection of all reactive chemicals. We explored the LC-MS/MS approach to generate kinetic data for 10 chemicals allowing further characterization of reactivity and a potentially more robust quantitative reactivity descriptor. Our ultimate aim is to integrate this dataset with available physicochemical data and outputs from other predictive assays, all addressing different key steps in the induction of sensitization, to help us make decisions about the safe use of chemicals without using animal tests. The epidermal protein target sites, modification of which may be immunogenic and lead to induction of skin sensitization, are currently unknown. Increasing the understanding of this process may help further refine in chemico reactivity assays as well as aid the interpretation of the reactivity data.

Characterization of p-Phenylenediamine-Albumin Binding Sites and T-Cell Responses to Hapten-Modified Protein

Exposure to p-phenylenediamine (PPD) is associated with the development of T-cell-mediated allergic contact dermatitis. The purpose of this study was to define the nature of the interaction of PPD with the protein and the antigenic determinant that stimulates T cells. Mass spectrometry was employed to show that PPD oxidation products bind irreversibly to cysteine (Cys, position 34) in human serum albumin (HSA). A modified tryptic peptide was characterized with an increase in mass of 106 Da, corresponding to the addition of PPD and not to the secondary products of self conjugation. Lymphocytes from 10 PPD-allergic patients, but not tolerant/naive individuals, were stimulated with PPD and PPD-modified HSA. A total of 70 PPD-specific and 10 PPD-HSA-specific CD4+, CD8+, and CD4+CD8+, Th2-secreting T-cell clones were generated from three allergic patients. In total, 40 clones were stimulated with both PPD and PPD-modified HSA. PPD-modified HSA triggered T-cell responses through a classical hapten mechanism involving processing. Presentation of PPD to several clones was dependent on protein complex formation (42 out of 48) and processing (32 out of 68); however, 12% of clones were triggered with PPD directly. These data identify Cys as the single target for PPD-HSA binding, and show that PPD protein adducts are antigenic determinants in patients with contact dermatitis.

A mechanistic investigation into the irreversible protein binding and antigenicity of p-phenylenediamine.

Exposure to the skin sensitizer p-phenylenediamine (PPD) is associated with allergic contact dermatitis; however, the ability of PPD to modify protein has not been fully investigated. The aims of this study were to characterize the reactions of PPD and the structurally related chemical 2,5-dimethyl-1,4-benzoquinonediamine with model nucleophiles, a synthetic peptide (DS3) containing each of the naturally occurring amino acids and His-tagged glutathione-S-transferase pi (GSTP), and to explore the effect of dimethyl substitution on PPD-specific T-cell responses using lymphocytes from allergic patients. The reductive soft nucleophiles N-acetyl cysteine and glutathione prevented PPD self-conjugation reactions and Bandrowskis base formation, but no adducts were detected. N-Acetyl lysine, a hard nucleophile, did not alter the rate of PPD degradation or form PPD adducts. With PPD and 2,5-dimethyl-1,4-benzoquinonediamine, only cysteine was targeted in the DS3 peptide. PPD and 2,5-dimethyl-1,4-benzoquinonediamine were also found to selectively modify the reactive Cys 47 residue of GSTP, which has a pK(a) of 3.5-4.2 and therefore exists in a largely protonated form. Glutathione formed mixed disulfides with the DS3 peptide, reducing levels of PPD binding. Lymphocytes from PPD allergic patients proliferated in the presence of PPD but not with 2,5-dimethyl-1,4-benzoquinonediamine. These results reveal that PPD and 2,5-dimethyl-1,4-benzoquinonediamine bind selectively to specific cysteine residues in peptides and proteins. Lymphocytes from PPD allergic patients were capable of discriminating between the different haptenic structures, suggesting that the hapten, but not the peptide moiety associated with MHC, is an important determinant for T-cell recognition.

Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin

Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization.

Proteomic analysis of the human skin proteome after in vivo treatment with sodium dodecyl sulphate.

Background Skin has a variety of functions that are incompletely understood at the molecular level. As the most accessible tissue in the body it often reveals the first signs of inflammation or infection and also represents a potentially valuable source of biomarkers for several diseases. In this study we surveyed the skin proteome qualitatively using gel electrophoresis, liquid chromatography tandem mass spectrometry (GeLC-MS/MS) and quantitatively using an isobaric tagging strategy (iTRAQ) to characterise the response of human skin following exposure to sodium dodecyl sulphate (SDS). Results A total of 653 skin proteins were assigned, 159 of which were identified using GeLC-MS/MS and 616 using iTRAQ, representing the most comprehensive proteomic study in human skin tissue. Statistical analysis of the available iTRAQ data did not reveal any significant differences in the measured skin proteome after 4 hours exposure to the model irritant SDS. Conclusions This study represents the first step in defining the critical response to an irritant at the level of the proteome and provides a valuable resource for further studies at the later stages of irritant exposure.

THE ROLE OF NON-COVALENT PROTEIN BINDING IN SKIN SENSITISATION POTENCY OF CHEMICALS

Skin sensitisation is a delayed hypersensitivity reaction caused by repeated exposure to common natural and synthetic chemical allergens. It is thought that small chemical sensitisers (haptens) are required to form a strong irreversible bond with a self protein/peptide and generate an immunogenic hapten–protein complex in order to be recognised by the immune system and stimulate T cell proliferation. The sensitisers are usually electrophilic chemicals that are directly reactive with proteins or reactive intermediates (metabolites) of chemically inert compounds (prohaptens). Sensitising chemicals are also capable of weak, non-covalent association with proteins and there is an ongoing debate about the role of weak interactions of chemicals and proteins in the chemistry of allergy. The non-covalent interactions are reversible and thus have a major impact on skin/epidermal bioavailability of chemical/reactive metabolites. We investigated the relationship between the relative level of non-covalent association to a model protein and their relative potencies as determined by the EC3 values in the murine local lymph node assay (LLNA) for a number of chemicals. Using human serum albumin as a model protein, we determined that no observable relationship exists between the two parameters for the chemicals tested. Therefore, at least for this model protein, non-covalent interactions appear not to be a key determinant of allergen potency.

Determination of Protein Haptenation by Chemical Sensitizers Within the Complexity of the Human Skin Proteome

Abstract Skin sensitization associated with the development of allergic contact dermatitis occurs via a number of specific key events at the cellular level. The molecular initiating event (MIE), the first in the sequence of these events, occurs after exposure of the skin to an electrophilic chemical, causing the irreversible haptenation of proteins within skin. Characterization of this MIE is a key step in elucidating the skin sensitization adverse outcome pathway and is essential to providing parameters for mathematical models to predict the capacity of a chemical to cause sensitization. As a first step to addressing this challenge, we have exposed complex protein lysates from a keratinocyte cell line and human skin tissue with a range of well characterized sensitizers, including dinitrochlorobenzene, 5-chloro-2-methylisothiazol-3-one, cinnamaldehyde, and the non (or weak) sensitizer 6-methyl coumarin. Using a novel stable isotope labeling approach combined with ion mobility-assisted data independent mass spectrometry (HDMSE), we have characterized the haptenome for these sensitizers. Although a significant proportion of highly abundant proteins were haptenated, we also observed the haptenation of low abundant proteins by all 3 of the chemical sensitizers tested, indicating that within a complex protein background, protein abundance is not the sole determinant driving haptenation, highlighting a relationship to tertiary protein structure and the amino acid specificity of these chemical sensitizers and sensitizer potency.

A bioinformatic tool for detailed interrogation of multiple iTRAQ datasets

literature to determine the most appropriate trigger in the numerous clinical situations that may be encountered. The transfusion of plasma has been studied more often, and, consistently, no increase in hemorhagic risk has been seen during a variety of interventional procedures up to at least a prothrombin time or activated partial thromboplastic time 1.5 times the midpoint of the reference range. A common practice of attempting to reduce the INR to 1.5 is without scientific justification. Furthermore, few studies have shown evidence that correcting an abnormal laboratory coagulation parameter improved patient outcome. Prophylactic platelet transfusion at 10000/mL has been shown to provide the same clinical effect as transfusion at 20000/mL. Children requiring lumbar punctures tolerated this procedure without increased risk of hemorrhage down to a platelet count of 10000/mL. However, the value of prophylactic platelet transfusion at all has not been assessed since before the era of providing leukoreduced components to multiply transfused patients, and the largest study before leukoreduction failed to show an overall benefit versus transfusing only for a direct therapeutic purpose. Therefore, while we have accumulated some useful data to indicate where transfusions may – or may not – be helpful in certain circumstances, we are left broad areas of clinical situations and laboratory parameters across which we must interpolate the data of clinical trials in order to estimate the value of transfusion. More studies are clearly needed. In the interim, careful assessment of a patient’s co-morbidities and a sober consideration of likely benefits and true risks of a transfusion should always be undertaken.