Sample 11
Homo sapiens
Protocols: In vivo patch test of peptide combination: The peptide formulation was constituted by dissolving 1% w/w of a 50:50 mixture of P1 (500ppm) and P7 (500ppm) in solvent excipients added to a simple oil in water cosmetic emulsion containing water, glycerine and thickened with ammonium acryloyldimethyltaurate /VP Copolymer, xanthan gum and preserved with phenoxyethanolphenoxyethanol; giving a final total peptide concentrations of either 10ppm or 30ppm. A vehicle was also formulated with identical composition, including the excipients but without the peptides. Eight healthy, white but photoaged volunteers with visible signs of photoageing were recruited (Fitzpatrick skin phototype I-III; 3 male, 5 female, age range: 72-84 years old) and subjected to an extended 12-day patch test as described by PMID: 18070204. Only three of these volunteers were studied by RNA-seq here. The assay uses occlusion to carry actives into the skin, so mimicking long-term use PMID: 18070204. The peptide formulation was applied for twelve-days, with reapplication every 4-days, alongside a vehicle control and an occluded but untreated area (baseline). The vehicle and peptide formulations (30 μl) were applied separately to the extensor photoaged forearm under standard 6mm diameter Finn Chambers (Epitest Ltd). ATRA (0.025%; Retin-A) was applied to skin for the final 4-days only (to avoid potential complications of irritation from extended occlusion) as a positive control. On day 12, the Finn chambers were removed and 3mm punch biopsies obtained under 1% lignocaine local anaesthesia from each test site. Biopsies were embedded in optimal cutting temperature compound (Miles Laboratories, Elkhart, IN, USA), snap frozen in liquid nitrogen and stored at –80°C, prior to cutting at 10μm thickness. The study was conducted in accordance with the principles of the Declaration of Helsinki, with written informed consent (Manchester University Research Ethics Committee reference: 13677). Immunofluorescence analysis was performed to assess fibrillin microfibril abundance (a dermal repair marker). For extraction of biopsy RNA, once obtained, biopsies were immediately stored in RNAprotect Tissue Reagent (76104, Qiagen) to stabilise RNA. Biopsies were incubated at 37°C for one hour with 20mM EDTA, then the epidermis was separated from the dermis using forceps. Epidermal and dermal samples were then processed for RNA extraction using the RNeasy Fibrous Tissue Mini Kit (74704, Qiagen) according to manufacturer's instructions. Briefly, samples were removed from the RNAprotect Tissue Reagent, and added to appropriate volumes of buffer RLT and homogenised at 50 Hz for 5 minutes using the TissueLyser LT (Qiagen), until all tissue was homogenised. Samples were then incubated with Proteinase K at 55°C for 10 minutes, prior to centrifugation to remove debris. A half-volume of ethanol was added, then samples were transferred to spin columns, where they were washed with buffer RW1. DNase I was then added to spin columns, and incubated at room temperature for 15 minutes. Samples underwent three further wash steps, once with buffer RW1, and the final two with buffer RPE. RNAse-free water was then used to elute RNA samples. Epidermal and dermal RNA samples were then combined and further processed to concentrate RNA and remove protein contaminants using RNeasy Mini Kit as above. Epidermal and dermal samples were then processed for RNA extraction using the RNeasy Fibrous Tissue Mini Kit (74704, Qiagen) according to manufacturer's instructions. Briefly, samples were removed from the RNAprotect Tissue Reagent, and added to appropriate volumes of buffer RLT and homogenised at 50 Hz for 5 minutes using the TissueLyser LT (Qiagen), until all tissue was homogenised. Samples were then incubated with Proteinase K at 55°C for 10 minutes, prior to centrifugation to remove debris. A half-volume of ethanol was added, then samples were transferred to spin columns, where they were washed with buffer RW1. DNase I was then added to spin columns, and incubated at room temperature for 15 minutes. Samples underwent three further wash steps, once with buffer RW1, and the final two with buffer RPE. RNAse-free water was then used to elute RNA samples. Epidermal and dermal RNA samples were then combined and further processed to concentrate RNA and remove protein contaminants using RNeasy Mini Kit as above. Stranded mRNA assay (Illumina)