tbuckley/cabot-search huggingface dataset:
P39 Using single-cell transcriptomics to investigate the potential role of the human hair follicle dermal papilla in controlling hair fibre pigmentation
Authors: Leah C Redmond, Summik Limbu, Francisco Jiménez, Chris D’Souza, Greg Williams, Claire Higgins
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims The human dermal papilla (DP) is a population of cells located at the base of the hair follicle, which signals to surrounding matrix cells facilitating their incorporation into the layers of the hair fibre and the hair follicle. The DP controls the process of hair follicle induction, reflecting its critical role in orchestrating processes integral in hair growth. We hypothesize that distinct functional subgroups exist within the structure, which would mean that there are different types of DP cells within each follicle. Methods To begin testing this, we reanalysed the single-cell RNA sequencing (RNA-Seq) dataset of the human anagen hair follicle used in the 2022 study by Shim et al. To expand on this, we are now generating single-cell RNA-Seq datasets of patient-matched pigmented and nonpigmented hair follicles (n = 4 patients). These datasets will be used to explore heterogeneity in the human DP, and to investigate which subpopulations are potentially changed in the case of pigmentation loss. This will allow us to explore a potentially undiscovered role of the DP in controlling hair fibre pigmentation. Results We found four distinct subpopulations in the DP with statistically significant marker genes in the Shim dataset. This confirms transcriptional heterogeneity in the human DP, hinting at clusters of DP cells with specialized functions within each hair follicle. One subcluster, which expressed S100B, endothelin-3 and bone morphogenetic protein (BMP)4, was of particular interest and gene ontology analysis of the genes in this cluster revealed terms associated with skeletal development and BMP signalling. We hypothesize that this DP cluster regulates melanin transfer from bulbar melanocytes to neighbouring keratinocytes, owing to the known involvement of the mentioned genes in both melanogenesis and melanin transfer. Conclusions The human DP is a heterogeneic structure, with a potentially unexplored role in controlling hair fibre pigmentation.
P04 How fibroblasts and azathioprine prepare the soil for cutaneous squamous cell carcinoma
Authors: Ioanna Kontou, Charareh Pourzand, Gernot Walko, Ute Jungwirth
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims DNA damage-inducing immunosuppressive drugs such as azathioprine (AZA) act synergistically with ultraviolet (UV)A radiation and photosensitize the skin of immunosuppressed individuals, such as organ transplant recipients. These individuals have an increased risk of developing cutaneous squamous cell carcinoma (cSCC). The specific combination of AZA and UVA causes oxidative DNA damage that leaves behind a characteristic mutational signature in tumour keratinocytes of patients treated with AZA. However, as UVA radiation can penetrate the dermis, the impact of AZA/UVA on cells of the tumour microenvironment, such as fibroblasts, also need to be addressed. Fibroblasts are crucial for cSCC development and progression as they can switch to a protumourigenic phenotype. Methods Cell viability and senescence assays were performed to investigate the impact of AZA in combination with or without UVA on neonatal (nonsun-exposed) and adult (sun-exposed) human dermal fibroblasts. In addition, the impact of AZA/UVA-treated fibroblasts on the clonal growth properties of different cSCC cell models was investigated using three-dimensional (co)culture models. Results We found that AZA and UVA synergistically reduce the viability of both UV-naïve neonatal and sun-exposed adult dermal fibroblasts. In addition, combined AZA and UVA treatments lead to an increased percentage of senescent fibroblasts. We hypothesize that the aberrant secretome of these senescent fibroblasts can impact the growth behaviour of tumour cells. In a soft agar assay, the more malignant cSCC cells (MET-1) formed more and larger clonal spheroids and were less dependent on growth factors compared with spheroids formed by premalignant (PM-1) keratinocytes. Conclusions AZA and UVA act synergistically and potentiate a UVA-induced senescent fibroblast phenotype. The changed characteristics of these fibroblasts can impact the stem cell-like properties from premalignant and malignant keratinocytes. Combining keratinocyte and fibroblast culture models enables us to study how AZA/UVA promote the initiation and progression of cSCC.
P38 Experimental evidence of ecto-5′-nucleotidase (CD73) modulation in psoriasis-like inflammation
Authors: Giacomo De Palma, Elisabetta Caiazzo, Armando Ialenti, Carla Cicala
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Adenosine is an endogenous purine nucleoside that, by binding to adenosine receptors, modulates inflammation and immune responses. Psoriasis is a chronic immune-mediated inflammatory skin disease characterized by keratinocyte hyperproliferation and abnormal differentiation. A2A and A2B adenosine receptors modulate keratinocyte proliferation and their expression was found to be altered in psoriatic epidermis. Ecto-5′-nucleotidase (CD73) is an enzyme that hydrolyses extracellular adenosine monophosphate and represents a key control point for extracellular adenosine accumulation. CD73 is involved in the regulation of local immune response and tissue barrier function. As changes in CD73 expression and activity have been associated with other autoimmune diseases, the aim of this study was to investigate CD73 involvement in skin inflammation using in vitro and in vivo approaches. Methods In vitro experiments were performed on human keratinocyte (HaCaT) cell lines stimulated with tumour necrosis factor-α, interleukin (IL)-17A, IL-6 and IL-1α (10 ng mL−1 each), and in vivo, on the mouse model of imiquimod-induced psoriasis-like inflammation on the right ear, which was also performed on CD73−/− mice and their wildtype counterpart. CD73 expression in cell lysates and ear homogenates were evaluated using Western blot analysis. Results We found that CD73 expression was significantly increased (N = 3 wells, P &amp;lt; 0.05) in cytokine-treated HaCaTs compared with unstimulated cells. In vivo experiments showed increased CD73 expression on psoriatic lesions (P &amp;lt; 0.001, N = 3). In CD73−/− mice, imiquimod-induced lesions were exacerbated compared with the development of lesions in wildtype mice as shown by ear thickness (0.190 ± 0.02 mm, knockout mice vs. 0.138 ± 0.02 mm, wildtype mice; P &amp;lt; 0.05, N = 5), erythema and scaling. Conclusions Our preliminary Results show involvement of CD73 in the development of psoriasis-like inflammation, suggesting that CD73 may represent a new therapeutic target in the treatment of skin diseases. Further investigations are needed to provide an in-depth study of CD73 localization in inflamed skin.
P22 A single-cell map of the epigenomic landscape in keratinocytes exposed to the cytokine milieu of atopic dermatitis
Authors: Laia Sadeghi, Michael Ardern-Jones
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims In atopic dermatitis (AD), induction of inflammatory responses regulated by cytokines such as of thymic stromal lymphopoietin exacerbate the disrupted integrity of the skin barrier. Cytokines, such as interleukin (IL)-4, IL-13 and IL-22, present in the skin milieu, bind to their corresponding receptors on the cell surface of keratinocytes, activating multiple signal transduction pathways that lead to changes in the gene expression profile of keratinocytes, in part, by modulating chromatin structure. Understanding the intricate interplay between cytokine-mediated signalling pathways in keratinocytes and the resulting epigenetic changes is essential for elucidating the pathogenesis of AD. In this study, we utilized high-throughput technology to examine the effects of cytokines on modulation of the epigenetic landscape at the keratinocyte single-cell level, and subsequent effects on the gene expression. Methods We utilized the reconstructed human epidermis model, an in vitro three-dimensional human epidermis model consisting of a polycarbonate membrane and keratinocytes mimicking the structure of native human epidermis. Following exposure to AD cytokine(s), the model gene expression and chromatin accessibility of target cells were analysed using single-cell multiomics approaches. Results Our preliminary data show that IL-4, IL-13 and IL-22 reduce the expression levels of the barrier-related gene filaggrin in keratinocytes. Conclusions Unlike genetic changes, epigenetic alterations are dynamic and can be restored to their normal state, which has major implications for the development of therapeutics for various diseases. This study will provide valuable insights into the epigenetic mechanisms underlying AD and present potential targets for epigenetic-related therapeutic interventions. These interventions can be used in combination with available compounds to reduce the recurrence of AD and improve the treatment efficiency.
O22 Unravelling the pathophysiology of KLICK syndrome to identify therapeutically targetable pathways
Authors: Hannah Cherry, Joe Rastrick, Joanna Jacków, Maddy Parsons, John McGrath
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Keratosis linearis with ichthyosis congenita and sclerosing keratoderma (KLICK) syndrome is an ultrarare genodermatosis associated with a homozygous pathogenic variant, c.-95delC in POMP, which encodes for proteasome maturation protein. Clinical manifestations include linear hyperkeratotic papules in the flexures, palmoplantar keratoderma and pseudoainhum. The pathophysiology of KLICK syndrome is poorly understood, resulting in nonspecific, limited treatment options. The aim of this work is to characterize KLICK syndrome at the cellular and molecular level, with the utilization of transcriptomic techniques for potential drug repurposing. Methods A lesional skin biopsy was obtained, following informed consent, from a 32-year-old female patient with KLICK syndrome. Histology and immunostaining were used to characterize the disrupted skin architecture. RNA sequencing was used to identify key dysregulated pathways and the L1000FWD reverse transcriptomics tool enabled the generation of a shortlist of potential therapeutics for KLICK syndrome. CRISPR editing techniques and induced pluripotent stem cell (iPSC) reprogramming Methods were used to generate cellular based models carrying the pathogenic POMP variant in order to support evaluation of the shortlisted therapeutic options. Results KLICK syndrome skin histology featured hyperkeratosis, hypergranulosis and delayed cellular flattening. Consistent with pathological features, pathway analysis of transcriptomic data revealed keratinocyte differentiation and epidermis development as key upregulated pathways in KLICK syndrome. Interestingly, the Panther pathway analysis tool suggested dysregulation in epidermal growth factor receptor (EGFR) signalling, and reverse transcriptomics identified erlotinib, an EGFR inhibitor, as a potential therapeutic treatment for KLICK syndrome. HaCaT keratinocytes were successfully edited using CRISPR-Cas9 to carry the KLICK patient POMP variant and a KLICK patient iPSC line was generated to support in vitro KLICK syndrome modelling. Conclusions KLICK syndrome is a hyperproliferative inflammatory skin disorder associated with a pathogenic variant in POMP. Reverse transcriptomics is a useful tool for shortlisting drugs with biologically relevant mechanisms of action with potential for treatment of rare genetic diseases such as KLICK syndrome.
O09 Microsatellite-unstable sporadic sebaceous and duodenal tumours are associated with loss of mismatch protein expression
Authors: Georgie Holt, Waleed Alfailakawi, Sam Cook, Thomas Ness, Rowen Coulthard, Claire Jones, Christine Hayes, Rachel Phelps, Ralf Kist, Mike Jackson, Mauro Santibanez-Koref, John Burn, Alastair Burt, Akhtar Husain, Chris Lamb, Richard Gallon, Neil Rajan
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Lynch syndrome (LS) is a common heritable cancer predisposition syndrome. Tumours from patients with LS demonstrate microsatellite instability (MSI-high), a genetic signature detectable by low-cost DNA sequencing assays that is currently utilized in colorectal cancer screening. The utility of MSI assays for LS detection has not been extensively studied in unselected sebaceous skin tumours or duodenal tumours, where currently immunohistochemical (IHC) expression of mismatch repair (MMR) proteins is used. In addition, it has recently been suggested that cutaneous squamous cell carcinoma (cSCC) could be linked to LS; however MSI status has not been studied in patients with early-onset cSCC. Methods We investigated MSI status in archival formalin-fixed paraffin-embedded cases with ethical approval. We examined 110 sebaceous tumours (STs) (45 female patients, 65 male patients; median age 77 years), 50 duodenal tumours (DTs) (28 female patients, 22 male patients; median age 64 years), and 148 cSCCs selected from patients under 50 years of age (69 female patients, 79 male patients; median age 49 years). Immunohistochemistry (IHC) was performed on each MSI-high sample to determine the expression of MMR proteins MLH1, MSH2, MSH6 and PMS2. Samples that passed quality control were included in the analysis. Results A total of 42 of 106 STs (39.6%) were MSI-high. All STs had loss of at least one MMR protein. Overall, 10 of 41 (24%) DTs were MSI-high. All DTs had loss of at least one MMR protein. We found that 12 of 148 (8.1%) cSCCs were MSI-high and 5 of 12 SCCs exhibited loss of at least one MMR protein expression (41%). Conclusions MSI-high status correlated well with loss of MMR protein expression in STs and DTs samples. Future studies will help establish MSI status in tumours initially selected by MMR loss on IHC, informing its use as an adjunct to MMR IHC in LS detection in patients with STs and DTs.
O16 Understanding the pleiotropic effects of the Rho-kinase inhibitor Y-27632 on human keratinocytes
Authors: Benjamin Flynn, Kelli Glallacher, Gernot Walko
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Successful epidermal transplant generation depends on sufficiently high numbers of self-renewing stem cells that give rise to clonal colonies termed ‘holoclones’. Treatment with Y-27632, a nonspecific Rho-kinase 1/2 inhibitor, conditionally immortalizes epidermal keratinocyte cultures. However, the pleiotropic signalling mechanisms downstream of Y-27632 remain largely unexplored. We have therefore characterized the effects of continuous long-term Y-27632 treatment on chromatin regulation, and investigated whether Y-27632 reprogrammes a holoclone-like state. Methods Neonatal foreskin-derived human epidermal keratinocytes were cultured from passage p5 to p30, with or without Y treatment. Samples were collected for RNA sequencing analysis from progenitor-enriched middle passage cells (p5, MPK), progenitor-depleted late-passage cells (p8, LPK) and Y-treated cells at p8 (LPK-Y). Samples were also collected for assay for transposase-accessible chromatin (ATAC)-Seq analysis to assess open and closed chromatin regions. Results Untreated cultures became depleted of proliferating cells by p11, whereas Y maintained a highly proliferative culture up to p30 and beyond. Y withdrawal at p11 resulted in rapid loss of proliferative capacity within a single passage. ATAC-Seq identified 196 differentially regulated chromatin sites in MPK vs. LPK. Y treatment maintained accessibility in approximately 40% of these MPK-enriched chromatin regions. Approximately 3000 differentially expressed genes (DEGs) were identified in MPK vs. LPK, while Y treatment induced approximately 11 000 DEGs. Activated genes in LPK-Y were enriched for a holoclone signature, with no evidence for activation of a precancerous transcriptome. Y treatment altered the expression of various genes associated with amino acid metabolism, enabling cells to overcome serine/glycine autotrophy. Conclusions Y acts by preserving accessibility of progenitor-enriched chromatin sites and further reprogrammes the keratinocyte transcriptome towards a more holoclone-like state. However, keratinocytes retain a ‘memory’ of their doubling number, which is conditionally suppressed by Y. The observed global transcriptomic changes are indicative of the highly pleiotropic nature of the Y-induced signalling changes, warranting further deconvolution.
P07 Mutagenicity of narrowband ultraviolet B
Authors: Joanna Fowler, George Coltart, Roshan Sood, Chester Lai, Noeline Nadarajah, Philip Jones, Eugene Healy
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Exposure to ultraviolet (UV) radiation causes DNA mutations in skin and development of skin cancer. Despite this, UV radiation in the form of narrowband UVB is commonly used by dermatologists to treat skin diseases such as psoriasis. However, the mutagenicity of narrowband UVB in human skin is unknown. In this study, we investigated the mutation burden resulting from a course of narrowband UVB in patients with psoriasis. Methods Skin biopsies were taken from uninvolved skin of sixteen patients with psoriasis before and after a treatment course of narrowband UVB. Following microdissection, DNA was extracted from the epidermis and sequenced using nanorate sequencing (NanoSeq), which detects mutations in single molecules of DNA with an error rate of fewer than five errors per billion base pairs. Whole-genome sequencing of blood DNA was employed as germline control. Comparison of mutations in prenarrowband UVB and postnarrowband UVB skin samples allowed quantification of mutation burden and mutational signatures owing to narrowband UVB. Results There was a significant increase in the mutation burden in skin following the narrowband UVB treatment course, with UV radiation signatures SBS7a, SBS7b and DBS1 documented in both nonsun-exposed skin and frequently sun-exposed skin samples. Comparison of the magnitude of the mutation burden resulting from narrowband UVB to that in keratinocyte cancers provided insight into the mutagenicity of narrowband UVB in relation to the potential numbers of narrowband UVB courses over a lifetime that might lead to skin cancer development. Conclusions Narrowband UVB treatment for psoriasis Results in significant numbers of UV-radiation-induced mutations in human skin.
P29 Long-read sequencing provides unprecedented insight into the skin and wound microbial landscape
Authors: Paisleigh Smythe, Holly Wilkinson
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims It is becoming increasingly evident that the microbial inhabitants of our skin play a key role in cutaneous health and disease. However, our understanding of the contribution of the complex skin microbiome to wound healing outcome remains incomplete. This is, in part, due to the historic challenges around traditional sampling and profiling Methods, which often fail to capture the abundance and diversity of the skin microbiome. Recent advances in microbiome sequencing technologies now enable more comprehensive characterization of skin bacterial composition and host-microbe interactions. Thus, the aim of this study was to utilize a novel long-read sequencing platform to provide species-level microbiome characterization and deliver important insight into the drivers of pathological skin repair. Methods Microbial DNA samples were collected from the skin and wounds of healthy volunteers and patients with chronic wounds (n = 40 per group). Long-read nanopore sequencing was used to profile microbiome samples, while RNA-Seq enabled generation of donor transcriptomic profiles. A multifaceted approach was used to integrate these datasets to investigate the complex interplay between host skin pathology and the microbiome. Results We first identified perturbations in the skin microbiome of patients with chronic wounds, which was significantly less diverse than healthy skin and colonized with key bacteria associated with pathogenicity. These alterations in the skin microbiome also correlated with transcriptomic changes, such as upregulation of genes associated with inflammation, stress and poor healing. Interestingly, long-read sequencing data displayed poor correlation with routine clinical microbiology swabs, while specific clinical characteristics (e.g. glycaemic control and wound duration) were strongly linked to wound microbiome composition. Conclusions Collectively, these data provide crucial new insight into the skin and wound microbial landscape, demonstrating a potential role for specific wound bacteria in modulating pathological skin repair.
Development of clinical tools to estimate the breathing effort during high-flow oxygen therapy: A multicenter cohort study
Authors: A. Protti, R. Tonelli, F. Dalla Corte, D.L. Grieco, E. Spinelli, S. Spadaro, D. Piovani, L.S. Menga, G. Schifino, M.L. Vega Pittao, M. Umbrello, G. Cammarota, C.A. Volta, S. Bonovas, M. Cecconi, T. Mauri, E. Clini
Journal: Pulmonology (2024)

Quantifying breathing effort in non-intubated patients is important but difficult. We aimed to develop two models to estimate it in patients treated with high-flow oxygen therapy.
Reply to Anwar R. Padhani, Frederic LeCouvet, Giuseppe Petralia, and Dow-Mu Koh’s Letter to the Editor re: Alonso Garcia-Ruiz, Carlos Macarro, Francesca Zacchi, et al. Whole-body Magnetic Resonance Imaging as a Treatment Response Biomarker in Castration resistant Prostate Cancer with Bone Metastases: The iPROMET Clinical Trial. Eur Urol. In press. https://doi.org/10.1016/j.eururo.2024.02.016
Authors: Alonso Garcia-Ruiz, Carlos Macarro, Joaquin Mateo, Raquel Perez-Lopez
Journal: European Urology (2024)
Re: Alonso Garcia-Ruiz, Carlos Macarro, Francesca Zacchi, et al. Whole-body Magnetic Resonance Imaging as a Treatment Response Biomarker in Castration-resistant Prostate Cancer with Bone Metastases: The iPROMET Clinical Trial. Eur Urol. In press. https://doi.org/10.1016/j.eururo.2024.02.016
Authors: Anwar R. Padhani, Frederic Lecouvet, Giuseppe Petralia, Dow-Mu Koh
Journal: European Urology (2024)