CycLing and EducATion (CLEAT): protocol for a single centre randomised controlled trial of a cycling and education intervention versus standard physiotherapy care for the treatment of hip osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a chronic degenerative joint disorder for which there is no known cure. Non-surgical management for people with mild-to-moderate hip OA focuses mainly on alleviating pain and maximising function via the National Institute for Health and Care Excellence (NICE) recommended combination of education and advice, exercise, and, where appropriate, weight loss. The CHAIN (Cycling against Hip pAIN) intervention is a group cycling and education intervention conceived as a way of implementing the NICE guidance. METHODS: CycLing and EducATion (CLEAT) is a pragmatic, two parallel arm, randomised controlled trial comparing CHAIN with standard physiotherapy care for the treatment of mild-to-moderate hip OA. We will recruit 256 participants referred to the local NHS physiotherapy department over a 24-month recruitment period. Participants diagnosed with hip OA according to NICE guidance and meeting the criteria for GP exercise referral will be eligible to participate. Primary outcome is the difference in Hip Disability and Osteoarthritis Outcome Score (HOOS) function, daily living subscale between those receiving CHAIN and standard physiotherapy care. Secondary outcomes include performance-based functional measures (40 m walking, 30s chair stand and stair climb tests), ability for patient to self-care (patient activation measure) and self-reported health-related resource use including primary and secondary care contacts. The primary economic endpoint is the number of quality adjusted life years (QALYs) at 24 weeks follow-up. The study is funded by the National Institute for Health Research, Research for Patient Benefit PB-PG-0816-20033. DISCUSSION: The literature identifies a lack of high-quality trials which inform on the content and design of education and exercise in the treatment of patients with hip OA and explore cost-effectiveness. CLEAT is a pragmatic trial which seeks to build further evidence of the clinical benefits of the CHAIN intervention compared to standard physiotherapy care within a randomised, controlled trial setting, and examine its cost-effectiveness. TRIAL REGISTRATION NUMBER: ISRCTN19778222. Protocol v4.1, 24th October 2022.

Proteomic Profiling of Archived Tissue of Primary Melanoma Identifies Proteins Associated with Metastasis.

Formalin-fixed paraffin embedded (FFPE) clinical tissues represent an abundant and unique resource for translational proteomic studies. In the US, melanoma is the 5th and 6th most common cancer in men and women, respectively, affecting over 230,000 people annually and metastasising in 5-15% of cases. Median survival time for distant metastatic melanoma is 6-9 months with a 5-year-survival of < 15%. In this study, 24 primary FFPE tumours which have metastasised (P-M) and 24 primary FFPE tumours which did not metastasise (P-NM) were subjected to proteomic profiling. In total, 2750 proteins were identified, of which 16 were significantly differentially expressed. Analysis of TCGA data demonstrated that expression of the genes encoding for 6 of these 16 proteins had a significant effect on survival in cutaneous melanoma. Pathway analysis of the proteomics data revealed mechanisms likely involved in the process of melanoma metastasis, including cytoskeleton rearrangement, extracellular changes and immune system alterations. A machine learning prediction model scoring an AUC of 0.922, based on these 16 differentially expressed proteins was able to accurately classify samples into P-M and P-NM. This study has identified potential biomarkers and key processes relating to melanoma metastasis using archived clinical samples, providing a basis for future studies in larger cohorts.

Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells.

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-ω. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells.

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10µM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.

Proteomic analysis of haptenation by skin sensitisers: Diphencyprone and ethyl acrylate.

The potential risk of skin sensitisation, associated with the development of allergic contact dermatitis (ACD), is a consideration in the safety assessment of new ingredients for use in personal care products. Protein haptenation in skin by sensitising chemicals is the molecular initiating event causative of skin sensitisation. Current methods for monitoring skin sensitisation rely on limited reactivity assays, motivating interest in the development of proteomic approaches to characterise the skin haptenome. Increasing our mechanistic understanding of skin sensitisation and ACD using proteomics presents an opportunity to develop non-animal predictive methods and/or risk assessment approaches. Previously, we have used a novel stable isotope labelling approach combined with data independent mass spectrometry (HDMSE) to characterise the haptenome for a number of well-known sensitisers. We have now extended this work by characterising the haptenome of the sensitisers Diphenylcyclopropenone (DPCP) and Ethyl Acrylate (EA) with the model protein Human Serum Albumin (HSA) and the complex lysates of the skin keratinocyte, HaCaT cell line. We show that haptenation in complex nucleophilic models is not random, but a specific, low level and reproducible event. Proteomic analysis extends our understanding of sensitiser reactivity beyond simple reactivity assays and offers a route to monitoring haptenation in living cells.

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

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.

Serum proteomic fingerprinting discriminates between clinical stages and predicts disease progression in melanoma patients.

PURPOSE: Currently known serum biomarkers do not predict clinical outcome in melanoma. S100-beta is widely established as a reliable prognostic indicator in patients with advanced metastatic disease but is of limited predictive value in tumor-free patients. This study was aimed to determine whether molecular profiling of the serum proteome could discriminate between early- and late-stage melanoma and predict disease progression. PATIENTS AND METHODS: Two hundred five serum samples from 101 early-stage (American Joint Committee on Cancer [AJCC] stage I) and 104 advanced stage (AJCC stage IV) melanoma patients were analyzed by matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (ToF; MALDI-ToF) mass spectrometry utilizing protein chip technology and artificial neural networks (ANN). Serum samples from 55 additional patients after complete dissection of regional lymph node metastases (AJCC stage III), with 28 of 55 patients relapsing within the first year of follow-up, were analyzed in an attempt to predict disease recurrence. Serum S100-beta was measured using a sandwich immunoluminometric assay. RESULTS: Analysis of 205 stage I/IV serum samples, utilizing a training set of 94 of 205 and a test set of 15 of 205 samples for 32 different ANN models, revealed correct stage assignment in 84 (88%) of 96 of a blind set of 96 of 205 serum samples. Forty-four (80%) of 55 stage III serum samples could be correctly assigned as progressors or nonprogressors using random sample cross-validation statistical methodologies. Twenty-three (82%) of 28 stage III progressors were correctly identified by MALDI-ToF combined with ANN, whereas only six (21%) of 28 could be detected by S100-beta. CONCLUSION: Validation of these findings may enable proteomic profiling to become a valuable tool for identifying high-risk melanoma patients eligible for adjuvant therapeutic interventions.

Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.

The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.

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.

A qualitative and quantitative proteomic study of human microdialysate and the cutaneous response to injury.

The extracellular fluid space is the site of intercellular communication and represents an important source of mediators that can shed light on the parenchymal environment. Sampling of this compartment using continuous microdialysis allows assessment of the temporal changes in extracellular mediators involved in tissue homeostasis and disease processes. However, novel biomarker identification is limited by the current need to utilize specific, targeted molecular assays. The aim of our study was to explore the use of qualitative and quantitative proteomic approaches to define the protein content of dermal dialysate. Timed dermal dialysate samples were collected from healthy human volunteers for 5 h following probe insertion, using a 3,000-kDa MWCO membrane perfused at a rate of 3 µl/min. Dialysate proteins were identified using GeLC-MS/MS and iTRAQ approaches and functions assigned according to the Gene Ontology classification system. More than 80 proteins (size range 11-516 kDa) originating from both extracellular and intracellular fluid space were identified using the qualitative approach of GeLC-MS/MS. Quantitative iTRAQ data were obtained for 27 proteins with relative change ratios between consecutive timed samples showing changes of >1.5-fold. Interstitial proteins can be identified and measured using shotgun proteomic techniques and changes detected during the acute inflammatory response. Our findings provide a platform from which to explore novel protein biomarkers and their modulation in health and disease.