Agenda of the 83Rd SSBC Business Meeting, Thursday 20Th May, Edinburgh University

Agenda of the 83rd SSBC Business Meeting, Thursday 20th May, Edinburgh University

Apologies:

Approval of Minutes of 82nd meeting:

Finances:

New members:

Matters arising:

AOCB:

Next meeting:

Minutes of the 82nd SSBC Business Meeting, Thursday 22ndOctober 2009, Glasgow Caledonian University.

Apologies:

Received from James Ferguson, Sally Ibbotson, Charlotte Proby, Alasdair Kerr, Irene Leigh, Adrianus Van Den Broek, Irwin Mclean, Peter Forsythe, Hilary Jackson, Andy South & Andrey Pantelyev.

Approval of Minutes of 81st meeting:

These were adopted. Proposed by Richard Weller; 2nd by David Greenhalgh.

Finances:

Total income from the Dundee meeting was £1270 (43 attendees; 24 dinner attendees; new membership fees).

Expenditure was £981.89.

As of 21st September 2009 the treasurer account contained £1065.

New members:

Karin Kroboth, Ulrike Gartner, Vikas Hegde and Adrian Mason were proposed by Frances Smith and Siao Pei Tan was nominated by Richard Weller. Seconded to floor.

Matters arising

The history of the SSBC is in the November issue of Clinical & Experimental Dermatology.

Website: we have agreed to pay Andy Cassidy for the maintenance of the website. We have agreed to pay on a per hour basis and also to pay to renew the domain name. It was discussed that someone within the club (RW) would attempt to take on the task.

AOCB:

JW suggested that the frequency of meetings should be reduced to once a year. This prompted some debate, but it was agreed that such a move would be a retrograde step. The meetings are considered to be an important training exercise for young researchers/students and also an important socialising event. It was suggested that if there is difficulty in obtaining submitted papers to fill the programme, then a review talk from a member (eg. an overview of the field or a teaching session) could be substituted.

Next meeting:

The next meeting will take place in May 2010 in Aberdeen or Edinburgh.

Programme for the 83rdSSBC, Thursday 20thMay, Edinburgh University

10.15-10.45:Registration & Coffee

10.45-11.00Business Meeting. Chair: Julie Woods

11.00-13.00:Morning Session: Chair: Dr Patricia Martin

11.00-11.20:Skin autofluorescence and non-invasive detection: Is there a ‘normal’ fingerprint?

K. Robinson, J. Woods, S. Ibbotson, Photobiology Unit, University of Dundee

11.20-11.40:Skin bound NO-related species mediate the beneficial health effects of sunlight.

D. Liu, J. Gallagher, M. Morris, R. Weller, Department of Dermatology, University of Edinburgh

11.40-12.00:Investigation of DNA repair proteins as prognostic biomarkers in cutaneous malignant melanoma using tissue microarray

L. Song, N.Anderson, T. Brenn, E. Brown, V. Doherty, D. Melton, MRC Human Genetics Unit, University of Edinburgh

12.15-13.00:Lunch

13.00-15.10:Afternoon Session: Chair: Dr Richard Weller

13.00-13.50:Invited Speaker: Dr Elizabeth Patton

Chemical and genetic approaches to melanocyte development and melanoma

Edinburgh Cancer Research Centre, University of Edinburgh

13.50-14.10:Immortalized keratinocytes derived from epidermolytic ichthyosis patients reproduce the disease phenotype: a useful in vitro model for testing new treatments.

JC. Chamcheu1,*, I. Pihl-Lundin1, C. Eteti Mouyobo1, T. Gester1, M. Virtanen1, A. Moustakas2, H. Navsaria3,A. Vahlquist1, H. Törmä1,

1Department of Medical Sciences/Dermatology and Venereology, Uppsala University, Uppsala, Sweden; 2 Ludwig Institute for Cancer Research, Biomedical Centre, Uppsala University, Uppsala, Sweden; 3 Centre for Cutaneous Research, ICMS, Queen Mary's School of Medicine and Dentistry, London, United Kingdom

14.10-14.30Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation: Involvement of heat shock proteins and MAP kinases

JC. Chamcheu1,H. Navsaria2,I. Pihl-Lundin1, M. Liovic3,4 , A.Vahlquist1, H. Törmä1

1Department of Medical Sciences/Dermatology and Venereology, Uppsala University, Uppsala, Sweden; 2 Centre for Cutaneous Research, ICMS, Queen Mary's School of Medicine and Dentistry, London, United Kingdom; 3 Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Slovenia; 4 National Institute of Chemistry, Ljubljana, Slovenia

Continued......

14.30-14.50:Identification and characterisation of DSPIa, a novel isoform of human desmoplakin

RM. Cabral1, H. Wan2, CL. Cole3, DJ. Abrams4, DP. Kelsell1,AP. South1,3

1Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science; 2Centre for Clinical and Diagnostic Oral Sciences, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, University of London, London, UK; 3Centre for Oncology & Molecular Medicine, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK. 4Department of Cardiac Electrophysiology, St Bartholomew’s Hospital, London, UK.

14.50-15-10A connexin26 G12R mouse model for Keratisis-icthyosis deafness (KID) syndrome

S. Donnelly, M. Hodgins, P.E. Martin, Department of Biological and Biomedical Sciences

Glasgow Caledonian University

Skin autofluorescence and non-invasive detection: Is there a ‘normal’ fingerprint?

K. Robinson, J. Woods, S. Ibbotson

The Photobiology Unit, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK

When skin is exposed to light, it absorbs and re-emits it as light of a different colour. This is known as autofluorescence (AF). Although first described almost a century ago it has recently been discovered that AF changes when the structure of the skin is altered, thus AF could potentially be exploited for medical use. Indeed, the AF signal from diseased tissue, such as skin tumours is distinct from that of normal skin. Thus imaging of AF by non-invasive methods could assist clinicians diagnostically, for example in the delineation of skin tumours from normal skin and other benign conditions. Despite its potential usefulness, there is incomplete understanding of how fluorescence of skin is linked to skin physiology and pathology. This is largely because the characteristics of normal in vivo skin AF have not been defined. Most studies have been in very small numbers of subjects, or have used ex-vivo biopsy material (thus omitting some of the factors such as blood flow etc., that make important contributions to AF). Preliminary observations indicate that AF is altered in skin cancer. Thus acquisition of high quality, reproducible data in normal subjects underpins all subsequent investigations and studies. We have obtained a state-of-the-art ‘SkinSkan’ device, which is a tunable, skin-enabled spectrofluorimeter capable of detecting human skin AF. However, because clinical data on AF are very limited and have generally involved measurement of the average AF signal, we are conducting a benchmark study to characterise in detail the AF fingerprint in healthy volunteers, which is excitation wavelength-dependent. This will enable us to determine inter- and intra-subject variability of the ‘normal’ fingerprint. These data will be essential in order to allow us to distinguish between normal and diseased skin AF, and in particular skin cancer, as we anticipate that this device will have application in the delineation of tumour margins. This approach may also have potential as a quantitative marker for a wide range of other clinical states.

Skin bound NO-related species mediate the beneficial health effects of sunlight.

D. Liu, J. Gallagher, M. Morris, R. Weller

Department of Dermatology, Lauriston Place, Lauriston Building, Edinburgh, EH3 9HA

Blood pressure level and the prevalence of hypertension correlates directly with latitude, being higher in populations living further from the equator (less sunlight).This may be due to various racial and environmental factors other than sunlight, but within the UK, all cause of mortality (of which cardiovascular is the major one) correlates linearly with latitude even after accounting for all known risk factors. We have previously shown significant stores of the cardiovascularly active molecule nitric oxide (NO), bound to skin in a UVA reversible manner1. Independently of vitamin D synthesised by UVB, we suggest that the improved cardiovascular health in populations living closer to equator is due to cleavage of skin bound nitro-species by UVA.

We irradiated 12 healthy volunteers (20-30 yrs) on one side of the body with 20J/cm2 UVA (8 Waldmann PUVA bulbs). Increased in circulating nitrite, decrease in circulating nitrate, and a drop of blood pressure was shown after active but not sham (similar temperature rise, but no UVA exposed) irradiation.

To demonstrate the drop of blood pressure is related to the dose of UVA delivered, we used a commercial tanning booth allowing 360º irradiation of volunteers (44 bulbs, 285-410 nm, peaking at 365 nm), we irradiated 9 healthy volunteers (30-66 yrs) at a dose of 12 J/cm2 on skin type II and 16/cm2 on skin type III individuals. The MAP (mean arterial pressure) fell by 2~8 mmHg after active irradiation, with the lowest BP at 30-40 minutes after irradiation. Circulating nitrosospecies are being analysed. Blood samples for vitamin D were taken before and 24 hour after PUVA irradiation from 9 volunteers, the results did not shown any significant change.

Mowbray M, McLintock S, Weerakoon R, Lomatschinsky N, Jones S, Rossi AG, et al. Enzyme-Independent NO Stores in Human Skin: Quantification and Influence of UV Radiation. Journal of Investigative Dermatology. 2009;129(4):834-42.

Investigation of DNA repair proteins as prognostic biomarkers in cutaneous malignant melanoma using tissue microarray

L. Song, N.Anderson, T. Brenn, E. Brown, V. Doherty, D. Melton

University of Edinburgh, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU

Cutaneous malignant melanoma is the eighth most common cancer in the UK, with more than 10,400 cases diagnosed in 2006. It is now the commonest cancer amongst young adults (aged 15-34) and incidence rates in the UK have more than quadrupled since the 1970’s. A further 73% rise is predicted in Scotland over the next 10-15 years, the largest increase for any solid tumour. Similar rapid increases are predicted worldwide. Early surgical removal of primary tumours is an effective treatment, but patients that go on to develop metastatic disease have a very poor prognosis and we urgently need to improve our understanding of the molecular pathogenesis to develop better prevention strategies, define new prognostic markers and identify new therapeutic targets. Expression of DNA repair pathways is elevated in metastatic melanoma, favouring accurate and rapid genome replication and probably also contributing to their extreme resistance to DNA-damaging therapeutics. We are investigating the hypothesis that levels of key DNA repair proteins can be used as prognostic biomarkers in primary melanoma. We are studying 10 genes which showed the highest over-expression in recurrent primary melanomas compared to non-recurrent primaries in a small gene expression microarray study carried out on frozen thick primary melanomas. This is being done by determining DNA repair protein expression in formalin-fixed and paraffin- embedded samples using our 500-sample melanoma tissue microarray and analysing the correlation with clinicopathologic factors and patient survival. So far we have identified two genes, replication factor C subunit 4 (RFC4) and excision repair cross-complementing 1 (ERCC1), which have significantly different expression levels between the different stages in melanoma progression. High expression of these two proteins in thick primary tumours was also correlated with significantly poorer survival, indicating that DNA repair proteins could prove to be useful prognostic biomarkers.

Immortalized keratinocytes derived from epidermolytic ichthyosis patients reproduce the disease phenotype: a useful in vitro model for testing new treatments.

JC. Chamcheu1,*, I. Pihl-Lundin1, C. Eteti Mouyobo1, T. Gester1, M. Virtanen1, A. Moustakas2, H. Navsaria3,A. Vahlquist1, H. Törmä1,

Background: Epidermolytic ichthyosis (EI) is an autosomal dominant skin fragility disorder caused by mutations in genes encoding suprabasal keratins 1 and 10. While the etiology of EI is known, model systems are needed for functional studies and development of novel therapies.

Objectives: To generate immortalized keratinocyte lines from EI patients for studies of EI cell pathology and effects of chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA), as putative therapies.

Methods: We derived keratinocytes from three patients with EI and one healthy control and established immortalized keratinocytes in serum-free medium using human papillomavirus 16 (HPV16-E6/E7). Growth and differentiation characteristics, ability to regenerate organotypic epidermis, keratin expression and formation of cytoskeletal aggregates, and responses to heat-shock with or without 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 (wildtype) 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 alsoinduced aggregates in 3% of EH11 cultures. Treatment with TMAO and 4-PBA reduced the number of aggregate-containing cells before and after heat-stress. Reconstituted epidermis from EI cells cornified and expressed typical markers of keratinization. Severely mutated cells produced suprabasal cytolysis, closely resembling the clinical phenotype.

Conclusions: Our well characterized immortalized cell lines represent a useful model for studying EI biology and novel therapies, exemplified herein by two chemical chaperones.

Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation: Involvement of heat shock proteins and MAP kinases

JC. Chamcheu1,H. Navsaria2,I. Pihl-Lundin1, M. Liovic3,4 , A.Vahlquist1, H. Törmä1

Epidermolysis bullosa simplex (EBS) is an inherited epithelial tissue fragility disorderdue to 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 some protein folding disorders such as cystic fibrosis and Alzheimer’s disease, chaperones and the ubiquitin-proteasome system modify disease severity, suggesting a novel therapeutic approach even for EBS. In this study, the effects of two chemical chaperones (trimethylamine-N oxide (TMAO) and 4-phenylbutyrate (4-PBA)) on heat stress-induced keratin aggregation responses were examined in newly and previously established immortalized control and EBS patient-derived keratinocyte cell lines. Heat-induced keratin-positive aggregates were observed in all EBS cells, which were most prominent in severe keratin-defective cell lines and less so in normal cells. The proportion of cells containing aggregates were dramatically reduced by TMAO and 4-PBA pretreatment. Furthermore, heat stress greatly induced MAP kinase activation (p38 and JNK) and increased Hsp70/Hsc70 expression, and TMAO was able to transiently modulate these effects. The results suggest that TMAO rescue may involve components of the endogenous chaperone and MAPK machineries, which may represent novel targets for the development of more effective treatments for EBS and other keratin-related genetic disorders.

Identification and characterization of DSPIa, a novel isoform of human desmoplakin.

RM. Cabral1, H. Wan2, CL. Cole3, DJ. Abrams4, DP. Kelsell1,AP. South1,3

Desmoplakin is a ubiquitous component of desmosomes and desmosome-like structures such as the cardiomyocyte area composita. Two major isoforms, desmoplakin I (DSPI) and desmoplakin II (DSPII) are encoded by alternative mRNA transcripts differentially spliced from the same gene. The resulting proteins are identical in amino acid sequence with the exception that DSPII contains only one third of the central alpha-helical rod domain present in DSPI. Here we describe a novel, minor isoform of desmoplakin also produced by alternative splicing of the desmoplakin gene which we name desmoplakin Ia (DSPIa). DSPIa is an alternatively spliced DSPI mRNA with a unique splice donor site 90% homologous to and downstream of the DSPII specific donor. The resulting DSPIa mRNA is in frame and encodes a protein with a central alpha-helical rod domain of intermediate size, 156 amino acids larger than DSPII and 443 amino acids smaller than DSPI. We demonstrate through recombinant expression and siRNA knockdown that DSPIa protein is readily detected, albeit at substantially lower levels than the dominant isoforms, DSPI and DSPII. DSPIa mRNA has similar tissue distribution to DSPI and DSPII.

Keywords: Desmosome, Desmoplakin, Isoform, splice site

A connexin26 G12R mouse model for Keratisis-icthyosis deafness (KID) syndrome

S. Donnelly, M. Hodgins, P.E. Martin, Department of Biological and Biomedical Sciences, Glasgow Caledonian University

Keratisis-icthyosis deafness (KID) syndrome is a rare and crippling disease causing cobblestone – like hyperkeratosis on the face, sensorineural hearing loss and vascularising keratitis. Patients also suffer from chronic gram positive bacterial (S. aureus) and fungal (C. albicans)infections. The disease is caused by dominant mutations, G12R, N14Y, S17F and D50N, in the gap junction protein connexin (Cx) 26. Connexins are transmembrane proteins that directly link the cytoplasm of neighbouring cells allowing the quick exchange of regulatory molecules up to 1KDa. Correct gap junction intercellular communication (GJIC) is essential to maintain the homeostasis within almost every tissue type. Cell transfection studies, using GFP linked Cx26 constructs, have shown that these mutations have the ability to target the plasma membrane but also show a high level of cytoplasmic accumulation. Analysis of ATP release form transfected cells showed a significant increase compared to Wt Cx26 and other non-KID syndrome mutations, suggesting that pathogenesis of the disease may be due to the formation of “leaky” hemichannels. To study the effects of these mutations in vivo a KID syndrome mouse model has been generated with the G12R mutation under the control of the keratin 10 promoter. The G12R mouse has a ruffled, thin coat developing hyperkeratotic plaques and they show signs of circumscribed erythametous hyperkeratotic plaques. The tip of the tail is also kinked and the mice are smaller when compared to their siblings. Histological studies show a hyperkeratotic epidermis, much thicker than the Wt. Although the epidermis is thicker it still maintains normal epidermal structure and displays all stages of differentiation from the basal layers to the corneum. Hair follicle distribution is sparse and the skin is smooth in comparison the undulating Wt sections. The dermis is also thickened and shows signs of increased inflammatory infiltrate. Based on these results we believe that the G12R mouse is an excellent KID syndrome model and a valuable tool in understanding the role of Cx26 in the skin.