Project description:the Enhanced Epidermal Antigen Specific Immunotherapy Trial -1 (EEASI) study was a two-centre, open-label, uncontrolled, single-group first-in-human Phase 1A safety study of C19-A3 GNP peptide in individuals with T1D (https://clinicaltrials.gov/ct2/show/NCT02837094). The investigational medicinal product (IMP) was C19-A3 GNP (Midacore™), which comprises Midacore™ GNPs (Midatech Pharma Plc, Cardiff, UK)(1, 20) of a size of less than 5 nm, covalently coupled to an 18-amino acid human peptide, the sequence of which is identical to the residues from position 19 in the C-peptide of proinsulin through to position 3 on the A-chain of the same molecule (GSLQPLALEGSLQKRGIV). The peptide is synthesised with a linker to facilitate binding to the GNPs: 3-mercaptopropionyl-SLQPLALEGSLQKRGIV 2 acetate salt (disulfide bond). The chemical composition of the IMP contained a ratio of 4 C19-A3 peptides: 11 glucose C2: 29 glutathione ligands as determined by 1H-NMR (proton nuclear magnetic resonance). A typical batch contained: [C19-A3 peptide] = 1.33 mg/ml; [gold] = 5.5 mg/ml; [glucose linker] = 0.6 mg/ml; [glutathione linker] = 1.79 mg/ml. As the drug substance was diluted 1:7 to 1:10, depending on the content of C19-A3 peptide per particle, and as 50 μl of the diluted solution was administered to the study participants, this corresponded to C19-A3 peptide: 10 μg; gold: 39 μg; glucose linker: 4.3 μg; glutathione: 12.7 μg. Participants diagnosed with T1D and confirmed to possess the HLA-DRB1∗0401 genotype, were given three doses of C19-A3 GNP at 4-weekly intervals (weeks 0, 4, and 8) in the deltoid region of alternate arms (2 doses in one arm and 1 dose in the other arm) via CE-marked 600 μm length MicronJet600™ hollow microneedles (NanoPass Technologies Ltd. Israel) attached to a standard Luer-lock syringe. The single-dose given in 50 μl volume was equivalent to 10 μg of C19-A3 peptide. Punch skin biopsies of the local area were performed under aseptic conditions and under local anaesthetic (Lidocaine Hydrochloride Injection BP 2%, w/v), using a 6-mm sterile disposable biopsy punch. Following the biopsy, samples were divided with half immediately placed in 10% formalin and transported to the laboratory and the other half placed in RNA laterTM (Fisher Scientific, Loughborough, UK) for bulk RNA sequencing. Control skin samples were obtained from female patients aged 19–82 years, following mastectomy or breast reduction after informed consent. Skin without obvious pathological findings, which was surplus to diagnostic histopathology requirements, was used in the experiments. Control punch biopsy samples were treated in the same manner as described above. After cutting skin samples into small pieces, samples were homogenised and lysed by using Tissue Raptor II and QIAzol® Lysis protocol (QIAzol® Handbook 2021, QIAgen, Crawley, UK). The quality of RNA was routinely assessed by determining the A260:A280 ratio using NanoDrop2000 (Thermo Scientific, UK). RNA libraries were created using TruSeq stranded Total RNA with ribozero GOLD (illumina, UK) and sequenced on an illumina HiSeq 2500 platform with 2 x 75 bp reads

Project description:A systems toxicology investigation comparing and integrating transcriptomic and proteomic results was conducted to pursue a holistic understanding of the effects of the explosives degradation product 2-amino-4,6-dinitrotoluene (2A-DNT) dosing on the health of the wildlife bird model, Northern bobwhite (Colinus virginianus). A subchronic 60d toxicology bioassay was leveraged where both sexes were dosed via daily gavage with 0, 3, 14, or 30 mg/kg-d 2-ADNT. Effects on global transcript expression were investigated in liver and kidney tissue using custom microarrays for C. virginianus in both sexes at all doses, while effects on the proteome expression were investigated in liver for both sexes and kidney in males, all at the 30 mg/kg-d dose. As expected, transcript expression was not directly indicative of protein expression in response to 2A-DNT. However, a high degree of correspondence was observed among platforms when investigating higher-order functional responses including statistically enriched gene networks and canonical pathways, especially when connected to toxicological outcomes of 2A-DNT exposure. Specifically, comparison of gene networks statistically enriched from differentially expressed transcripts and for proteins showed common responses including inhibition of programmed cell death and arrest of cell cycle in liver tissues at 2A-DNT doses that caused liver necrosis and death in females. Additionally, both transcript and protein expression in liver tissue was indicative of induced phase I and II xenobiotic metabolism potentially as a mechanism to detoxify and excrete 2A-DNT. Nuclear signaling, transcript expression and protein expression assays each implicated PPAR nuclear signaling as a primary molecular target in the 2A-DNT exposure with significant downstream enrichment of PPAR-regulated pathways including various lipid metabolic pathways and gluconeogenesis suggesting impaired bioenergetic potential. Although the relative expression of transcripts and proteins within enriched pathways was at times divergent, transcript expression assays identified many critical metabolic pathways involved in 2A-DNT toxicity as well as impaired PPAR signaling, a key molecular initiating event known to be affected in di- and tri-nitrotoluene exposures.

Project description:A systems toxicology investigation comparing and integrating transcriptomic and proteomic results was conducted to pursue a holistic understanding of the effects of the explosives degradation product 2-amino-4,6-dinitrotoluene (2A-DNT) dosing on the health of the wildlife bird model, Northern bobwhite (Colinus virginianus). A subchronic 60d toxicology bioassay was leveraged where both sexes were dosed via daily gavage with 0, 3, 14, or 30 mg/kg-d 2-ADNT. Effects on global transcript expression were investigated in liver and kidney tissue using custom microarrays for C. virginianus in both sexes at all doses, while effects on the proteome expression were investigated in liver for both sexes and kidney in males, all at the 30 mg/kg-d dose. As expected, transcript expression was not directly indicative of protein expression in response to 2A-DNT. However, a high degree of correspondence was observed among platforms when investigating higher-order functional responses including statistically enriched gene networks and canonical pathways, especially when connected to toxicological outcomes of 2A-DNT exposure. Specifically, comparison of gene networks statistically enriched from differentially expressed transcripts and for proteins showed common responses including inhibition of programmed cell death and arrest of cell cycle in liver tissues at 2A-DNT doses that caused liver necrosis and death in females. Additionally, both transcript and protein expression in liver tissue was indicative of induced phase I and II xenobiotic metabolism potentially as a mechanism to detoxify and excrete 2A-DNT. Nuclear signaling, transcript expression and protein expression assays each implicated PPAR nuclear signaling as a primary molecular target in the 2A-DNT exposure with significant downstream enrichment of PPAR-regulated pathways including various lipid metabolic pathways and gluconeogenesis suggesting impaired bioenergetic potential. Although the relative expression of transcripts and proteins within enriched pathways was at times divergent, transcript expression assays identified many critical metabolic pathways involved in 2A-DNT toxicity as well as impaired PPAR signaling, a key molecular initiating event known to be affected in di- and tri-nitrotoluene exposures. The animal exposure design is presented in a study by Quinn et al (2010). Five treatment groups consisting of 12 male and 12 female bobwhites each were administered 2A-DNT at 0, 0.5, 3, 14, or 30 mg/kg/day via oral gavage for 60 days. Controls received a volume of corn oil equivalent to the mean volume given to birds in the highest dose group without compound. Microarray hybridizations were conducted using completely randomized design experiments including a 2 x 4 factorial treatment arrangement to investigate the conditions: sex (male and female) and 2A-DNT dose (control, 3, 14, and 30 mg/kg-d) for both liver and kidney tissues. All conditions included 4 biological replicates. REFERENCE: Quinn Jr. MJ, McFarland CA, LaFiandra EM, Bazar MA, Johnson MS (2010) Acute, subacute, and subchronic exposure to 2A-DNT (2-amino-4,6-dinitrotoluene) in the northern bobwhite (Colinus virginianus) Ecotoxicology 19:945-952.

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