Chamcheu J.C.,Uppsala University |
Chamcheu J.C.,University of Wisconsin - Madison |
Navsaria H.,Center for Cutaneous Research |
Pihl-Lundin I.,Uppsala University |
And 4 more authors.
Journal of Investigative Dermatology | Year: 2011
Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies. © 2011 The Society for Investigative Dermatology.
Nassab R.,Whiston Hospital |
Myers S.,Center for Cutaneous Research |
Frame J.,Center for Cutaneous Research
Aesthetic Surgery Journal | Year: 2011
The cosmetic surgery market is a rapidly growing sector of healthcare, and the use of marketing strategies is now an integral part of any cosmetic surgery practice. In this study, the authors review 50 Web sites from practitioners in London and New York to quantify the utilization of online marketing, comparing results between the United Kingdom and the United States. © 2011 The American Society for Aesthetic Plastic Surgery, Inc.
Tan D.W.M.,Wellcome Trust Medical Research Council Cambridge Stem Cell Institute |
Jensen K.B.,Wellcome Trust Medical Research Council Cambridge Stem Cell Institute |
Trotter M.W.B.,Celgene |
Connelly J.T.,Center for Cutaneous Research |
And 5 more authors.
Development (Cambridge) | Year: 2013
SUMMARY Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signaling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. © 2013. Published by The Company of Biologists Ltd.
Jones M.,University College London |
Dry I.R.,University College London |
Frampton D.,University College London |
Singh M.,Center for Cutaneous Research |
And 8 more authors.
PLoS Pathogens | Year: 2014
Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread. © 2014 Jones et al.
Matin R.N.,Stoke Mandeville Hospital |
Szlosarek P.,Barts Cancer Institute |
McGregor J.M.,Center for Cutaneous Research |
Cerio R.,Blizard Institute |
Harwood C.A.,Center for Cutaneous Research
Clinical and Experimental Dermatology | Year: 2013
An increased frequency of renal carcinoma in men with melanoma has been reported in population based-studies. We report the clinicopathological findings of five cases of synchronous renal cell carcinoma (RCC), identified after routine radiological staging for cutaneous malignant melanoma (MM) between October 2006 and October 2008. The five patients (three men and two women, with a mean age of 62.4 years), presented with six melanomas of varying subtypes. The mean Breslow thickness was 1.87 mm. There was no family history of cancer in any of the cases. Routine radiological staging identified a mass arising from the left kidney in three cases and the right kidney in two cases. All patients underwent radical nephrectomy, and histology in each case confirmed RCC of the clear-cell subtype. Mean follow-up was 3 years. Although the simultaneous occurrence of RCC and MM may be coincidental, there are several plausible aetiological links. Further analysis of the synchronous occurrence of MM and renal cancer may provide therapeutic insights into these two important tumours. © The Author(s). CED © 2012 British Association of Dermatologists.
Ruge F.,University of Cardiff |
Glavini A.,University of Cardiff |
Gallimore A.M.,University of Cardiff |
Richards H.E.,University of Cardiff |
And 4 more authors.
Journal of Investigative Dermatology | Year: 2011
Defolliculated (Gsdma3 Dfl /) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3 Dfl / mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1/ mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies. © 2011 The Society for Investigative Dermatology.
Patel I.I.,Lancaster University |
Harrison W.J.,Center for Cutaneous Research |
Kerns J.G.,Lancaster University |
Filik J.,Diamond Light Source |
And 7 more authors.
Analytical and Bioanalytical Chemistry | Year: 2012
Normal function and physiology of the epidermis is maintained by the regenerative capacity of this tissue via adult stem cells (SCs). However, definitive identifying markers for SCs remain elusive. Infrared (IR) spectroscopy exploits the ability of cellular biomolecules to absorb in the mid-IR region (λ=2.5-25 μm), detecting vibrational transitions of chemical bonds. In this study, we exploited the cell's inherent biochemical composition to discriminate SCs of the inter-follicular skin epidermis based on IRderived markers. Paraffin-embedded samples of human scalp skin (n=4) were obtained, and 10-μm thick sections were mounted for IR spectroscopy. Samples were interrogated in transmission mode using synchrotron radiation-based Fourier-transform IR (FTIR) microspectroscopy (15×15 μm) and also imaged employing globarsource FTIR focal plane array (FPA) imaging (5.4× 5.4 μm). Dependent on the location of derived spectra, wavenumber-absorbance/ intensity relationships were examined using unsupervised principal component analysis. This approach showed clear separation and spectral differences dependent on cell type. Spectral biomarkers concurrently associated with segregation of SCs, transit-amplifying cells and terminally-differentiated cells of epidermis were primarily PO2 - vibrational modes (1,225 and 1,080 cm-1), related to DNA conformational alterations. FPA imaging coupled with hierarchical cluster analysis also indicated the presence of specific basal layer cells potentially originating from the follicular bulge, suggested by co-clustering of spectra. This study highlights PO2 - vibrational modes as potential putative SC markers. © Springer-Verlag 2012.
Chamcheu J.C.,Uppsala University |
Pihl-Lundin I.,Uppsala University |
Mouyobo C.E.,Uppsala University |
Gester T.,Uppsala University |
And 5 more authors.
British Journal of Dermatology | Year: 2011
Summary Background: Epidermolytic ichthyosis (EI) is a skin fragility disorder caused by mutations in genes encoding suprabasal keratins 1 and 10. While the aetiology of EI is known, model systems are needed for pathophysiological studies and development of novel therapies. Objectives To generate immortalized keratinocyte lines from patients with EI for studies of EI cell pathology and the effects of chemical chaperones as putative therapies. Methods We derived keratinocytes from three patients with EI and one healthy control and established immortalized keratinocytes using human papillomavirus 16-E6/E7. Growth and differentiation characteristics, ability to regenerate organotypic epidermis, keratin expression, formation of cytoskeletal aggregates, and responses to heat shock and chemical chaperones were assessed. Results The cell lines EH11 (K1-p.Val176-Lys197del), EH21 (K10-p.156Arg>Gly), EH31 (K10-p.Leu161-Asp162del) and NKc21 (wild-type) currently exceed 160 population doublings and differentiate when exposed to calcium. At resting state, keratin aggregates were detected in 9% of calcium-differentiated EH31 cells, but not in any other cell line. Heat stress further increased this proportion to 30% and also induced aggregates in 3% of EH11 cultures. Treatment with trimethylamine N-oxide and 4-phenylbutyrate (4-PBA) reduced the fraction of aggregate-containing cells and affected the mRNA expression of keratins 1 and 10 while 4-PBA also modified heat shock protein 70 (HSP70) expression. Furthermore, in situ proximity ligation assay suggested a colocalization between HSP70 and keratins 1 and 10. Reconstituted epidermis from EI cells cornified but EH21 and EH31 cells produced suprabasal cytolysis, closely resembling the in vivo phenotype. Conclusions These immortalized cell lines represent a useful model for studying EI biology and novel therapies. © 2011 British Association of Dermatologists.
Upton J.H.,Center for Cutaneous Research |
Hannen R.F.,Center for Cutaneous Research |
Bahta A.W.,Center for Cutaneous Research |
Farjo N.,Farjo Medical Center |
And 2 more authors.
Journal of Investigative Dermatology | Year: 2015
Dermal papilla cells (DPCs) taken from male androgenetic alopecia (AGA) patients undergo premature senescence in vitro in association with the expression of p16 INK4a, suggesting that DPCs from balding scalp are more sensitive to environmental stress than nonbalding cells. As one of the major triggers of senescence in vitro stems from the cell "culture shock" owing to oxidative stress, we have further investigated the effects of oxidative stress on balding and occipital scalp DPCs. Patient-matched DPCs from balding and occipital scalp were cultured at atmospheric (21%) or physiologically normal (2%) O 2. At 21% O 2, DPCs showed flattened morphology and a significant reduction in mobility, population doubling, increased levels of reactive oxygen species and senescence-associated β-Gal activity, and increased expression of p16 INK4a and pRB. Balding DPCs secreted higher levels of the negative hair growth regulators transforming growth factor beta 1 and 2 in response to H 2 O 2 but not cell culture-associated oxidative stress. Balding DPCs had higher levels of catalase and total glutathione but appear to be less able to handle oxidative stress compared with occipital DPCs. These in vitro findings suggest that there may be a role for oxidative stress in the pathogenesis of AGA both in relation to cell senescence and migration but also secretion of known hair follicle inhibitory factors. © 2015 The Society for Investigative Dermatology.
Fox J.L.,University of Oxford |
Ismail F.,Center for Cutaneous Research |
Azad A.,University of Oxford |
Ternette N.,University of Oxford |
And 6 more authors.
EMBO Journal | Year: 2010
Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis. © 2010 European Molecular Biology Organization.