Name of conference: ASPCR-ASDR 2013
Date: 17-19th May 2013
Venue: Mercure Sydney Hotel, Sydney, Australia
Name of conference: ASPCR-ASDR 2013
Date: 17-19th May 2013
Venue: Mercure Sydney Hotel, Sydney, Australia
University of Cincinnati, Cincinnati, OH, USA
We had reported that the paracrine/autocrine factor α-melanocortin (α-MSH) reduces UV-induced DNA damage in melanocytes by enhancing the repair of DNA photoproducts, reducing the generation of reactive oxygen species, activating antioxidant enzymes and basic excision repair. These effects are critical for melanoma prevention, and are evident prior to increased pigmentation. The effects of α-MSH are mediated by activating the melanocortin 1 receptor (MC1R) and explain why loss-of-function allelic variants of the MC1R gene increase melanoma susceptibility. We observed that the keratinocyte-derived endothelin-1, a potent mitogen and melanogenic factor for melanocytes, inhibits the generation of ROS immediately after UV irradiation. Recently, we found that 1,25(OH)2vitamin D3 (vit D3), also synthesized by keratinocytes upon UVB exposure, enhances the repair of DNA photoproducts, reduces ROS generation, and increases the protein levels of enzymes involved in base excision repair in primary cultures of human melanocytes. Vitamin D3 reduced the burden of UV-induced DNA photoproducts in human skin explants, evident as profound reduction in DNA photoproducts in the entire epidermis, and in melanocytes. Melanocytes express the endothelin B receptor and vitamin D receptor. Both endothelin 1 and vitamin D3 up regulate the expression of the MC1R gene, which is predicted to increase the expression of the MC1R on melanocytes and the response to α-MSH. These results suggest that the paracrine network in the skin inhibit melanoma formation by reducing UV-induced DNA damage by a mechanism that involves up regulation of MC1R expression.
S. A. Ainger1, S.-S. Wong1, H. J. Leonard2, R. A. Sturm1
1University of Queensland, St Lucia, Qld, Australia2Queensland Institute of Medical Research, Brisbane, Queensland, Australia
The MC1R gene is highly polymorphic in European populations, with three common alleles highly penetrant for red hair, fair skin, inability to tan, and increased risk of developing skin cancers (RHC alleles). Carriers of low penetrant alleles (r) show intermediate responses to UV compared to MC1R-WT and RHC variants. Notably the induction of Dopachrome tautomerase (DCT) is compromised in RHC variant human melanocyte strains grown in culture. MC1R-WT melanoblast (MB) cells treated with DCT-siRNA and UVR showed reduced cell survival. Expression of p53/pp53 increased after DCT siRNA treatment, UVR, or both. MC1R-RHC variant MB strains showed negligible responses including reduction in DCT, with subsequent increases in p53/pp53. The effect of knockdown of DCT expression using lentivirus particles containing hairpin shRNA against human DCT in co-cultures of MC1R-WT MB cells and keratinocytes showed the reduction in DCT protein levels, changes in cell survival, and levels of p53, p38 and other proteins were not as great as that seen in monoculture. It is possible that keratinocytes conferred a protective function to the MB in co-culture, allowing increased survival post-UVR, and reduced DNA damage. Assay of H2O2 found an increase in MC1R-WT MB cells after DCT-siRNA and UVR in monoculture. Without functional DCT protein, the cells were more sensitive to oxidative as well as DNA damage. Loss of DCT results in reduced increases in DNA damage response proteins, indirectly contributing to the accumulation of mutations that may eventually lead to melanoma.
Department of Dermatology, Keio University School of Medicine, Shinjuku, Tokyo, Japan
Classic atopic dermatitis is complicated by asthma, allergic rhinitis, and food allergies, cumulatively referred to as atopic diseases. Recent discoveries of mutations in the filaggrin gene as predisposing factors for atopic diseases have refocused investigators’ attention on epidermal barrier dysfunction as a causative mechanism. The three musketeers of the epidermal barrier are as follows: the stratum corneum (air-liquid barrier), tight junctions (liquid-liquid barrier), and the Langerhans cell network (immunological barrier). Clarification of the molecular events underpinning epidermal barrier function and dysfunction should lead to a better understanding of the pathophysiological mechanisms of atopic diseases.
To evaluate the impact of filaggrin deficiency on skin barrier function, we generated filaggrin knockout (KO) mice. Filaggrin KO mice exhibited dry and scaly skin, but did not develop any spontaneous dermatitis. Permeability assay using calcein-encapsulating liposomes demonstrated that filaggrin-deficiency allows its penetration through SC. Barrier defect in filaggrin KO mice led to enhanced hapten-induced contact hypersensitivity responses and humoral responses to topically immunized protein antigens. Using 3D visualization method of epidermal TJs in mouse ear skin, we demonstrated that activated Langerhans cells (LC) extend their dendrites beyond tight junctions (TJ) to capture external antigens. We, then, demonstrated preemptive immunity is evoked against the captured antigens with a murine model for staphylococcal scaled skin syndrome (SSSS), a severe blistering disease caused by skin infection of exfoliative toxin (ET)-producing Staphylococcus aureus. Percutaneous humoral responses elicited by LCs highlight an efficient mechanism by which immunity to potentially pathogenic skin-surface microbes is provided without disrupting tight junction barriers, and demonstrate an important role for LCs in host defense in vivo.
H. Aoki, T. Kunisada
Gifu University Graduate School of Medicine, Gifu, Japan
Ionizing radiation (IR)-induced hair graying is caused by the ectopic differentiation of melanocyte stem cells (MSCs) in their niche located at the bulge region of the hair follicle. However, little is known about the relationship between MSC differentiation and keratinocytes during IR-induced hair graying.
We investigate the target of IR-induced hair graying.We found that both follicular MSCs and hair follicle keratinocyte stem cells (KSCs) were affected by IR by using immunohistochemical detection of γH2AX as a genotoxicity marker. We also found that KSCs prepared from irradiated mice were functionally affected by IR as indicated by their reduced colony forming activity in culture and the delayed hair cycle in vivo, while these effects were temporal. On the other hand, MSC population, which proliferated and differentiated to mature melanocytes, were persistently maintained after irradiation. In addition, the irradiated keratinocyte lineage cells suppressed the colony formation of MSCs in vitro. Furthermore, in the presence of irradiated keratinocytes, pigmented hairs were not regenerated by using hair reconstitution assay in vivo, while the irradiated MSCs can contribute to the pigmentation of the reconstituted hair.
The severe reduction of MSCs was thus attributed to the temporal loss of the niche activity of KSCs. These results provide a new insight that the primary target of IR sufficient to induce hair graying is not MSCs but follicular KSCs.
L A Skin and Aesthetic Clinic, New Delhi, India
Macular amyloidosis (MA) presents as poorly delineated grayish-brown macules with a rippled pattern associated with deposition of amyloid material in the papillary dermis and mild pigmentary incontinence. It is a chronic disease, whose treatment is disappointing. Most of the treatments are directed towards relief of pruritus which include antihistamines, topical corticosteroids, calcipotriol, UVB, etretinate or acitretin. But Q-switched NdYAG laser and fractional photothermolysis respectively reduce the melanin debris of upper dermis and shunt out the amyloid deposits and melanophages and seem promising treatments. There is paucity of studies on use of these modalities in treatment of MA.
To determine the efficacy of Q-switched Nd:YAG laser 1064 nm alone or in combination with Fractional Photothermolysis in reducing pigmentation in MA.
A pilot study was carried out on four subjects with clinically diagnosed and biopsy proven MA. They were treated with Q-switched NdYAG laser 1064 nm alone or Q switched NdYAG laser followed by Fractional Photothermolysis. A physician based quartile scoring system was followed to assess the efficiency, based on digital photographs before laser therapy and 8 weeks after treatment.
Q-switched 1064 Nd:YAG and Fractional photothermolysis are effective in reducing pigmentation in MA. Ninety percent of cases treated by 1064 nm and fractional photothermolysis had good or very good response, and for the 1064 nm-treated patches, 60% of cases had good or very good response.
Initial results of the pilot study are encouraging; with more improvement in MA with combined Q-switched NdYAG laser and fractional photothermolysis than 1064 laser alone.
P. Banan, L. L. Lin, D. Lambie, T. Prow, H. P. Soyer
School of Medicine, University of Queensland, Woolloongabba, Qld, Australia
Over the years diagnostic dermatology has relied on a precise history and an accurate clinical examination. However, in many cases skin biopsies, skin scrapings, blood tests or genetic tests are necessary to reach a final diagnosis. Punch biopsy is one of the primary procedures clinicians use frequently to obtain full-thickness skin samples to aid with histopathological diagnosis but due to the invasive nature of this procedure it is not embraced by patients. We have developed a minimally invasive skin biopsy device for small tissue collection and molecular diagnosis which can help early detection of melanomas and non-melanoma skin cancers as well as a refined diagnosis of inflammatory skin disease. It does not need any local anaesthetic or sutures and does not leave any visible scar behind. This miniature skin biopsy device causes minimal damage to the epidermis and superficial papillary dermis and therefore is not believed to interfere with the histopathologic diagnosis of the lesion. To confirm this we demonstrate the effects of microbiopsies on the histopathological diagnosis of five melanocytic lesions based on the evaluation of an experienced dermatopathologist.
K. A. Beaumont1, W. Weninger1,2,3, N. K. Haass1,2,3,4
1Centenary Institute, Newtown, NSW, Australia2Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia3Discipline of Dermatology, University of Sydney, Camperdown, NSW, Australia4Diamantina Institute, TRI, University of Queensland, Woolloongabba, Qld, Australia
Melanoma is the most aggressive and deadly form of skin cancer. Although early stage melanoma is curable by surgical removal, metastatic melanoma is notoriously difficult to treat. Understanding melanoma progression is therefore important. A recent theory proposes that melanoma cells undergo a reversible switch from proliferative to invasive phenotypes, and that this phenotype switch is responsible for driving the tumorigenic (proliferative) and disseminating (invasive) phases of disease progression. Microphthalmia-associated Transcription Factor (MITF), a master regulator of gene expression in melanocytic cells, is known to be responsible for the phenotype switch. However, the downstream MITF target genes directly responsible for mediating changes in melanoma cell behaviour are less well known. In a previous study Rab27a (an MITF target gene) was identified as a tumour ‘driver’ gene. Rab GTPases are a family of proteins that regulate vesicular transport. Altered expression of Rabs and aberrant protein trafficking has been associated with several types of cancer.
Our data revealed increased expression of Rab27a in melanoma cells with a proliferative phenotype. We show that shRNA knockdown of Rab27a inhibits growth and increases cell death, consistent with a role for Rab27a in melanoma cell proliferation and perhaps also programmed cell death. Rab27a knockdown altered cell morphology – suggesting a potential role in cell movement, however 2D migration was unaffected. Notably, Rab27a knockdown decreased invasion in our 3D melanoma spheroid model. In contrast, MITF knockdown resulted in increased invasion, while also decreasing proliferation, consistent with its role in phenotype switching. This suggests that while Rab27a may be involved in MITF-mediated proliferation, other MITF target genes mask the effect of Rab27a loss on invasion in MITF-depleted cells. Given that loss of Rab27a decreased both melanoma growth and invasion, this protein would be an attractive target for melanoma therapies.
D. C. Bennett1, C. J. Cairney2, L. S. Goodwin1, D. M. Kallenberg1, W. N. Keith2
1St George's, University of London, London, UK2CRUK Beatson Laboratories, University of Glasgow, Glasgow, UK
Cell senescence is a permanent arrest of cell division following excessive proliferation or cellular stresses. It has emerged as a major physiological mechanism for tumour suppression, and increasing evidence implicates it in normal and abnormal aspects of ageing. The commonest known gene for familial melanoma is CDKN2A, encoding p16, a key mediator of senescence; and benign moles (naevi) appear to be senescent melanocyte clones following oncogene activation. Senescence has even been suggested to explain aspects of vitiligo. Clinical aspects of cell senescence will be reviewed. Data will then be discussed from our microarray analysis of gene expression changes in human melanocytes cultured to reach replicative senescence, including melanocytes deficient in p16. Unsurprisingly, a prominent theme is the downregulation of cell cycle-related genes, less pronounced in p16-deficient cells. We also see a signature previously reported in other senescent cells, known as the SASP, or senescence-associated secretory phenotype. Here, a range of secreted cytokines, proteases and angiogenic factors become expressed, mimicking processes seen in inflammation. Signatures of interferon and TGFβ signalling are found, with some differences between melanocyte strains. Perhaps less predictably, the senescent cells also show signatures of neural differentiation, especially in p16-deficient strains. Possible clinical implications and parallels of the findings will be discussed.
A. Beverdam1,2, C. Claxton1, X. Zhang3,4, G. James1, K. F. Harvey3,4, B. Key1
1University of Queensland, St Lucia, Qld, Australia2School of Medical Sciences, Sydney, NSW, Australia3Sir Peter MacCallum Cancer Centre, Melbourne, Vic., Australia4Department of Pathology, University Melbourne, Melbourne, Vic., Australia
Tissue renewal is an ongoing process in the epithelium of the skin. We have begun to examine the genetic mechanisms that control stem/progenitor cell activation in the postnatal epidermis. The conserved Hippo pathway regulates stem cell turnover in arthropods through to vertebrates. Here we show that its downstream effector, yes-associated protein (YAP), is active in the stem/progenitor cells of the postnatal epidermis. Overexpression of a C-terminally truncated YAP mutant in the basal epidermis of transgenic mice caused marked expansion of epidermal stem/progenitor cell populations. Our data suggest that the C-terminus of YAP controls the balance between stem/progenitor cell proliferation and differentiation in the postnatal interfollicular epidermis. We conclude that YAP functions as a molecular switch of stem/progenitor cell activation in the epidermis. Moreover, our results highlight YAP as a possible therapeutic target for diseases such as skin cancer, psoriasis, and epidermolysis bullosa.
R. E. Boissy
University of Cincinnati College of Medicine, Cincinnati, OH, USA
imm_Tyrosinase inhibitors have been developed to partially interfere with melanin synthesis and have been utilized both medicinally for the amelioration of lesions in numerous hyperpigmentary diseases (melasma, lentigos, post inflammatory/wound healing hyperpigmentation, etc.) and cosmetically for general skin lightening. However, current commercially available inhibitors are ineffective or may cause various side effects (erythema, scarring, vitiligo) and possibly carcinogenic. Therefore, deoxyArbutin (dA) has been developed as an effective and safe tyrosinase inhibitor. The hydroxyl groups on the polar tail ring of arbutin were removed to create dA, thus facilitating its transepidermal penetration. dA was a more effective inhibitor of mushroom tyrosinase than hydroquinone, exhibiting a 10-fold lower Ki. In cultures of normal human melanocytes, tyrosinase activity was reduced 20% after 5 days of dA treatment and normalized 5 days after removing treatment. In a pigmented skin Guinea Pig model, the L-value was significantly raised in a dose dependant manner by 3 weeks of dA application and skin color normalized 8 weeks after halting application. Various metabolic and safety analyses were performed with optimal results. The efficacy of dA on human skin was demonstrated in three models; human zenografts on SCID mice, a clinical study assessing constitutive skin color, and a clinical study assessing facultative skin color. Second generation molecules of dA, including Fluro-dA and the L isoform of dA, were more effective than dA as tyrosinase inhibitors. dA has recently been brought to market.
E. Coates1,2, F. J. Moloney1,2, P. Guitera1,2,3, N. K. Haass1,2, K. Ho1,2, R. Khoury1, R. O'Connell4, L. Raudonikis5, G. L. Mann3,6, S. W. Menzies1,2
1Discipline of Dermatology, University of Sydney, Sydney, NSW, Australia2Sydney Melanoma Diagnostic Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia3Melanoma Institute Australia, North Sydney, NSW, Australia4Clinical Trials Centre, Unviersity of Sydney, Sydney, NSW, Australia5Raudonikis Database Services, Mount Colah, NSW, Australia6Westmead Millennium Insitute, University of Sydney, Westmead, NSW, Australia
A small number of individuals have a disproportionately higher new melanoma risk. Although early detection is crucial, there is no clear consensus for optimal screening in this cohort.
To determine the role of total body photography (TBP) and sequential digital dermoscopy imaging (SDDI) in detecting new primary melanoma in a high risk melanoma cohort over 5 years.Individuals fulfilling ≥1 of the following criteria were included: (i) CDKN2A/CDK4 mutation; (ii) ≥2 previous invasive melanomas; (iii) ≥1 previous invasive melanoma with Dysplastic Naevus Syndrome or (iv) ≥3 1st/2nd degree relatives with prior melanoma. Three hundred and twelve patients were screened including TBP at least 6-monthly over 5 years with short-term (approximately 3 months) and long-term (≥6 months) SDDI used.
Seventy-seven primary melanomas were detected, 16 at baseline and 61 subsequently over a median follow-up time of 3.5 years. TBP detected 37.7% and SDDI 39.3%. Post-baseline median Breslow thickness was in-situ (IQR = In-situ-0.60 mm), with five thick (>1 mm) melanomas identified. The overall benign/malignant excision ratio was 1.6:1 and 4.2:1 for melanocytic lesions.
Sixty-nine percent of non-baseline primary melanomas were detected within the first 2 years, with a cumulative new primary melanoma risk of 12.8% by year 2 and 18.7% by year 4. The melanoma incidence during the last three follow-up years was 51% of that observed during the first 2 years (P = 0.013).
Monitoring high risk patients with TBP and SDDI assisted with early primary melanoma identification. Use of these diagnostic techniques in this subpopulation is crucial to ensure optimal treatment and reduced associated mortality.
E. Coates1, S. N. Chee1, P. M. Lowe1,2
1Department of Dermatology, Royal Prince Alfred Hospital (RPAH), Camperdown, NSW, Australia2Discipline of Dermatology, University of Sydney, Sydney, NSW, Australia
Solid organ transplant recipients (SOTRs) have an increased skin cancer incidence up to 65 times that of non-transplant patients, as well as more aggressive variants1. Further research into this at-risk cohort in high ultraviolet radiation regions such as Australia is crucial to facilitate local evidence-based practice.One hundred and eighty-eight SOTRs attended the Royal Prince Alfred Hospital transplant dermatology clinic over an 18-month period from 2011 to 2013. Data obtained from attendances was collated in a Filemaker Pro© database specifically developed to sequentially record core patient information for subsequent analysis.
One hundred and twenty-two males and 66 females were evaluated with a median age of 58 (20–81). Sixty-four percent were born in Australia. One hundred and ten were renal, 79 liver and one lung SOTRs, with a median of 7.2 years post-transplantation at inclusion. Sixty-eight percent had seborrhoeic keratoses, 54% actinic keratoses and 25% warts at baseline attendance.
Thirty-three percent had a past history of ≥1 SCC (0–25), 32% ≥1 BCC (0–60), and 3% ≥1 melanoma. Histology from procedures undertaken demonstrated 38 SCCs, 54 SCC-in-situ, 60 BCCs and one melanoma. The overall SCC:BCC ratio was 1.5:1, with ratios of 1.5:1 for renal and 1.6:1 for liver SOTRs.Standardised incidence ratios were 26:1 for non-melanoma skin cancers (NMSCs), and were 31:1 for SCCs and 23:1 for BCCs. Twenty-one percent had their quality of life significantly impacted by skin concerns.
SOTRs in Australia are burdened with a high incidence of NMSCs, especially SCCs, as well as associated dermatoses and quality of life concerns.
D. Damian1, C. Andrew1, B. Thompson1, A. Martin2, B. Choy1, P. Fernandes-Penas1, R. Dalziell1, G. Halliday1
1University of Sydney, Camperdown, NSW, Australia2NHMRC Clinical Trials Centre, University of Sydney, Sydney, NSW, Australia
Skin cancer is four times as common in Australia as all other cancers combined, and its incidence continues to increase. Nontoxic, affordable chemopreventive agents are urgently needed to reduce skin cancer rates, especially in high risk groups. Nicotinamide (vitamin B3) is inexpensive, widely available and non-toxic. It inhibits phocarcinogenesis in murine models and mice and protects from ultraviolet radiation-induced immunosuppression in humans. Phase 2 studies have shown significant reductions in AKs and nonmelanoma skin cancer in sun-damaged volunteers. Nicotinamide seems to act by replenishes cellular energy and enhances repair of photodamaged DNA.
A Phase 3 randomised double blinded controlled trial is currently underway to determine whether oral nicotinamide can reduce basal cell and squamous cell cancers in individuals at high skin cancer risk. The ONTRAC study (Oral Nicotinamide To Reduce Actinic Cancer) is underway at Sydney's Royal Prince Alfred and Westmead Hospitals. Three hundred and eighty-six high-risk patients are randomised 1:1 to nicotinamide 500 mg or placebo twice daily for 12 months with skin checks every 3 months. The primary outcome is new nonmelanoma skin cancers by 12 months.
This study aims to evaluate the efficacy of nicotinamide as a safe, inexpensive, accessible and convenient chemopreventive agent against NMSC that is instantly translatable to clinical practice.
J. R. Davey1,2, B. Fergie1,2, L. M. Rhodes1,2, S. Hwang1,2, B. S. Daniels1,2, G. Mangos1,3, S.-J. Ho1,4, D. F. Murrell1,2
1University of New South Wales, Kensington, NSW, Australia2Department of Dermatology, St George Hospital, Sydney, NSW, Australia3Department of Renal Medicine, St George Hospital, Sydney, NSW, Australia4Department of Haematology, St George Hospital, Sydney, NSW, Australia
Renal failure has been reported as the second leading cause of death in Recessive Dystrophic Epidermolysis Bullosa (RDEB) and is also known to occur in Junctional Epidermolysis Bullosa (JEB). Common types of renal disease in EB include IgA mesangial cell nephropathy, post-streptococcal nephritis, and renal amyloidosis. Despite this, there are currently no studies identifying the prevalence of renal impairment in EB in Australia.
Using a dataset of 366 Australian EB patient charts, 142 available haematology, blood urea/creatinine and urine tests were analysed for evidence renal impairment. Patient data was used to determine the prevalence of renal impairment in the sample, and estimate the prevalence in the Australian EB population.
9.15% of patients had renal impairment with seven of 13 being RDEB or JEB sufferers. There were also five EB Simplex patients and one Dominant Dystrophic EB patient. Thus the prevalence in the overall Australian EB population could be estimated at 3.55%. Of the 13 patients with renal impairment, four were deceased and at least one of those deaths was due to renal failure. Of the remaining living patients, two are undergoing peritoneal dialysis, with one being considered for a live donor transplant.
Renal impairment can develop in severe EB subtypes at an early age. Therefore routine screening is important. Timely detection and treatment may not only increase life expectancy, but also improve wellbeing and quality of life in EB patients.
Queensland Institute of Medical Research, Oncogenomics Laboratory, Brisbane, Qld, Australia
Melanoma of unknown primary (MUP) is an unusual phenomena whereby a patient will present with metastatic melanoma, but upon clinical examination the original primary site is never identified. Currently there is minimal evidence to explain their mechanism of development. To address this issue, we have compiled exome sequencing data on 33 MUP (11 early passage cell lines in addition to 22 tumours from publically available datasets) with mutation profiling on an independent panel of 92 FFPE and fresh frozen MUP screening for frequently occuring hotspot mutations in genes including BRAF, NRAS, KIT, GNAQ and GNA11. The majority of MUP exhibited a high somatic mutation rate and high ratio of C>T/G>A transitions, indicative of a carcinogenic UV damage signature. Mutation profiling revealed a high rate of BRAF (45 of 103, 43.7%) and NRAS (32 of 103, 31.1%) mutation with a distinct lack of mutations associated with non-cutaneous melanomas (KIT, GNAQ, GNA11). Our results indicate that MUP have a mutation profile consistent to that of cutaneous sun exposed melanomas; this data suggests a significant proportion of MUP arise from regressed or unrecognized primary cutaneous melanomas or through de novo development in sentinal lymph nodes from migrating nevus cells.
B. Gabrielli1, S. Pavey1, M. Wigan1, A. Pinder1, N. Cloonan2, A. Burgess3, S. Wong4, R. A. Sturm4
1Translational Research Institute, The University of Queensland Diamantina Institute, Brisbane, Qld, Australia2Queensland Institute of Medical Research, Brisbane, Qld, Australia3Garvan Institute of Medical Research, Sydney, NSW, Australia4Institute for Molecular Biosciences, The University of Queensland, Brisbane, Qld, Australia
Ultraviolet radiation (UVR) is a major environmental factor in the development of melanoma and defects in mechanisms that respond to UVR damage in melanocytes must be major contributors to the development of melanoma. The recently sequenced melanoma genomes have revealed high level of UV signature mutations but there is little evidence that known DNA repair mechanisms responding to UV-induced damage are commonly defective in melanomas. We have identified a novel cell cycle response to suberythemal doses of UVR that is commonly defective in melanomas. Suberythemal UVR exposure has several effects on the cell cycle of cells in basal layer of the epidermis. It is strongly mitogenic, driving quiescent basal cells into cycle where they subsequently arrest in G2 phase checkpoint arrest. The G2 phase checkpoint arrest has utilises ATR/Chk1 checkpoint signalling activated by RPA foci associated single strand DNA (ssDNA) which accumulate during S phase as a consequence of WRN helicase-dependent bypass of a small number of unrepaired UVR-induced DNA lesion. WRN is required to prevent replication fork stalling and collapse when the fork machinery encounters these unrepaired lesions. The ssDNA gaps are repaired by a RAD18 dependent mechanism during G2 phase. Loss of this G2 phase response results in increased UV signature mutations. In an effort to fully define the checkpoint and repair responses we have used microarray analysis of polysome preparations from UV-induced G2 phase checkpoint arrested cells to identify genes differentially loaded onto polysomes in the checkpoint arrested cells, indicating genes that are being actively translated into protein, and also performed a SILAC proteomics screen to identify proteins whose levels are significantly altered in the checkpoint arrested cells. These screens have identified several classes of gene/proteins; checkpoint related, repair related, cytoskeletal and mitochrondrial/apoptosis. We have initially pursued the checkpoint and repair genes, and will report the analysis of these and their potential roles in the G2 phase checkpoint and repair response. Identification of all the genes contributing to this response will be critical to understanding the defects in this response in melanoma, and whether it is loss of this response that underlies the increased UV signature mutations found in melanomas.
D. Galloway, J. Carter, K. Paulson, O. Asanaviev, J. Ayer, A. Chapuis, C. Yee, P. Nghiem
Fred Hutchinson Cancer Research Center, Seattle Cancer Care Alliance and the University of Washington, Seattle, WA, USA
Merkel Cell Carcinoma (MCC) is a rare but deadly skin cancer. Its incidence is increasing in the elderly and in immunosuppressed populations. In 2008 a polyomavirus, MCPyV, was identified as a likely causal agent, a finding replicated in many laboratories. We showed that the seroprevalence of MCPyV was 65–80% in the general population. However, antibodies to the MCPyV T antigens were restricted to cases with MCC. Importantly a rise in the titer of these antibodies could predict recurrence. Some patients were shown to have CD8+ T cells directed towards T antigen epitopes. Expansion of tetramer-isolated MCPyV specific T cells was developed for immunotherapy. We will present data for one patient with increasing T antigen titers who was shown to have pancreatic metastases. Following treatments to upregulate HLA expression and infusions of MCPyV specific T cells metastatic lesions regressed. These data indicate that immunotherapeutic approaches to eliminate MCPyV expressing tumor cells can improve patient outcomes.
E. Y. Gan, B.-K. Goh
National Skin Centre, Singapore, SGP
Noncultured cellular grafting is an advanced surgical technique for treatment of vitiligo refractory to conventional therapy. The aim of our study was to evaluate the long-term results of vitiligo patients who underwent noncultured cellular grafting at our centre over a 3-year period.
Medical records were reviewed for clinico-epidemiological characteristics and repigmentation data. Repigmentation of vitiligo was based on clinical assessment and standardized digital photography and was graded as ‘poor’, ‘fair’, ‘good’ and ‘excellent’, corresponding to 25% intervals on a scale of 0–100% repigmentation.
There were 83 patients (46 females, 37 males) with a mean age of 35 years and 89% were Fitzpatrick skin type IV. Mean duration of vitiligo prior to grafting was 100 months. Forty-nine patients (59%) had non-segmental, 33 (40%) had segmental and one had mixed vitiligo. A total of 112 grafting sessions were performed. Of these, 90 were primary, 21 were repeated and there was one staged grafting. The most commonly grafted sites were face (49%), trunk (17%) and neck (11%). Good to excellent repigmentation was seen in 58%, 64%, 71% and 68% of patients at 3-, 6-, 12- and 24-month follow-ups respectively. In the face and neck subgroups, more than 70% achieved good to excellent repigmentation at 12 months, compared to only 50% in the scalp and acral subgroups. More patients with segmental vitiligo (83%) achieved good to excellent repigmentation at 12 months compared to non-segmental vitiligo (64%). Loss of initially achieved repigmentation occurred in seven patients (8%) due to active disease. No patients experienced significant scarring or adverse reactions.
Noncultured cellular grafting is a safe procedure. More than 70% had good to excellent repigmentation at 12 months, with most patients achieving maximal repigmentation by then. Better repigmentation rates were seen on the face, neck and in segmental vitiligo.
Dr. D.Y. Patil Medical College & Hospital, Navi Mumbai, Mahar, India
Melasma is a common, acquired, circumscribed hypermelanosis of sun- exposed skin. It presents as symmetric, hyperpigmented macules having irregular, serrated, and geographic borders. The most common locations are the cheeks, upper lips, the chin, and the forehead, but other sun-exposed areas may also occasionally be involved. The main causative factors among the male melasma patients appeared to be sun-exposure and family history. Mustard oil application could also contribute to this condition. Most Indian patients do not like to use sunscreens due to oily sticky nature. Chemicals in sunscreens absorb light energy and release heat as exothermic reaction. This causes heat on face which is not well tolerated by patients. Physical sunscreens do not release heat but whitening on face is a disadvantage. Sunscreens can be mixed with calamine lotion or zinc to minimize these side effects.We studied new triple combination of 2% hydroquinone, 0.05% Tretinoin and 0.01% fluocinolone acetonide cream with glycolic acid peels, in the treatment of melasma. The results were noted after four peels, in the form of more than 50% improvement in pigmentation in five out of 10 patients. Two patients showed more than 75% improvement.
Dr. D.Y. Patil Medical College & Hospital, Navi Mumbai, Mahar, India
Sun exposure has a pathogenic effect on the skin in the form of photo aging, melasma, tanning and others.
The aim of this study was to evaluate the awareness of Indian population regarding effects of sun exposure, as well as to study their sun-protective attitudes and practices.
A cross-sectional population-based survey using specially devised questionnaire on a stratified random sample of general population in Navi Mumbai, Maharashtra was done between January and February 2012.
Five hundred persons participated in the study, out of which 255 were females and 245 were males. Their average age was 33.5 years. Out of 500, 224 (44.8%) subjects were aware of the damage caused by sun exposure. Also, 82.2% subjects were aware about sunscreens. Despite that, only 69 (13.8%) participants used sunscreens. Commonest factor for non-usage of sunscreens was its cost-ineffectiveness, as said by 265 (61.48%) participants and side effects such as acne (68.13%).
This study has indicated a low rate of awareness regarding sun exposure. Also various factors like cost, sticky consistency, need for multiple applications and climatic conditions limit widespread use of sunscreens by the general population, despite sufficient awareness. This necessitates the need for development of sun awareness policies and newer sunscreen preparations.
B. K. Goh
Skin Physicians Pte Ltd, Mount Elizabeth Medical Centre, Singapore
Melasma, one of the most common acquired hyperpigmentary disorders among Asians, remains a therapeutic challenge. Although much is known about its predisposing and aggravating factors, including female gender, genetics, sun-exposure and hormonal influence, its pathogenetic mechanisms are not fully understood; however much progress has been made in recent years.
What began as an immunohistopathological understanding of this condition, from the increase in melanogenesis and melanocytosis to the disturbance of the basement membrane and increase in dermal vasculature and mast cells, has evolved into molecular elucidation of the signaling pathways. Upregulation of melanin biosynthesis-related genes and melanocyte markers demonstrated through transcription analysis of melasma lesions, participation of non-coding RNA like H19 gene, involvement of the Wnt signaling pathways (reduced WIF-1 expression), and overexpression of VEGF are some examples. However these innovative findings have not or are yet to be translated into clinical interventions.
Skin lightening agents and sun-protection still remain as the therapeutic mainstay for melasma. Combination therapy based on Kligman formula has achieved better results than monotherapy. However a subset of patients with melasma remains refractory to this intervention. Addition of oral tranexamic acid can be helpful, and its benefits have been consistently reproduced by different investigators. How this antifibrinolytic agent works in reducing melanogenesis specific to melasma still bewilders researchers, but its effects on mast cells and dermal vasculature have been demonstrated. The demand for laser and light interventions for treatment of melasma is high, driven partially by the economic returns of such procedures. The results from intense pulse light are inconsistent, but low-fluence, repeated ‘laser toning’ has achieved satisfactory lightening of melasma but with possible risks of rebound hyperpigmentation and guttate leukoderma. Fractional laser ablation, whether carbon dioxide or thulium fiber, has also shown to be beneficial.
Despite all these interventions, melasma relapses and continues to vex patients and challenge clinicians. It remains an art to treat melasma.
G. J. Goodman
Dermatology Institute of Victoria, South Yarra, Vic., Australia
The major endogenous pigment in the skin is melanin and tattoo ink the major exogenous pigment. Melanin, unlike other skin tissue chromophores does not have defined absorption peaks; instead increasingly absorbing shorter wavelengths from 1000 nm through to the ultraviolet spectrum. Thus, a variety of laser and light wavelengths can be used to target melanin, although none is entirely specific for this chromophore: Many lasers in the wavelength band width of 500–800 nm (532 nm-KTP, 595 nm-PDL, 694 nm-Ruby, 755 nm-alexandrite) and even the 1064 nm QS Yag laser may be used..
The band of wavelength that needs to used depends how superficially the pigment resides. Superficial (epidermal) pigment is encountered in solar lentigines, ephelides, café-au-lait macules and seborrhoeic keratosis and may be selectively targeted with long pulsed as well as Q Switched lasers. Water seeking infrared ablative lasers such as Erbium and CO2 and the non ablative fractional Thulium lasers are all useful in the treatment of widespread pigmentation when the pigment is within reach. Similarly, IPL is an effective ‘broad brush’ approach for many cases of widespread epidermal pigmentation and dyschromias.
However, deep (dermal) pigment is a feature of melanocytic naevi, blue naevi, naevi of Ota/Ito, Hori's naevus, drug-induced hyperpigmentation requires both long wavelengths to achieve the depth required without superficial absorption and short pulse duration to target pigment specifically and safely without drift to unintended targets. Melasma remains a conundrum for all laser users and will be one of many conditions that will be highlighted as those not well treated by lasers.
E. Hacker1,2, M. Kimlin2, Z. Boyce2, L. Wockner1, T. Pollak1, N. Hayward1, D. Whiteman1
1Genetics & Population Health Division, Queensland Institute of Medical Research, Brisbane, Qld, Australia2Queensland University of Technology, Kelvin Grove, Qld, Australia
Australia has the highest rate of skin cancer in the world. Over 434 000 non melanoma and 10 000 melanoma skin cancers are removed in Australia each year. This costs the Australian health system $300 million annually. Melanoma, the most lethal form of skin cancer, arises from melanocytes. It is postulated that the proliferative response of melanocytes following sun exposure may play a role in melanoma susceptibility. Whether this proliferative response is the same for all people, and whether the effect is modified by topical sunscreens, is unknown. In a clinical trial designed to improve our understanding of the interplay between sun exposure, genetic susceptibility and melanoma risk, we recruited 57 participants and used immunohistochemistry to compare the proliferative response of skin cells in vivo. We found that 14 days after exposure of human skin to two MED solar simulated ultraviolet radiation (SS-UVR), there were twice as many epidermal melanocytes compared with unirradiated skin. We observed that sunscreen protected against the proliferative response of skin cells following SS-UVR. This study quantified the proliferative response of melanocytes to SS-UVR among people with different phenotypes and genotypes, and found people carrying germline MC1R variants had a 50% lower melanocyte response 14 days after SS-UVR than people with wild-type MC1R. This study confirms the role of UVR in initiating melanocytic proliferation, demonstrates the effectiveness of sunscreen in preventing these responses, and implicates MC1R as a key mediator in this process.
G. M. Halliday1, P. W. Sou1, N. M. M. Hassan1, R. A. Scolyer2, N. Di Girolamo3, G. Lyons1
1Dermatology Bosch Institute, Royal Prince Alfred Hospital and University of Sydney, Sydney, NSW, Australia2Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, NSW, Australia3School of Medical Sciences, University of NSW, Sydney, NSW, Australia
SWI/SNF unravels DNA from its tightly compacted chromatin structure to make it accessible for processes such as transcription and DNA repair. The energy for this process derives from ATP and all SWI/SNF complexes contain one of two mutually exclusive ATPase subunits, Brm or Brg-1. Brm, like other subunits of SWI/SNF is therefore a master regulator of multiple cellular processes. We identified a novel hotspot mutation in Brm in human skin cancer, and also found that both Brm and Brg-1 protein have low expression in human skin cancers. Mice were exposed to a low dose ultraviolet (UV) irradiation protocol that caused few skin tumours in wild-type mice. Brm−/− mice with both p53 alleles intact had an increased incidence of skin and ocular tumours compared to Brm+/+p53+/+ controls. Brm loss in p53+/− mice did not further enhance skin or ocular cancer incidence beyond the increased photocarcinogenesis in p53+/− mice. However, the skin tumours that arose early in Brm−/−p53+/− mice had a higher growth rate. Unexpectedly, Brm−/− inhibited UV-induced immunosuppression, which would be predicted to reduce rather than enhance photocarcinogenesis. In conclusion, the absence of Brm increased skin and ocular photocarcinogenesis. Even when one allele of p53 is lost, Brm has additional tumour suppressing capability. As the Brm gene is mutated in human skin cancer and protein expression is reduced, and it protects from the damaging effects of UV, it may be important in human skin and ocular carcinogenesis.
S. Hames, S. Sinnya, J.-M. Tan, P. Soyer, T. Prow
Dermatology Research Center, School of Medicine, University of Queensland, Brisbane, Qld, Australia
Actinic keratoses are important precursor lesions associated with the development of squamous cell carcinoma. We propose and test a method of automatically locating actinic keratoses in high quality clinical photographs. The method focuses on detecting the erythema as peaks in a colour space that combines the blue and red chrominance of the input image. Test images were acquired from two groups of six volunteers – one group with severe photodamage and the other group with low photodamage. Images were acquired of each dorsal hand and forearm as well as front, left and right views of the face. Performance was assessed by comparing the automated method with an expert dermatologists assessment of the same digital image files. It was found that there was good colocalisation between the automated method with the automated method matching 65% of the dermatologist identified lesions, though there were significant numbers of false positives, and lesions characterised by scale and horn formation were not identified by the automated method. Comparison of the automatic method on low and high photodamage groups showed a significantly (P < 0.05) lower number of lesions in the low photodamage group, indicating that while this method does not ideally match the expert in all cases, automated detection is consistent with the level of photodamage.
L. Hang1, L. L. Lin1, A. P. Raphael1, D. Sundh1,2, H. P. Soyer1, T. W. Prow1
1Dermatology Research Centre, University of Queensland, Brisbane, Qld, Australia2Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
Zinc oxide nanoparticles (ZnO-NP) are commonly used in sunscreens and their use is currently debated due to potential toxicity. The removal of topically applied nanoparticles is an area that has been unexplored, but is relevant due to safety concerns, and the lack of suitable data to support ZnO-NP safety on damaged skin. We aimed to investigate the penetration and removal of ZnO-NPs from intact and wounded in vivo and ex vivo human skin.
Ex/in vivo human skin was damaged by tape-stripping and/or microneedling followed by application of 2 mg/cm2 ZnO-NP (approximately 60 nm in size). After 2-h incubation the skin was washed three times using soap and water. Multiphoton tomography was used to assess ZnO-NP signal before and after each wash.
Washing once removed more than 85% of the detectable ZnO-NP signal (P < 0.05) from ex vivo intact skin and 83% from ex vivo tape-stripped skin (P < 0.05). However, only 28% of ZnO-NP signal (P = 0.5) was removed from puncture sites. A similar trend was found in vivo with removal of 85% of the detectable ZnO-NP signal (P < 0.05) from intact skin and 93% from tape-stripped skin (P < 0.05).
Soap and water washing is effective for the removal of ZnO-NPs from the superficial layers of intact and tape-stripped (×40) skin, but not from puncture wounds.
P. Hersey, S. Gallagher, B. Mijatov, T. Becker
University of Sydney, Sydney, NSW, Australia
Development of resistance to therapy is common in melanoma irrespective of whether the treatment is with BRAF/MEK inhibitors, immunotherapy or chemotherapy. We have shown previously that pan histone deacetylase inhibitors (HDACi) can overcome, at least partially, resistance to TRAIL and to selective BRAF inhibitors like Vemurafenib in assays of apoptosis. This was associated with upregulation of the pro apoptotic BH3 only protein Bim and downregulation of XIAP, Bcl-X and Mcl-1. The development of resistance in melanoma to selective BRAFi and immunotherapy with T cells can, in some melanoma, be attributed to paracrine or autocrine stimulation of resistance pathways. We show that this appears related to activation of NF-kB and its downstream target genes. We further show that this activation is dependent on co activation of NF-kB and its target genes by Bromodomain and extraterminal domain (BET) protein containing complexes that read and inhibit acetylated histones in chromatin. The role of the ATPase BRG1 in this inhibition is under study but may implicate the SWI/SNF complex in these interactions. Treatment of melanoma with BET protein inhibitors I-BET151 or JQ1 have proven effective inhibitors of NF-kB and trigger apoptosis of melanoma by upregulation of Bim and regulation of other apoptosis regulators. We suggest that understanding the interplay between transcription factors and chromatin co-activators provides treatment approaches that may be effective against a range of inhibitory mechanisms in melanoma.
S. Hodgson, Z. Neufeld, K. Khosrotehrani
University of Queensland, Qld, Australia
Wnt signalling and its importance in hair follicle development and differentiation has been largely described at the individual cell or hair follicle level. We aimed to generate a model for in vivo quantitative monitoring of beta catenin activity in the skin to follow wnt signalling in mouse back skin at the macroscopic level.
We generated mice harbouring a luciferase reporter gene under the control of beta catenin binding sites TCF/LEF inducible promoter. Using in vivo bioluminescence imaging, we were able to track beta catenin activity in the skin from P1 in rostro-caudal waves. The signal peaked at P9 with >400 fold increase. Histological assessment allowed attributing signal levels to specific phases of the hair follicle cycle. Furthermore, we were able to reproduce all macroscopic features of hair follicle biology, such as propagating waves, border stability, refractory telogen and random initiation points. In adults, hair plucking or cyclosporine predictably induced beta catenin activity with an intense signal appearing 6 days after initiation.
We took advantage of the possibilities offered by this model to assess the level of beta-catenin activity in situations of health and disease. During pregnancy, the peak of beta-catenin activity was not modified. However, there was a delay in the natural entry into anagen. Similarly wound healing and models of defective hair growth affected the level of bioluminescence. Finally, mathematical modelling of beta-catenin signalling based on a generic dynamical mechanism for producing oscillatory behavior in activator-inhibitor system allowed to reproduce the characteristic patterns of hair follicle progression and cycling in a two dimensional grid.
In conclusion, tracking Wnt signalling macroscopically in the mouse back skin allows a detailed understanding of hair cycle progression and could be used for screening drugs or molecular targets.
MelanoSite, New Delhi, India
With conventional non cultured epidermal suspension transplantation procedure especially with warm trypsinization one can manage patches with size ≤300 cm2 in a single sitting.
To establish that successful repigmentation is possible in patches ≥500 cm2 size with single sitting non cultured epidermal suspension transplantation using cold trypsinization.Twenty-three stable vitiligo patients (17 females, six males; age range 18–29 years) with ≥2 year stability were included. Thirty-four patches ≥500 cms size (single or multiple patches with total area) selected and treated in single sitting. Non cultured epidermal cells obtained from split thickness skin graft after the overnight cold trypsinization method was suspended in phosphate-buffered saline (PBS). This suspension was transplanted to the dermabraded recipient area with a modified procedure using chlorhexidine gauze, PBS soaked gauze and Tegaderm. Collagen dressing was not used in the procedure. During 1 year follow-up period results were assessed based on the extent of repigmentation, colour match and adverse events.Average area treated per procedure was 636.74 cm2. Repigmentation was successful (repigmentation >75%) in 33/34 lesions (97.05%). Color match at 1 year was excellent in 32/34 lesions (94.11%). No significant adverse events were reported.
This study shows that with the overnight cold trypsinization method of non cultured epidermal suspension transplantation procedure it is possible to treat patches of size ≥500 cm2 in a single sitting. Higher yield of cells with cold trypsinization could play a significant role. Non cultured epidermal suspension transplantation is an effective and safe option for larger areas.
University of Hong Kong, Hong Kong
In East Asian culture, lighter skin tone is more preferable among the female population. Moreover, irregular hyperpigmentations such as solar Lentigines, melasma, and freckles are of concern in both Asia and western countries. Due to the high demand and potential for skin whitening products, the search for safe and effective depigmenting agents has become a popular research area. Hypopigmentation agents, also known as skin-whitening agents, are widely used to lighten the skin tone or treat abnormal hyperpigmentation by inhibiting the melanogenesis process. Cudrania tricuspidata, a deciduous tree growing in East Asia, is rich in isoprenylated xanthones and flavonoids. According to the previous research from our laboratory, oxyresveratrol and trans-dihydromorin purified from the twigs of Cudrania tricuspidata, are potent mushroom tyrosinase inhibitors, and may effectively inhibit the melanogenesis process in melanocyte. In this study, two cell cultures are exploited: B16 cell and melan-a cell. B16 murine cell line is selected as it is widely used in current research for the search of melanogenesis inhibitors. However, as B16 cells are highly metastatic tumor cells, a normal epidermal cell-based system should also be considered. Thus, Melan-a cell line, a normal epidermal melanocyte is utilized. The hypopigmenting effects of oxyresveratrol and trans-dihydromorin in these two cell lines are evaluated by measuring the melanin content in SRB assay. Their mechanisms of action are also identified by examining the inhibitory effects on cellular tyrosinase activity, l-dopa oxidative activity, as well as the related gene mRNA expression and protein expression.
Y. Ishii-Osai1, T. Yamashita1, Y. Tamura2, N. Sato2, A. Ito3, H. Honda4, K. Wakamatsu5, S. Ito5, E. Nakayama6, M. Okura1, K. Jimbow1
1Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan2Department of Pathology 1, Sapporo Medical University School of Medicine, Sapporo, Japan3Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka, Japan4Department of Biotechnology, School of Engineering, Nagoya University, Nagoya, Japan5Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Japan6Faculty of Health and Welfare, Kawasaki University of Medical Welfare, Kurashiki, Japan
Because melanogenesis is inherently toxic and uniquely expressed in the melanin-containing cells, therapy focusing on melanogenesis can become a specific molecular target to melanoma cells without significant systemic side effects. N-propionyl-4-S-cysteaminylphenol (NPr-4-S-CAP) is selectively incorporated into melanoma cells for the substrate of tyrosinase and causes cytotoxicity against them. We have previously demonstrated that NPr-4-S-CAP selectively induces apoptosis to pigmented melanomas accompayed by caspase 3 activation and DNA fragmentation and how reactive oxygen species (ROS) were generated in this's process (Ishii-Osai Y et al: J Dermatol Sci, 2012). It has been reported that ROS mediates inductions of p53, the redox sensor Trx-ASK1 and the Fas/Fas ligand that may result in the apoptotic cell death.
In this study, we aimed to elucidate the detection of the molecules and the analysis of molecular mechanisms related to apoptosis by NPr-4-S-CAP. Western blot analysis and real-time RT-PCR did not detect increased Nuclear factor E2-relted factor 2 (Nrf2) or Heme Oxygenase-1 (HO-1) in two human melanoma cell lines cultured in the presence of NPr-4-S-CAP. We, then, analyzed cDNA species induces in the NPr-4-S-CAP treated 70W melanoma cells by using cDNA microarray. Results indicated that expressions of 13 candidate genes includinding TNF receptor- and p53-associated proteins were increased in association with NPr-4-S-CAP-mediated apoptosis. This suggests that in the process of NPr-4-S-CAP-mediated apoptosis, several cellular genes related to apoptosis induced but protective Nrf2/HO-1 genes were not induced in human melanoma cells.
Y.-M. Jeong1, H.-S. Jeong1, J. S. Shin1, H. A. Kim1, K.-C. Park2, K. J. Baek1, N. S. Kwon1, H.-Y. Yun1, D.-S. Kim1
1Department of Biochemistry, Chung-Ang University College of Medicine, Seoul, Korea2Department of Dermatology, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, Korea
Recently, we demonstrated that leucine-rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the role of LGI3 on melanocytes still remains unknown. The present study showed that LGI3 could serve as a stimulator for melanogenesis without affecting cell viability and cell proliferation. To determine the effects of LGI3 on melanin synthesis, Mel-Ab cells were treated with recombinant LGI3, and the melanin content was measured. Interestingly, LGI3 promoted the melanin synthesis in Mel-Ab cells. Western blot analysis demonstrated that LGI3 induced Akt activation and glycogen synthase kinase 3β (GSK3β) phosphorylation. Moreover, upregulation of microphthalmia-associated transcription factor (MITF) and tyrosinase mRNA and protein expression was exhibited in Western blotting and RT-PCR. Our results suggest that LGI3 promotes melanogenesis through the upregulaion of MITF and tyrosinase expression.
L. N. Jones, R. D. Sinclair
Department of Dermatology, Epworth Research Institute, Richmond, Vic., Australia
Publish consent withheld.
J.-W. Jung1, P. J. Taylor2, F. Simpson1, I. H. Frazer1, J. W. Wells1
1UQ Diamantina Institute, Brisbane, Qld, Australia2Department of Clinical Pharmacology, The University of Queensland, Brisbane, Qld, Australia
Organ transplant recipients have an increased risk of non-melanoma skin cancer development. Immunosuppressive agents, given to these patients to prevent organ rejection, are widely recognised to play a key role in the increased incidence of malignancy. However, different immunosuppressive agents, such as rapamycin and tacrolimus, which both prevent organ rejection, appear to convey different risks of tumour formation. Using a UV-induced murine model of squamous cell carcinoma (SCC); HPV38 E6E7, we hope to determine whether these differences are due to a direct influence of these drugs on tumour cells, or an indirect influence mediated via differences in mechanisms of immune suppression. When added to mice diet at a concentration of 20 mg/kg over 8 weeks, rapamycin did not result in any gross health problems. A steady state concentration of 3–4 ng/ml of rapamycin in the blood could be detected by LC-MS/MS analysis; however adoptively transferred transgenic T-cell proliferation and the rate of allogenic skin graft rejection appeared unaffected in these mice. We will shortly begin testing the addition of 100 mg/kg of rapamycin in the diet to see whether we can reach the immunosuppressive threshold, which in humans is 15–20 ng/ml. We will also begin work to establish optimal UV dosing schedules for SCC development. Through this project, we hope to expand our knowledge base regarding mechanisms of SCC formation in immunosuppressed patients.
H. Kakavand1,2, O. Crainic3,4, T. Lum3, J. F. Thompson1,2,5, G. V. Long1,2, R. A. Scolyer1,2,3
1University of Sydney, Camperdown, NSW, Australia2Melanoma Institute Australia, North Sydney, NSW, Australia3Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia4Canterbury Health Laboratories, Canterbury, NSW, Australia5Department of Melanoma and Surgical Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
BRAF V600E mutations are considered an early event in the development of melanocytic tumours with reported rates of up to 82% in common acquired naevi. About, 30–50% of melanomas are reported to arise within a pre-existing naevus.The aims of this study were to determine the frequency of BRAF V600E mutations in this group and the concordance between the primary melanoma and the associated naevus.
Formalin-fixed, paraffin-embedded (FFPE) tissue samples from 30 patients containing primary melanomas arising in association with a naevus were identified. Two 4 μm sections were cut and a haematoxylin and eosin slide was assessed for the presence of primary melanoma and naevus cells within the same FFPE block. Immunohistochemical staining was carried out with the VE1 antibody (Spring Bioscience) as per the manufacturer's instructions. The VE1 slides were scored by two pathologists (OC and RS) as either positive or negative for the BRAF V600E mutation.
Sixty-three percent of cases stained positive for BRAF V600E, and all cases were concordant between the primary melanoma and the associated naevus. In the VE1 positive cases the intensity of staining was markedly higher in the melanoma cells compared with the naevus.
This is the first study, as far as we are aware, assessing immunohistochemical staining of BRAF V600E in melanomas arising in association with a naevus. The concordance between the primary and the naevus in mutation status suggests that in these cases the melanoma arose from the naevus and these melanomas have a high frequency of BRAF V600E mutations.
H. Kawasaki1, K. Nagao1, A. Kubo1,2, T. Hata1,3, H. Mizuno4, T. Yamada5, M. Amagai1
1Dermatology, Keio University School of Medicine, Tokyo, Japan2The Center for Integrated Medical Research, Keio University School of Medicine, Tokyo, Japan3KOSÉ Corporation, Tokyo, Japan4Brain Science Institute, RIKEN, Wako, Japan5Pathology, Keio University School of Medicine, Tokyo, Japan
Loss-of-function mutations in filaggrin gene are major predisposing factors for atopic dermatitis (AD). Although various reports suggest that filaggrin contributes stratum corneum (SC) barrier formation, the lack of filaggrin-null animal model has hampered progress to elucidate the pathogenic mechanism. Flaky tail/matted (ft/ma) mice have been used as a model of filaggrin deficiency, however these mice do not present complete loss of filaggrin and have unknown gene mutation ‘matted’. Therefore, we generated filaggrin-null (Flg−/−) mice and evaluated the impact of filaggrin loss in vivo. Flg−/− mice showed dry and scaly skin, but did not develop spontaneous dermatitis, in contrast to ft/ma mice. Despite of decreased natural moisturizing factor level in Flg−/− mice, which are degradation products of filaggrin, water content and transepidermal water loss were not affected in Flg−/− SC, indicating that hydration status of SC might not correlate with dry skin manifestation and natural moisturizing factor level. Histological observation suggested the premature detachment of cornified layer in Flg−/− mice, and tape stripping analysis demonstrated that Flg−/− SC were more fragile and more susceptible to physical stress. Loss of keratin pattern, which is important for maintaining SC integrity, was observed in Flg−/− SC using electron microscopy, and it is likely attributable to fragility in Flg−/− SC. Increased penetration of substances into SC was confirmed by permeability assay using liposome-encapsulated calcein. Barrier defect in Flg−/− mice led to enhanced hapten-induced contact hypersensitivity responses and humoral responses to topically immunized protein antigen, but the responses in ft/ma mice were constantly stronger, suggesting an additional role for matted. Our Flg−/− mice represent pure filaggrin deficiency, and should provide a valuable tool to dissect the function of SC, especially in the context of AD.
M. S. Kim1, T. J. Park2, H. R. Kim1, S. Y. Park1, J. Y. Park1, W. J. Choi1, K. C. Park3, J. P. Ortonne4, H. Y. Kang1
1Dermatology, Ajou University School of Medicine, Suwon, Korea2Biochemistry, Ajou University School of Medicine, Suwon, Korea3Dermatology, Seoul National University Bundang Hospital, Seongnam, Korea4Dermatology, Archet-2 Hospital, Nice, France
Wnt signaling plays a role in differentiation as well as development of human melanocytes. Using microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor-1 (WIF-1) compared to perilesional normal skin. In this study, the expression and functional role of WIF-1 on melanocytes were investigated. WIF-1 was expressed in both melanocytes of normal human skin and melanocytes in culture. The up-regulation of WIF-1 on cultured normal human melanocytes significantly induced the expressions of MITF and tyrosinase which was associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF-1, WIF-1 siRNA reduced melanogenesis of the cells. Moreover, the treatment of WIF-1 on ex vivo cultured skin showed the increased pigmentation of the epidermis. These findings suggested that melanocytes express WIF-1 constitutively in vivo and in vitro and WIF-1 promotes the melanogenesis in normal human melanocytes.
M. S. Kim1, J. Y. Son2, I. Jou2, H. Y. Kang1
1Dermatology, Ajou University School of Medicine, Suwon, Korea2Department of Pharmacology, Ajou University School of Medicine, Suwon, Korea
Publish consent withheld.
H. Kudo, M. Jinnin, H. Ihn
Kumamoto University, Kumamoto, Japan
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. There is an urgent need to develop new therapeutic approaches against skin fibrosis. Although intravenous immunoglobulin (IVIG) may be one of the promising treatments, the mechanisms by which IVIG improves the fibrosis of SSc remain unknown.
To compare the cytokine profile in the sera and skin of SSc patients before and after IVIG administration, and try to clarify the mechanism of the effect of IVIG.
Each three patients received 5-day administration of IVIG, or the same dose of physiologic saline for placebo. Cytokine levels were determined by ELISA array, immunostaining, and real-time PCR.
Cytokine array revealed that the serum levels of IFN-g and IL-12, representative Th1 cytokines, were increased by IVIG treatment, but not by placebo. The percentage of IFN-g- and IL-12-positive cell number/CD4-positive T lymphocyte cell number was also significantly increased by IVIG in SSc skin. Furthermore, mRNA expression of IFN-g and IL-12 in SSc skin tissue was significantly up-regulated after IVIG treatment.
The expression of Th1 cytokine is reported to be decreased in SSc. Our study suggested IVIG recovered the suppressed levels of Th1 cytokines, and that the treatment improves skin fibrosis by correcting the Th1/Th2 balance. In order to facilitate the clinical use of IVIG for SSc, it is necessary to perform a lager study in the future.
P. K. Kumar
DIRDS, Faridkot, Punjab, India
Periorbital Hyperpigmentation or Dark circles around eyes occur due to a combination of reasons.Common causes of dark circles around eyes
1. Thinning skin
2. Allergies and Hayfever
4. Fluid Retention
5. Lack of sleep
6. Iron Deficiency Anemia
Management of dark circles around eyes There are some points that may help to temporarily diminish the appearance of dark circles under the eyes, and prevent dark circles from worsening.
1. Be sure to drink enough water.
2. Wear sunscreen with a minimum of SPF 30 under the eyes to prevent skin weakening caused by sun damage.
3. Get plenty of rest.
4. Apply cool cucumber slices over closed eyes for 15 min.
5. Be careful not to consume too much dietary salt.
6. Use light coloured eye makeup
1. Treat the underlying cause with the appropriate therapy.
2. Treat secondary causes appropriately
3. Avoid aggravating or causative factors
4. Chemical peels
Improves skin texture, elasticity and dermal thickness; reduces pigmentation and improves absorption of topical agents. Glycolic acid, Kojic Acid, Phytic Acid, Azelaic Acid.
5. Depigmenting agents
Reduce melanin synthesis, storage and deposition. Kligman regimen is the most effective treatment. It consists of Topical Tretinoin, Hydroquinone and mild steroid.
6. Treat underlying allergies or eczema
7. Skin laxity or Excess Skin
Excess skin can be removed surgically with a blepharoplasty.Skin tightening devices using either radiofrequency or infra-red light source.
8. For Deep set or Sunken Eyes: Use Filling agents and Fat repositioning
R. Kumar, D. Parsad, S. Bhardwaj, D. Kaul, A. J. Kanwar
Post Graduate Institute of Medical Education and Research, Chandigarh, UT, India
Vitiligo is characterized by disappearance of melanocytes and one of the important goal of treatment is replenishing the melanocytes from existing reservoirs. Epidermal melanocytes are differentiated cells with low proliferative capacity. The low proliferative capacity of melanocytes contributes significantly to the difficulty of repigmenting vitiligo skin lesions. Dermal stem cells from human skin are multipotent and are capable of differentiating into melanocytes. These dermal stem cells are a reservoir for melanocytes in the more protective dermis of the skin.
Skin grafts were collected from lesional and uninvolved skin of vitiligo patients selected at the outpatient clinic of the Department of Dermatology, Post Graduate Institute of Medical Education and Research, Chandigarh with their written informed consent and this part of the study was approved by the Institute Ethics Committee. Dermal stem cells were cultured, characterized and than differentiated in to functional melanocytes. NGFRp75 positive cells were analysed in dermal cell suspension by FACScan.
In this study we found that multipotent dermal stem cells are present in the vitiligo patient's uninvolved skin, lesional skin as well as glabrous lesional skin. These dermal stem cells, grown as three-dimensional spheres, displayed a capacity for self-renewal and expressed NGFRp75, OCT4 and nestin markers. These dermal stem cells were differentiated into functional melanocytes.
Our results demonstrate that stem cells in the dermis of vitiligo skin can become mature epidermal melanocytes and play an important role in the repigmentation of vitiligo lesions. Stimulating these dermal stem cells to differentiate into melanocytes and followed by their migration to lesional skin would be an ideal therapy for the repigmentation of vitiligo lesions.
T. Kunisada, T. Motohashi, H. Aoki, N. Yoshimura
Gifu University Graduate School of Medicine, Gifu, Japan
Melanocytes are pigment-producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. In a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. We show that a NCC population that is not derived from Sox1+ dorsal neuroepithelial cells but are derived from Sox1- cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1- cells clearly segregate from cells that originated from Sox1+dorsal neuroepithelial cell-derived NCCs. The possible derivation of Sox1- cells from epidermal cells also strengthens their non-neuroepithelial origin.
C.-C. E. Lan
Kaohsiung Municipal Ta-Tung Hospital, Kaoshiung Medical University, Kaohsiung, Taiwan
Solar irradiation consists of light at ultraviolet (UV), visible, and infrared spectrum. Since ancient time, sun exposure has been used for treating various diseases. Currently, phototherapy is an essential part of dermatology practice for treating various skin conditions including for regenerative purposes. The proposed mechanisms responsible for phototherapy, particular those in the UV region, mostly focus on immune suppressive effects of lights. Little is known regarding the mechanisms involved in visible light therapy. It is recognized that specific absorption of photon energy by chromophore, or photoacceptor, is responsible for light and tissue interaction. Although non-thermal, low energy visible light has been used to promote wound healing and rejuvenate photodamaged skin clinically, the molecular mechanisms involved remained unclear. In 2003, we proposed and demonstrated that low-energy laser (Helium-Neon laser) emitting visible light at 632.8 nm induces vitiligo repigmentation. Subsequently, we demonstrated that He-Ne laser has multifaceted biological effects on keratinocytes and melanoblast that create a favorable environment for vitiligo repigmentation. More recently, we showed that Helium-Neon laser significantly increases the mitochondrial DNA copy number, induces the expression of regulatory genes for mitochondrial biogenesis, and upregulated the antioxidant enzyme expression of primitive melanoblasts. Mechanistically, we demonstrated that Helium-Neon laser initiated mitochondrial retrograde signaling via a Ca2+-dependent cascade. In summary, visible light phototherapy induces vitiligo repigmentation via mitochondrial retrograde signaling. Combination of visible light with other spectrum of light may have important clinical applications in the other aspects of regenerative medicine including wound healing and skin rejuvenation.
E. B. Lane, J. Common, C. Badowski, T. S. Tan, I. Szeverenyi, G. Sundaram, P. Sampath
A*STAR Institute of Medical Biology, Immunos, Singapore
Three diverse projects have recently converged in our institute, which have taken different approaches to an old problem of understanding cancer and wound healing. The first study looked at epidermolysis bullosa simplex (EBS), a very rare, incurable, skin fragility disorder caused by mutations in keratin intermediate filament proteins, and carrying an increased risk of basal cell carcinoma. We set out to investigate the downstream consequences of mutant keratin expression, in the search for an entry point for cheaper therapeutic approaches to rare diseases like these. Cell models were generated expressing wild-type and mutant keratin proteins coupled to GFP and used for live cell imaging. When these cells were subjected to stress in tissue culture, it emerged that the constitutive stressed state of these mutant cells can mimic stresses such as wounding, raising questions about the wound signals that initiates keratinocyte migration in vivo. An unrelated study on rare inherited self-healing keratoacanthomas had recently focused our attention on TGFbeta signaling, known to be essential for wound healing. When TGFBR1 receptor was lost, individuals became susceptible to multiple self-healing squamous epithelioma (MSSE). Meanwhile a third study, using the culture wounding model systems set up for EBS, has now uncovered a unique miRNA switch mechanism by which TGFbeta signaling triggers wound healing migration in keratinocytes, and fails to do so in diabetic wounds. The unexpected synergy between these projects has demonstrated how studies on rare diseases can better position us to learn about more common medical challenges.
J. Mosenson1, A. Zloza2, J. A. Guevara-Patino1, I. C. Le Poole1
1Loyola University Chicago, Maywood, IL, USA2Rush University, Chicago, IL, USA
Heat shock proteins protect stressed cells from apoptosis. We reasoned that an environmental stressor inducing HSP70 expression may be associated with greater secretion by melanocytes. Extracellular stress proteins can bind dendritic cells and induce immune response to peptides bound to these molecular chaperones. Meanwhile, intracellular colocalization with melanosomal proteins explains the immunogenic cargo that leads to vitiligo development. We showed that HSP70i is central to disease development in mouse models; knockout mice do not depigment in response to antigenic challenge, whereas HSP70i alone is sufficient to induce disease in a transgenic mouse model of spontaneous depigmentation. Thus, we hypothesized that targeting HSP70i in autoimmune vitiligo is suited to halt depigmentation. We confirmed that dendritic cell profiles in human patients mimic those observed in response to HSP70i in mice. The c-terminus and a peptide within it proved crucial to depigmentation. A single amino acid modification introduced by site directed mutagenesis was sufficient to prevent human dendritic cell activation and inhibit depigmentation in mice. Mutant HSP70i drove expansion of a regulatory subset of APC, in contrast to the inflammatory DC subset observed in response to wildtype HSP70i. Treatment was associated with reduced T cell influx to the skin, and circulating T cells maintained a memory profile not observed in response to the wildtype molecule. Mutant HSP70i likewise affected profiles of immune cells collected from cultured human skin explants. Taken together, our data support the remarkable treatment potential of mutant HSP70i for vitiligo.
Department of Dermatology, Dongguk University, Issan Hospital, Gyeon, Korea
Melasma is one of the most common patterns of hyperpigmentation, particularly during the reproductive lifespan of women. In terms of pigmentation, melasma does not differ from other conditions with hyperpigmentation, however, the outcome is different from the hyperpigmentation caused by inflammation or by UV exposure, suggesting different regulatory mechanisms being involved in these conditions. Recently, we performed microarray analysis to compare hyperpigmented with normally pigmented skin specimens from patients with melasma to identify specific factors associated with the hyperpigmentation of melasma. Down-regulation of H19 RNA was one of the identified factors associated with melasma. There was also a twofold or greater down-regulation in the expression of the Wnt inhibitory factor-1 (WIF-1) gene in the hyperpigmented skin of melasma patients. The real time-PCR and immunohistochemistry using anti-WIF-1 antibody confirmed the significant reduction of WIF-1 gene expression in the hyperpigmented skin from melasma patients, but not in healthy controls, regardless of UV irradiation.
Wnts, a family of secreted glycoproteins, regulate a vast array of biological processes, including skin pigmentation through either the canonical or the non-canonical pathways. In the canonical pathway, β-catenin plays a critical role, which is also called the Wnt/β-catenin pathway; the binding of Wnts to their receptor generates a signaling pool of β-catenin and translocate β-catenin to the nucleus to stimulate transcription of target genes, whereas β-catenin without the binding is degraded by the destruction complex, which is composed of four proteins including GSK3 (glycogen synthase kinase 3). Non-canonical Wnt pathway is β-catenin-independent and involves a diverse signal transduction, such as JNK or calcium flux, the latter is called the Wnt/calcium pathway which activates PKC (protein kinase C) and NFAT (nuclear factor of activated T cells). The extracellular antagonists of the Wnt signaling pathway can be divided into two classes, the secreted Frizzled-related protein class (sFRP) and the Dickkopf class. WIF-1 belongs to the sFRP class family, and inhibits both canonical and non-canonical Wnt pathways.
WIF-1 is expressed in cultured skin keratinocytes and fibroblasts, but not in melanocytes. Melanocyte growth and differentiation is affected by neighboring fibroblasts as well as by keratinocytes. Therefore, we examined whether WIF-1 knockdown in neighboring keratinocytes and fibroblasts plays a role in melasma. For the examination, keratinocyte-melanocyte co-cultures were used to examine the effect of WIF-1 down-regulated keratinocytes or fibroblasts on normal melanocytes. Additionally, the effect of WIF-1 over-expression on the amelioration of hyperpigmentation was examined by the treatment of cultured melanocytes with rhWnt-1. WIF-1 knockdown, either in fibroblasts or in keratinocytes significantly stimulated tyrosinase expression and melanosome transfer, whereas melanocytes with WIF-1 over-expression, significantly reduced those parameters. The WIF-1 knockdown decreased GSK-3β, β-catenin and NFATc2 phosphorylation and increased MITF expression as in melanocytes with Wnt-1 over-expression, whereas the WIF-1 over-expression reversed the results. Expression of Wnts, both canonical and non-canonical, was increased in the hyperpigmented skin of melasma patients. Collectively, WIF-1 down-regulation, which may occur in epidermal keratinocytes and in dermal fibroblasts, is involved in melasma development due to the stimulation of melanogenesis and melanosome transfer through up-regulation of the canonical and the non-canionical Wnt signaling pathway.
M.-H. Lee1, H.-K. Lim1, M. K. Shin1, G. J. Lee2, Y.-K. Uhm3
1Kyung Hee University Hospital, Seoul, Korea2Department of Biomedical Engineering, School of Medicine, Kyung Hee University, Seoul, Korea3Department of Pharmacology, School of Medicine, Kyung Hee University, Seoul, Korea
It is well known that α-melanocyte stimulating hormone (α-MSH) induces melanization, but structural changes of melanocytes after α-MSH exposure is not well known.
The aim of this study was to investigate the serial morphologic change of human cultured melanocyte after stimulation with α-MSH using atomic force microscopy (AFM).
Cultured human melanocytes were used for study and the cells were treated with α-MSH for 7 days. Contact mode AFM images were obtained using a NANOstation II and fixed melanocytes were scanned in PBS solution at a resolution of 512 × 512 pixels, at a scan speed of 0.4 line/s. The surface roughness and pore-like structures of the melanocytes were measured from the topographic images using the Scanning Probe Imaging Processor.Surface roughness of cell body did not show significant change over time. All three roughness parameters (Sa, Sq, Sz) inside the pore-like structures on the dendrites were significantly increased. The number of pore-like structures increased and the length and the breadth of pore-like structures were also increased gradually. The change of pore-like structures was statistically significant.
Pore-like structures were main structure which showed statistically significant change in response to α-MSH stimulation. Further study will be needed whether the nature of pore-like structures is related to movement of melanosome or not.
H. Li1, J. Kim1, H.-S. Jeong1, K.-C. Park2, K. J. Baek1, N. S. Kwon1, H.-Y. Yun1, D.-S. Kim1
1Department of Biochemistry, Chung-Ang University College of Medicine, Seoul, Korea2Department of Dermatology, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, Korea
The purpose of this study was to demonstrate the effects of KHG26792, a potential new skin-whitening agent and to identify the mechanisms of the action. KHG26792 significantly reduced melanogenesis via activation of extracellular signal-regulated kinase (ERK) in a dose- and time-dependent manner. Also, microphthalmia-associated transcription factor (MITF) was downregulated through phosphorylation of ERK, but tyrosinase, the rate-limiting enzyme was not inhibited directly. Moreover, we showed the hypopigmentary effects of KHG26792 in the pigmented skin equivalents established with Cervi cornus Colla (deer antler glue). It was found that the color of pigmented artificial skin became lightly after treatment of KHG26792. Our findings suggest that KHG26792 is a novel candidate compound for a skin whitening agent.
L. L. Lin, A. P. Raphael, H. P. Soyer, T. W. Prow
Dermatology Research Centre, School of Medicine, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia
Traditional skin biopsies are employed to diagnose questionable lesions and to provide an accurate prognosis of disease where present. However, these techniques are painful and render the lesion unsuitably damaged for further study. One major problem is that patients can present with hundreds of suspicious actinic keratosis lesions or nevi. In addition, these lesions commonly present on cosmetically sensitive areas, e.g. the face. This decreases acceptance of conventional biopsy strategies and exemplifies the clinical need for less invasive method of biopsy.We have developed a microbiopsy platform as a technology that can enable repeated, minimally invasive sampling without the need of local anesthesia and without pain and scarring – while providing enough tissue for molecular detection of disease and live-cell assays. The application of the microbiopsy device induces a barely visible puncture site with subtle to no irritation. We have successfully collected micro-sized skin biopsies containing 1000–3000 skin cells from volunteers. We have isolated 5–20 ng of DNA and RNA, and approximately 0.4 μg of protein from these microbiopsies.
This new biopsy platform will help to fill both clinical and research needs. It will obtain targeted molecular pathology data to enable improved molecular diagnoses of difficult cases, without inflicting pain and scars. Additionally, this device will set the framework for advancing clinical research for neoplastic skin diseases.
C. C. H. Loh1,2, J. Kim2, J. Su3, B. S. Daniel4, S. S. Venugopal5, L. Rhodes2, L. R. A. Intong2, M. Law6, D. F. Murrell2
1Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia2St George Hospital, Sydney, NSW, Australia3Department of Paediatrics, University of Melbourne, Melbourne, Vic., Australia4Department of Medicine, St Vincent's Hospital, Sydney, NSW, Australia5Deparment of Dermatology, Westmead Hospital, Sydney, NSW, Australia6The Kirby Institute, University of New South Wales, Sydney, NSW, Australia
There is a lack of validated outcome measures for epidermolysis bullosa (EB) which measure disease activity separately from damage. We present a novel EB Disease Activity and Scarring Index (EBDASI), which scores activity responsive to therapy separately from scarring, which we have validated.
MethodContent validity was established by including all physical complications of EB and adapting the scoring methodology from another validated blistering disease severity score, the Pemphigus Disease Area Index, to create the EBDASI. We compared the reliability and validity of the EBDASI against the Birmingham EB Scale (BEBS), using the Physician's Global Assessment Scale (PGA) as a reference measurement. Sixteen patients of different EB subtypes and severities were scored separately by five EB experts using the EBDASI, BEBS and PGA on the same day.
For inter-rater assessment, the intra-class correlation coefficients (ICCs) for overall total score were: EBDASI 0.964, BEBS 0.852, and PGA 0.873. For intra-rater reliability, the ICCs (95% CI) were: EBDASI 0.994 (0.976–0.998), BEBS 0.926 (0.748–0.981), and PGA 0.932 (0.764–0.982). The EBDASI correlated better with PGA (ρ = 0.871) than the BEBS with PGA (ρ = 0.852). Scatter-plots showed that EBDASI had better correlations with increased severity scores than BEBS. Nine other EB patients were also assessed with ad-hoc scores ranging from 85 to 212/506 (RDEB) and 94 to 113/506 (JEB).
The EBDASI demonstrated excellent reliability and validity, which is superior to BEBS.
MAP kinase inhibitors, including BRAF and MEK inhibtors, are the first class of small molecule targeted agents to improve the overall survival of patients with metastatic melanoma. Although almost all patients respond, the extent of response, pattern of disease progression and time to progression are variable, and complete responses are rare. Understanding the clinical heterogeneity of response and progression, and molecular mechanisms behind them is critical to improve outcomes further. For example, when is it appropriate to treat with the MAPK inhibitor beyond progression, and will drug-targeting of one diagnosed resistance mechanism help the patient? Results from a large systematic patient screen of melanoma tissue taken before and during MAP kinase inhibitor therapy, as well as at progression will be presented in the context of clinical heterogeneity of response and progression. The implications for systemic therapies and patient outcomes will be discussed.
1st Affiliated Hospital, Nanjing Medical University, Nanjing, JS, China
Vitiligo is an acquired pigmentary disorder of unknown etiology that is clinically characterized by the development of white macules related to the selective loss of melanocytes. Recent data have suggested an altered functionality of mitochondria in vitiligo patients. To better understand whether a defective ultrastructure of mitochondria is involved in the pathogenesis of vitiligo, we have performed an ultrastructural study on normal and perilesional skin from 10 healthy volunteers and 20 patients with active or stable disease by on microscope. The resulting images of cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups including volume density (Vv), surface density (Sv) and numerical density (Nv). We found that mitochondria were abundant with normal structure and cristae mitochondriales concentrated either in keratinocytes or melanocytes from volunteers. A large number of melanosomes in melanocytes of control group mainly stage III and IV were detected. However, mitochondrial swelling and vacuolization as well as fragmentation of the mitochondrial cristae were commonly observed in vitiligo group. Stage III melanosomes decreased significantly compared with normal group. Besides, no mitophagy was observed in the least, whereas autophagosomes could be occasionally noticed in normal melanocytes. The stereology results showed that Vv and Sv of mitochondria measured in vitiligo were significantly elevated compared with the normal, but Nv was markedly decreased. Remarkable differences were noted between active and stable stage of vitiligo for the Vv, Sv and Nv. These data provide further evidences for an altered mitochondrial ultrastucture and functionality in vitiligo patients, and the mitochondrial damage was closely correlated with active status of the disease.
R. Mahajan, A. J. Kanwar
PGIMER, Chandigarh, India
Numerous therapeutic options have been tried in the management of melasma. This prospective randomized study was planned to assess the efficacy of triple combination cream (TC-hydroquinone 2%/tretinoin 0.05%/flucinolone 0.01%) vs glycolic acid peels/azelaic acid 20% cream (GA/AA) combination in melasma.
Forty patients with melasma were recruited into this study and randomized into two groups. Group A consisting of 20 patients received TC cream once daily for night time application for 3 months. Group B comprising of 20 patients received GA/AA 20% cream combination for 3 months. The disease severity was monitored with digital photography, melasma area and severity index (MASI) score which was calculated at baseline, 6 and 12 weeks, and visual analogue scale (VAS) score which was calculated at baseline and 12 weeks.
Of 40 patients, 38 completed the study. A significant reduction in MASI and VAS was recorded after 6 and 12 weeks of treatment in both group A and group B (P = 0.001). However, there was no difference in the mean MASI scores between the two groups at baseline, 6 and 12 weeks. Similarly, there was no difference in the mean VAS scores between the two groups at baseline and 12 weeks. Four patients in group A and three in group B experienced adverse effects like irritation, dryness and photosensitivity.
Both TC cream and GA/AA 20% cream combination are equally effective in treating melasma among Indian patients.
V. Mar1, S. Wong1, J. Li1, R. Scolyer2, C. McLean3, A. T. Papenfuss4, R. Tothill1, H. Kakavand2, G. J. Mann5, J. F. Thompson6, A. Behren7, J. Cebon7, R. Wolfe8, J. W. Kelly9, A. Dobrovic1, G. McArthur1
1Peter MacCallum, East Melbourne, Vic., Australia2Pathology, Melanoma Institute Australia, Sydney, NSW, Australia3Anatomical Pathology, Alfred Hospital, Melbourne, Vic., Australia4Bioinformatics, WEHI, Melbourne, Vic., Australia5Oncology, Melanoma Institute Australia, Sydney, NSW, Australia6Surgical Oncology, Melanoma Institute Australia, Sydney, NSW, Australia7Molecular Oncology, Ludwig Institute, Melbourne, Vic., Australia8Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic., Australia9Victorian Melanoma Service, Melbourne, Vic., Australia
The mutation load in melanoma is high compared to other tumor types due to extensive UV damage. Translation of exome sequencing data into clinically relevant information is therefore challenging. Melanoma exome sequencing studies have predominantly concentrated on mutations identified in cell lines generated from melanoma metastases. This study sought to characterize mutations identified in primary cutaneous melanomas and correlate these with clinico-pathologic features.DNA was extracted from 34 fresh frozen primary cutaneous melanomas and matched peripheral blood. Tumor histopathology was reviewed by two dermatopathologists. Exome sequencing was performed and mutation rates were correlated with age, sex and tumor site and histopathologic variables. Differences in mutations between categories of solar elastosis, pigmentation and BRAF/NRAS mutational status were investigated.
The average mutation rate was 12 per megabase, similar to published results in metastases. Tumors arising in severely sun-damaged (SSD) skin (P = 0.003) and BRAF/NRAS wild-type tumors (P = 0.0001) had higher mutation loads. There was an inverse correlation between mutation rate and tumor thickness (Spearman r = −0.4, P = 0.02). Tandem CC>TT/GG>AA mutations comprized 70% of all dinucleotide substitutions and were more common in tumors arising in SSD skin (P = 0.0008) and in BRAF/NRAS wild-type tumors (P = 0.0007). Targetable and potentially targetable mutations in wild-type tumors, including NF1, KIT and NOTCH1, were spread over various signaling pathways.
Melanomas arising in SSD skin have higher mutation loads and contain a spectrum of molecular subtypes compared to BRAF and NRAS-mutant tumors. It is likely that a number of genomic insults are required for melanoma progression in this group. Potentially targetable mutations in wild type tumors are spread over different signaling pathways and this may have implications for therapeutic approaches in the future. Classification of melanoma into BRAF mutant, NRAS mutant and high mutation load groups may be helpful for identification of patients more likely to respond to combined drug therapies.
G. A. McArthur
Peter MacCallum Cancer Centre, East Melbourne, Vic., Australia
Strong focus on understanding the cellular and molecular biology of melanoma and the role of anti-tumour immunity in melanoma have generated unprecedented therapeutic opportunities in the disease. Over 60% of melanomas have mutations that activate the RAS/RAF/MEK/ERK pathway that has led to identification of systemic therapies that improve overall survival in the advanced disease setting, and potentially offer even more significant impact in the adjuvant setting. We have recently showed that primary melanomas without mutations in these driving oncogenes have extraordinarily high numbers of sequence variants consistent with chronic UV damage. These melanomas harbor multiple potential drivers suggesting single agent therapies will be of limited value. We propose the greatest potential to reduce mortality from melanoma, a disease characterised by the development of metastases from small primary tumours, is the combination of systemic therapies particularly in the adjuvant setting. The new systemic therapies for melanoma coupled with ongoing understanding of the biology of the disease is poised to further accelerate novel therapeutic opportunities.
M. Martinez1,2, A. Fernandez1,2, D. Seruggia1,2, C. Vicente1,2, M. Cantero1, L. Montoliu1,2
1National Centre for Biotechnology (CNB-CSIC), Madrid, Spain2Sensorineural Pathology, CIBERER-ISCIII, Madrid, Spain
Albinism is a rare genetic condition associated to a variable hypopigmentation phenotype, which can affect the pigmentation of only the eyes or both the eyes and the skin/hair, resulting in ocular (OA) or oculocutaneous albinism (OCA). Four forms of OCA and one of OA are known, associated to TYR (OCA1), OCA2 (OCA2), TYRP1 (OCA3), SLC45A2 (OCA4) and GPR143 (OA1) loci, respectively. Syndromic forms of albinism are the Hermansky-Pudlak syndrome (HP1-9) and the Chediak-Higashi syndrome (CHS1). Currently, at least 15 genes are associated to albinism. Murine counterparts exist, and, hence, mouse mutant models of all these different types of albinism have been detected and/or produced and have contributed to our understanding of the albinism phenotype and the associated common visual deficits. In addition, for mouse models of OCA1, hearing deficits have been also reported. Several observed spontaneous and genetically-modified mouse mutant mice produced will be reviewed and discussed, focusing in those that have mostly contributed to our current knowledge of the albino condition.
In addition, and greatly facilitated by the international collaborative efforts within the Pigment Cell community that have been instrumental in the validation phases of the experiments, we have initiated an approach to eventually attempt to genotype all known genetic mutations associated to albinism in any of the 15 loci mentioned above. Genetically diagnosed samples, from albino patients from France (in Bordeaux: Alain Taïeb, Benoît Arveiler; in Marseille: Robert Aquaron), Italy (in Milan: Vittoria Schiaffino), Spain (in Madrid: Carmen Ayuso) and Japan (in Yamagata: Tamio Suzuki), have served to define a set of mutations that can now be easily detected thanks to sophisticated innovative equipments, in collaboration with the laboratory of Angel Carracedo (University of Santiago de Compostela, Spain). Examples of these efforts will be presented and discussed.
National Centre for Vitiligo & Psoriasis, Riyadh, SA, Saudi Arabia
Vitiligo is a chronic disorder, primarily believed to be of autoimmune origin. The natural course of the disease is characterized by periods of progression and remission, which remains unpredictable in spite of many advances in its pathogenesis and treatment. Medical therapies are considered as first choice to treat all types of vitiligo. However, it is increasingly known and documented that segmental vitiligo, lesions located on glabrous skin and with leukotrichia respond poorly to medical therapies. Surgical methods are useful to restore pigment in such cases and are being used more often. Surgical methods can be divided into tissue grafting such as punch graft, blister graft, and split thickness skin graft and cellular grafting which include cultured and non-cultured cell transplantation. Noncultured melanocyte-keratinocyte transplantation (MKTP) has many advantages over other methods and gives excellent cosmetic results in most of the treated cases. It is available only at select centers due to regulatory issues and lack of training. In addition to vitiligo, post burn leukoderma, piebaldism, chemical leukoderma, post DLE depigmentation, halo nevus were treated successfully with MKTP. The technique is being practiced for about 17 years at our center.
D. F. Murrell1, B. Dréno2, J.-J. Grob3, T. Jouary4, N. Basset-Seguin5, J. Hansson6, P. Ascierto7, C. Garbe8, L. Mitchell9, M. Starnawski9, A. Hauschild10, R. Kunstfeld11
1Department of Dermatology, St George Hospital, Kogarah, NSW, Australia2CHU Nantes-Nantes Hospital University, Nantes, France3Service de Dermatologie et Cancérologie Cutanée, Hopital de la Timone, Marseille, France4Unité de Cancérologie Cutanée, Service de Dermatologie, Bordeaux, France5PUPH, Hôpital Saint-Louis Paris, Paris, France6Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden7Istituto Nazionale Tumori Fondazione G. Pascale, Napoli, Italy8Department of Dermatology, Universität Tübingen, Tübingen, Germany9F. Hoffmann-La Roche Ltd, Basel, Switzerland10Universitätsklinikum Schleswig-Holstein, Kiel, Germany11AKH Wien, Klinische Abteilung für Allgemeine Dermatologie, Vienna, Austria
While most cases of BCC can be managed by surgery, some progress to advanced stage where surgery is inappropriate. Vismodegib, first-in-class Hedgehog-pathway inhibitor, has demonstrated efficacy in advanced BCC. STEVIE is a safety study of vismodegib in advanced BCC. Patients received oral vismodegib 150 mg, once-daily until progressive disease, unacceptable toxicity or withdrawal. Safety assessed by Common Terminology Criteria for Adverse Events (AEs) v4.0. Overall response rate assessed according to Response Evaluation Criteria in Solid Tumours, (v1.1). Recruitment is ongoing. This analysis (cutoff-17 May 2012) included 150 patients with locally advanced (n = 138) or metastatic (n = 12) BCC with potential for ≥3-month follow-up. Locally advanced patients had lesions considered inoperable-54.3% or surgery contraindicated-45.7%. Median treatment duration-144 days. Most common treatment emergent AEs (TEAEs; ≥20% of patients) included muscle spasms-53.3%, alopecia-42.7%, dysgeusia-36.0%, ageusia-27.3%, and asthenia-26.7%. Most TEAEs were mild/moderate severity. Serious TEAEs occurred in 22 patients (14.7%). Patients discontinued treatment (25.3%) due to AEs (n = 10). Deaths were due to disease progression (n = 2) or AEs considered by investigators unrelated to study drug. Initial preliminary best overall response was confirmed for 124 patients; 19.4%-complete responders, 55.6%-partial responders, 21.8%-stable disease and 3.2%-progressive disease. This interim analysis from STEVIE confirms a similar vismodegib safety profile to ERIVANCE BCC.
This study was funded by Roche-Genentech. Support for medical writing assistance was provided by F. Hoffmann-La Roche.
H. Sohn, J. Y. Kim, D. S. Kim, S. H. Oh
Yonsei University College of Medicine, Seoul, Korea
Keratinocytes secrete many mitogenic or melanogenic molecules for melanocytes. Among several keratinocyte-derived cytokines, SCF and ET-1 are regarded as main mediators involved in skin pigmentation. Protease-activated receptor (PAR)-2 is a member of G-protein coupled seven transmembrane receptor family. PAR-2 is known to be involved in the production of inflammatory mediators as well as melanosomes transfer. In this study, we would like to investigate whether direct activation of PAR-2 by PAR-2 agonist can induce the production of SCF and ET-1 in keratinocytes.
The expression of SCF and ET-1 in HaCaT cells was examined after PAR-2 activation with PAR-2 activating peptide (PAR-2 AP, SLIGKV-NH2) and kallikrein 5 in western blotting and real time RT-PCR. After treatment of PAR-2 AP and kallikrein 5, SCF and ET-1 in HaCaT cells were increased in the levels of protein and mRNA. To confirm if the increase of SCF and ET-1 by PAR-2 AP and kallikrein 5 was directly associated with PAR-2 activation, incubation of PAR-2 AP and kallikrein 5 after pretreatment of PAR-2 antagonist and knockdown of PAR-2 expression by PAR-2 siRNA technique were performed. After treatment with PAR-2 antagonist, increased SCF and ET-1 after kallikrein 5 was not observed. In addition, treatment with PAR-2 AP did not increase SCF and ET-1 in PAR-2 siRNA-treated HaCaT cells contrary to scrambled siRNA-treated HaCaT cells.In conclusion, our study suggested that PAR-2, which is known to act as a gate of melanin transfer might directly mediate skin pigmentation via production of keratinocyte-derived mediators such as SCF and ET-1.
S. J. Palmer1, C. P. Canales1, P. Carmona-Mora1, F. Tomasetig1, P. Kaur2, I. Smyth3, P. W. Gunning1, E. C. Hardeman1
1Cellular and Genetic Medicine Unit, University of New South Wales, Sydney, NSW, Australia2Epithelial Stem Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, Vic., Australia3Biochemistry and Molecular Biology, Monash University, Melbourne, Vic., Australia
Williams-Beuren syndrome (WBS) is a developmental disorder resulting from a hemizygous microdeletion of chromosome 7, causing a characteristic set of neurological and physical abnormalities. WBS is typified by a distinctive craniofacial appearance that involves an enlargement of specific soft tissue features such as the lips. Genotype/phenotype correlations in patients with atypical deletions suggest that the facial gestalt, maps to a pair of genes that encode the evolutionarily-related transcriptional regulators GTF2IRD1 and GTF2I.
To test these mapping data and to determine molecular cause, we have generated Gtf2ird1 knockout mouse lines, which show some similar facial features. Knockout mice show extreme thickening of the skin around the nose and lip region1 associated with an expansion of the basal proliferative zone. Concordantly, expression of Gtf2ird1 is localised to a similar zone but only during development. During adulthood, expression declines.Our studies on the molecular function of GTF2IRD1 show that it is part of a larger gene silencing complex that lays down epigenetic marks during development. In the epidermis, it seems that these marks permanently set the epidermal proliferation rate in specific facial zones.
K.-C. Park, H.-R. Choi, S.-H. Yang, K.-M. Nam, H.-S. Lee
Department of Dermatology, Seoul National University, College of Medicine, Seoul, Korea
Keratinocytes can be divided into stem cells, transit amplifying cells, and postmitotic differentiating cells. Epidermal stem cells can contribute to the maintenance of the epidermis via their self-renewing ability. It was suggested that cell-ECM interactions influence these interactions. Thus, strategies for manipulating cell-ECM interactions hold promise in controlling skin disease or preventing aging effects. It is possible that epidermal cells are under the control of surrounding environment. For decade, we have been studying the effects of environmental factors for interfollicular epidermal stem cells. The effect of dermal sheath cells (DSC) was found to be mediated by IGFBP-2. Furthermore, IFGBP-3 contributes survival of interfollicular epidermal stem cells in skin equivalent models. In addition, it was reported by us that adipose tissue derived stem cells have stemness regulating effects. All these findings clearly showed that stemness of interfollicular stem cells can be regulated by surrounding factors and these stem cell activators can be useful in controlling skin aging and skin disease. Recently, our results have been well applied for the development of anti-aging cosmetics. Then, ‘stem cell activator whether it is internal or external’ will be promising strategy for the development of therapeutic or cosmetic agents.
PGIMER, Chandigarh, India
The development of effective treatment for vitiligo depends on understanding the mechanisms of depigmentation and repigmentation. The basic pathogenesis of vitiligo in general, or for any of the putative subsets of vitiligo, is not fully known although substantial strides have been made in pathogenesis and the treatment of vitiligo. Since the etiopathogenesis of depigmentation in vitiligo is still obscure, the source of pigmentation in the repigmentating lesion and its stability is also not fully known.
An approach combining clinical investigations and focused research on the cellular and molecular mechanisms of vitiligo repigmentation will lead to finding better treatments for this disease. The mechanistic aspects of melanocyte repopulation in vitiligo and of other factors that trigger and influence melanocyte growth, maturation and survival have been little explored. A better understanding of vitiligo repigmentation will provide new therapeutic perspectives leading to development of an ideal weapon against vitiligo.
Understanding basic mechanism of repigmentation in vitiligo and its co-relation with various treatment modalities seems to be the key to successful treatment. Recently we successfully demonstrated presence of dermal stem cell in depigmented vitiligo lesion. Next question to be answered is how to stimulate these dermal stem cells which will be an ideal treatment of vitiligo.
J. Patel, E. Seppanen, N. M. Fisk, K. Khosrotehrani
UQ Centre for Clinical Research, Herston, Qld, Australia
Reconstitution of dermal components of the skin upon wounding is essential to avoid hypertrophic scarring and proper wound closure. To date, MSC therapy results in improved healing, but injected cells do not engraft. Moreover, therapies often fail to address improving perfusion via angiogenesis. Our aim was to improve cellular engraftment and trigger new blood vessel formation.
Endothelial colony forming cells (ECFC), a rare highly proliferative subset of EPC, are regarded as the gold-standard for their in vivo vessel forming capacity. ECFC and MSC were readily isolated from human term placentas. Nu/nu mice were injected subcutaneously, 8 h after wound formation, with either single populations of ECFC, MSC or dual combination of ECFC/MSC (10 to 1 ratio). Assessment of wound healing was conducted 7 days post injection.
Consistent with previous data, MSCs did not persist in wounds. Local administration of single ECFC and MSC doses showed no significant increase in wound acceleration in comparison to controls. However, improved engraftment of ECFC and de novo vessel formation in wound was observed when co-injected with MSC demonstrating possible synergistic effects.
The human term placenta is an accessible and abundant supply of ECFC, which have been shown to have promising effects in improving perfusion in a wound healing environment when in co-therapy with MSC.
V. Maresca, E. Flori, G. Cardinali, D. Kovacs, B. Bellei, M. Picardo
San Gallicano Dermatology Instutite, Roma, Italy
Alfa Melanocyte Stimulating Hormone (α-MSH) is the key factor in controlling melanogenesis through the activation of the G-protein coupled receptor MC1R. The interaction with the receptor activates the cAMP/PKA pathway and subsequently the Microphthalmia-associated transcription factor (MITF). Polymorphisms of the receptors are associated with fair skin. However α-MSH exerts several other functions on the skin including control of cell differentiation, inflammatory response and oxidative stress and it also acts on non pigmented cells. Previously we have shown a correlation between the type and the amount of melanin synthesis and the expression of the antioxidant enzyme catalase. Moreover, we have shown how intermediates of eumelanogenesis induce keratinocyte differentiation whereas pheomelanogenesis is associated with an alteration of gap junction. These data suggest a role of melanogenesis in control of skin barrier functions. We have also recently discovered a new pathway of α-MSH activity which involves the Peroxisome proliferator activated receptor -γ (PPAR-γ). This link exerts an influence on melanogenesis, proliferation and antioxidant expression and it is PI(4,5)P2/PLC beta pathwayβ dependent. Lipid composition of the plasma membrane seems to possess a relevant role in this mechanism. These results suggest that skin phototype is the result of several mechanisms and it can be considered a biochemical fingerprint of individuals. The interaction of α-MSH with nuclear receptors can explain the complexity of the process.
T. J. Piva, V. Muthusamy, R. Ravi
RMIT University, Bundoora, Vic., Australia
Ultraviolet (UV) light is main carcinogen involved in the formation of skin cancer. UV radiation is a potent inducer of TNFα and cytokine gene expression in human keratinocytes. TNFα plays an important pro-inflammatory role in the skin, and is cleaved from its precursor by the action of Tumor Necrosis Factor α Converting Enzyme (TACE). TACE is cleaved from its preproform by furin, a proprotein convertase. The effect UV radiation has on the intracellular signalling pathways and that of furin's expression and/or activity in keratinocytes is not known. The major signalling pathways activated by UV radiation are p38 MAPK, JNK and NF-κB. We investigated the effect 1 MED UV radiation [UVA (40 kJ/m2), UVB (2 kJ/m2) and UVAB (40 + 2 kJ/m2)] had on the expression of p-p38, p-JNK, NFκB as well as the activity of furin, and secretion of TNFα were examined using human primary keratinocytes (HEK), immortalised HaCaT cells as well as the squamous cell carcinoma Colo16 cell line. Colo 16 cells were more sensitive to high dose UVB and UVAB radiation than were HaCaT or HEK cells, though neither cell line was sensitive to UVA radiation. p38 MAPK was the major signalling pathway activated by UVA and/or UVB in all cell lines, though the level of activation differed. Only in the presence of IL1α did UVB and UVAB radiation induce maximal secretion of TNFα from HEK cells with lesser amounts secreted from the other cells. Of interest was that UV radiation increased furin expression in Colo16 and HaCaT cells which did not correspond to that of TNFα released from these cells. The differences observed between HaCaT cells and that of HEK cells raise questions about the use of these cells in studies on keratinocyte cell biology, the implications of which will be discussed.
T. W. Prow, H. P. Soyer
The University of Queensland, School of Medicine, Woolloongabba, Qld, Australia
Skin cancer diagnosis is moving toward more accurate, non-invasive approaches with in vivo imaging techniques at the forefront (e.g. reflectance confocal microscopy [RCM], multiphoton tomography4 and optical coherence tomography). We have begun developing automated RCM analysis approaches that enable objective photoageing analysis. This effort has also lead to our developing novel approaches for identifying signs of precancerous lesions in clinical photographs. Ideally, skin cancer risk could be defined through non-invasive imaging. Practically, minimally invasive microneedle-based biopsies could hold the key for molecular detection of skin cancer. We have developed a stacked 3D microneedle device for instantaneously capturing small pieces of skin from suspicious lesions. We have also examined the effects this type of sampling my have on downstream histopathological assessment. This microbiobiopsy approach has the potential to enhance lesion diagnosis through minimally invasive molecular analysis. Topical treatment is an attractive approach to lesion management once a precancerous lesion is diagnosed. Unfortunately, many potentially therapeutic drugs have poor skin penetration profiles. We have developed and patented a novel platform for field-directed drug delivery, Foroderm™. This technology is based on unique microparitcles that were engineered to only penetrate the stratum corneum and viable epidermis. These microparticles are applied by massaging the material, with the active ingredient, into the skin in a manner similar to sunscreen. Our in vivo pig and volunteer data show attractive microparticle penetration profiles and enhanced drug delivery. Together, these technologies are the first step toward step change improvements in how skin cancer is detected and treated.
Punjab Health Systems Corporation, Ferozepur, Ludhiana, Punjab, India
Melasma is a commonly acquired hypermelanosis occuring on sun-exposed areas.
We selected 30 patients of melasma for the study. The patients were divided into two groups of 15 patients each. Group I patients were subjected to four sessions of erbium glass fractional laser. In group II patients four sessions of chemical peeling with 70% glycolic acid were performed.
Epidermal type of melasma was seen in 70% patients, dermal was seen in 10% patients and mixed melasma was seen in 10% patients. Amongst the various side effects seen, the commonest side effect seen was erythema seen in 20% patients in laser group and 16.6% patients in the peels group. Burning sensation was seen in 13.3% patients in both the groups, pain was seen in seen in 16.6% patients in laser group and none of the patients in the peels group, hypopigmentation was seen in 3.3% patients in laser group and 10% patients in the peels group, post inflammatory hyperpigmentation was seen in seen in 6.6% patients in laser group and and 10% patients in the peels group. After 12 weeks of treatment, percentage reduction in MASI score was seen in 62.9% in the laser group and 58.7% in the peels group.
The pulsed radiation of the Q-swithed Nd:YAG laser has been shown to remove pigmentation by selectively destructing pigment cells with short pulse and low fluence.
A. P. Raphael, C. A. Primiero, H. P. Soyer, T. W. Prow
Dermatology Research Centre, School of Medicine, Translational Research Institute, Princess Alexandra Hospital, The University of Queensland, Brisbane, Qld, Australia
Delivery of therapeutic and cosmetic agents into skin is hindered by the epidermal barriers. Many physical and chemical approaches have been developed to overcome the skin's barriers. Chemical approaches often involve the complete disruption or removal of the epiderms resulting in irritation and inflammation, and physical approaches are restricted to the size of the delivery device – both limiting the delivery to focused areas of skin instead of much larger field delivery.
To overcome the skin's barriers for the treatment of skin diseases, we have developed a novel cutaneous delivery method capable of field treatment using elongate microparticles.We compared elongate microparticles with topical only and microneedle-enhanced delivery of sodium fluorescein, vitamins B3/E and 5-aminolevulinate. Elongate microparticle delivery results in a continuous delivery profile across the application area (field-delivery). Elongate microparticle-enhanced sodium fluorescein, Vitamins B3/E and 5-aminolevulinate delivery resulted in 7.1 (P = 0.001), 8.8 (P = 0.0001), 8.5 (P = 0.0017) and 2.6 (P = 0.0018) fold increase compared to topical alone. Furthermore, the passive and minimal invasive nature of elongate microparticle delivery results in a faster reduction in redness within the treated area compared to microneedles which require healing of the puncture sites. Finally, it was observed that the elongate microparticles are naturally removed through transepidermal elimination.
J. Rothnagel, A. Nouwens, L. Friend, R. Smith
University of Queensland, St Lucia, Qld, Australia
Recent work has identified a new component of the proteome; the translation of small open reading frames (sORFs) located with the untranslated and coding regions of mRNAs and a variety of non-coding transcripts. In this study we have focused on the coding potential of upstream open reading frames (uORFs). We hypothesize that some uORFs encode bio-active peptides which we have termed uPEPs (uORF encoded peptides). Significantly, there are reports showing uORF mutations associated with gene disorders including Marie Unna hereditary hypotrichosis.
We used an in-house sequence comparison program (uPEPperoni) to identify approximately 500 uPEP sequences that show a high level of conservation between human and other vertebrate transcripts. We cloned several of these into GFP expression vectors and expressed them in HeLa cells. uPEP expression was visualized by confocal microscopy and five exhibited organelle-specific localization. Additionally, we confirmed the localization of three uPEPs using synthetic peptides tagged with a fluorescent label. We complemented these studies with mass spectrometry to confirm the expression of peptides derived from uORFs. Lysates from HeLa and 293 cells were enriched for low molecular weight proteins, digested with trypsin and analysed by nanoLC-MS/MS. The resulting MS/MS data was searched against SwissProt and then all unmatched spectra were searched against the uPEPperoni output. MS/MS spectra matching peptides encoded by uORFs were identified from both cell types. In particular, multiple peptides matching an 11.1 kDa uPEP were identified. Bioinformatic analysis showed that this uPEP is conserved across a number of species including human, mouse, orangutan, rat, zebra fish and salmon.
Our study has shown that uORF-derived peptides are expressed in human cells and that a significant number of them are conserved across several species, indicating possible bio-activity. It now remains to determine what roles these peptides play and what the consequences are if they are altered by mutation.
Department of Medicine, Epworth Research Institute, St Vincent's Hospital, Melbourne, Vic., Australia
Wool follicles from sheep have long been a subject of biological research. In addition to their relevance to the wool industry, ovine follicles provide an interesting comparison to follicles from humans and other species. We have developed two cell culture models for studying aspects of wool follicle biology. In the first, keratinocytes are grown from explants comprising the follicle germinative epithelium, still attached to the adjacent dermal papilla. Preservation of attachment between the two tissues is necessary for the initiation of keratinocyte growth. However once these keratinocytes are established in culture, they can be cloned by limiting dilution and will continue to proliferate extensively. The behaviour of these cells suggests that the germinative epithelium of the follicle bulb is not composed solely of transit amplifying cells with inherently limited proliferative potential, but that their growth characteristics are strongly influenced by their culture environment.
In the second model, serially-passaged dermal papilla cells spontaneously aggregate to form papilla-like structures in vitro. The size of these aggregates can be experimentally manipulated with bioactive compounds that impinge on signalling pathways implicated in follicle development. Lithium chloride, dorsomorphin and SU5402 (which act via Wnt, inositol phospholipid, phosphotyrosine phosphatase, BMP, FGF, VEGF and PDGF signalling) all induced dose-dependent reductions in aggregate size. The alopecia drug, minoxidil, reversed the effect of lithium chloride. Dysregulation of papilla size is thought to underlie follicle miniaturisation in alopecia and follicle enlargement in hypertrichosis. These ovine papilla cells provide a new model for studying the morphogenetic mechanisms that regulate papilla size.
Department of Dermatology, Maulana Azad Medical College, New Delhi, India
Melasma is a rather recalcitrant condition to treat. Superficial and medium depth peels are recommended for treatment of melasma in dark skinned patients. Conventional peels such as glycolic acid peels, trichloroacetic acid peels, Jessner's peel and salicylic acid peels have been used successfully over several decades for melasma. However, they are also associated with several complications, such as postpeel hyperpigmentation. There is always a scope for newer combinations of peels, which may improve the tolerance of these agents. Several alpha hydroxy acids are used in combination along with chelating agents and antioxidants. Newer innovations also including self neutralizing and more cosmetic elegant peels. However, the actual peeling effect of some of these may be questionable due to the addition of several cosmeceutical ingredients as well.
N. Saunders, M. Hazar-Rethinam, L. M. De Long, O. Gannon
University of Queensland, Diamantina Institute, Translational Research Institute, Woollongabba, Qld, Australia
There is unequivocal data implicating the E2F family of transcription factors as key regulators of proliferation, differentiation and neoplasia. In addition, we recently showed that the ratio between activating E2Fs (e.g. E2F1) and inhibitory E2Fs (e.g. E2F7) may regulate apoptotic responses (1,2). We now present the results of our recent studies that identify an important, unique and non-redundant role for E2F7 in suppressing sensitivity to cytotoxic stimuli. Dysregulation of responses to UV or chemotherapy-induced cytotoxicity are fundamental to the development of squamous cell carcinoma (SCC) and its resistance to chemotherapeutics. Thus, identification of the downstream effectors that regulate E2F7-dependent sensitivity to the major causative factor in skin carcinogenesis (UV) or sensitivity to the major chemotherapeutic agents will be of significant clinical and biological interest.
Using a series of knockout and overexpression strategies we show that E2F7 antagonises E2F1-dependent apoptosis in response to UV and doxorubicin in keratinocytes and SCC cell lines. This is likely to have clinical implications since E2F7 is overexpressed in human SCC approximately 200 fold. We go on to show that cytotoxic-sensitive SCC cell lines have a low E2F7/E2F1 ratio and insensitive SCC cell lines have a high E2F7/E2F1 ratio. Using these sensitive and insensitive cell lines, coupled with E2F7 knockdown strategies, we conducted an -omics based screen and identified four genes that may act as downstream effectors of E2F1/7 mediated cytotoxic sensitivity.
D. R. Menon1, S. Das1, C. Krepler2, H. Knausz1, B. Rinner1, E. Bonyadirad1, J. Villanueva2, B. Gabrielli3, G. Hoefler1, M. Herlyn2, H. Schaider1
1Medical University of Graz, Graz, Austria2The Wistar Institute, Philadelphia, PA, USA3Diamantina Institute, Brisbane, Qld, Australia
Acquired drug resistance is a major reason for limited success of multiple drugs including recently introduced molecular targeted therapies, like that of BRAF inhibitors in melanoma. Exposing melanoma cells to different concentrations of PLX4032 resulted in transformation of parent cells into slow proliferative multiple drug tolerant cells characterized by high expression of CD271 and an altered chromatin state. These cells presented with upregulation of SOX10 and downregulation of melanoma antigen markers like Melan-A and Tyrosinase. A sustained rewiring of signaling cascades exhibited by this cell population, termed adaptive drug tolerant cells (ADTCs), renders them multiple-drug tolerant. Exposure to combinations of different drugs, like PLX4032 with inhibitors for IGF1-R, HDAC or Pi3K did not affect survival of ADTCs compared to parent cells. ADTCs did not originate from a particular subpopulation, since sorting of parent cells into CD133+/− or CD271+/− always resulted in high expressing CD271 ADTCs. Under prolonged drug exposure ADTCs re-establish homeostasis in signaling leading to the formation of permanently drug resistant cells with loss of sensitivity towards the drug they primarily were exposed to, but susceptible to other drugs. This shift was observed to be coupled to the loss of CD271 and higher proliferation. Nevertheless the capability to exhibit the trait of transformation into ADTCs is preserved even in permanently drug resistant cells, exemplifying a relentless nature to drug resistance. ADTCs represent a new phenotype of early multiple drug tolerant cancer cells amenable for therapeutic intervention.
A. Sevinc1, H. Turna2, M. Ozdogan3, S. Buyukberber4, N. M. Mandel5, O. G. Demir6, E. Gokmen7, S. Paydas8, H. Akbulut9, I. Celik10
1Gaziantep University, Gaziantep, TR, Turkey2Medical Oncology, Istanbul University, Istanbul, Turkey3Medical Oncology, Akdeniz University, Antalya, Turkey4Medical Oncology, Gazi University, Ankara, Turkey5Medical Oncology, Koc University, Istanbul, Turkey6Medical Oncology, Bilim University, Istanbul, Turkey7Medical Oncology, Ege University, Izmir, Turkey8Medical Oncology, Cukurova University, Adana, Turkey9Medical Oncology, Ankara University, Ankara, Turkey10Medical Oncology, Hacettepe University, Ankara, Turkey
Ipilimumab, an anti-CTLA-4 monoclonal antibody has been shown to enhance immune responses and induce durable clinical responses in patients with metastatic melanoma. The authors report retrospectively on metastatic melanoma patients treated under Expanded Access Program.
Patients with metastatic melanoma were treated with ipilimumab 3 mg/kg for four doses. Response evaluation was done on week 12.
A total of 96 patients enrolled with 73 evaluable. Ipilumumab treatment was given as 2nd, 3rd and 4th line in 19.1%, 65.7% and 15.1% of patients, respectively. The numbers of patients receiving ipilimumab for 1, 2, 3 and 4 doses were 5, 9, 12 and 47, respectively.
G1/2 adverse events were reported in 36.9% (27/73) of patients whereas G3/4 was 16.4% (12/73). There were eight G3 side effects (diarrhea in five, muscle weakness in one, hypothyroidism in one and GI bleeding in one patient). There was one reversible G4 thrompocytopenia and two deaths were associated with hepatic AEs.
Fourty-six of 73 patients (63.0%) completed four doses. The disease control rate (DCR) was 36.9% (PR: 15.2% and SD 21.7%). Median time to progression (TTP) was 3.6 months (95% CI, 2.2–5.8 months). Median overall survival was 8.7 months (95% CI, 5.3–12.1 months) with 24.8% of patients surviving within 24 months of follow up period.
Ipilimumab treatment resulted in favorable clinical benefit for metastatic melanoma patients. Data generated by our 10 centers revealed an efficacy and safety profile consistent with the currently avaliable knowledge.
C. Singh, D. Parsad, A. Kanwar, S. Dogra
Postgraduate Institute of Medical Education and Research, Chandigarh, India
Publish consent withheld.
A. Smith, R. Kim, A. Metcalf, M. Harrison, W. Lim, R. Sturm
University of Queensland, St Lucia, Qld, Australia
Recent GWAS evidence has revealed the presence of a SNP within the fouth intron of the Interferon Regulatory Factor 4 gene (IRF4), which is associated with pigmentation, nevus formation, UV sensitivity and melanoma suceptibility.1 The rs12203592_T SNP, which is associated with a lighter pigmentation phenotype, is only found in around 16% of Caucasian populations, whilst the C allele variant is homozygous in Chinese, Japanese and African populations. While this association has been identified in a number of independent GWAS the functional effect of the SNP and role of IRF4 in melanocyte cell function is unknown. IRF4 and other members of the IRF family have been recognised as key mediators of differentiation and cellular function in other systems and are crucial regulators of interferon responses in these biological contexts. Interestingly, an interferon (IFN)-gamma-dependent crosstalk between melanocytes and macrophages has recently been reported that allows UVB-irradiated melanocytes to evade immunosurveillance, which ultimately contributes to melanomagenesis.2 Using primary human melanocytes, we have demonstrated differential expression of IRF4 between individuals homozygous for the T and C allele, with C/C individuals having significantly higher IRF4 expression. Analysis of the cis-regulatory activity of the IRF4 intron 4 suggests the SNP directly effects the transcriptional regulation of the IRF4 gene. To gain further insight into the function of IRF4 in melanocytes, transcriptome profiling of primary human melanocytes following siRNA-mediated IRF4 knock-down was performed, in the presence or absence of IFN-gamma. Validation of selected candidate genes in melanocytes of known IRF4 genotype provides strong evidence for a role for IRF4 in mediating melanocytic interferon responses. Current investigations are aimed at further understanding the role of IRF4 in human pigmentation and exploring the link between differential IRF4 expression and the response of melanocytes to IFN-γ signalling.
E. J. Song1, M. S. Rybchyn1, G. M. Halliday1, D. L. Damian2, C. Gordon-Thomson1, R. S. Mason1
1The University of Sydney, Sydney, NSW, Australia2Dermatology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
Ultraviolet (UV) radiation causes potentially mutagenic DNA damage in skin cells. Our previous studies in human subjects showed photoprotective effects of topical 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), applied before irradiation, on thymine dimers (TD), but the mechanisms were unclear. The aim of this study was to assess the activity of post-UV 1,25(OH)2D3 in human volunteers and to determine if one DNA repair mechanism (nucleotide excision repair- NER) is modulated by 1,25(OH)2D3. Volunteers were exposed to solar-simulated UV close to the MED, followed immediately by treatment with topical 1,25(OH)2D3, or vehicle. In punch biopsies taken 2 h later, DNA damage and expression of DNA repair enzymes were analysed by immunohistochemistry and image analysis using antibodies to three types of DNA damage: TD, 8-oxo-7,8-dihydroguanine (8-oxodG), and 8-nitroguanosine (8-NG), and two types of NER proteins, Xeroderma Pigmentosum complementation group C (XPC) and DNA damage binding protein 2 (DDB2).
1,25(OH)2D3 reduced UV-induced TD by 57%, 8-oxodG by 51%, and 8-NG by 41%. At the same time, 1,25(OH)2D3 increased XPC by 146% and DDB2 by 97%. The data is consistent with the proposal that improved efficiency of NER may be one of the mechanisms explaining the photoprotective effects of 1,25(OH)2D3 in skin.
S. Sonthalia1, R. Sarkar2, J. Bharti1
1SKINNOCENCE, Haryana, India2Maulana Azad Medical College, LNJP Hospital, New Delhi, India
Publish consent withheld.
H. P. Soyer
Dermatology Research Centre, School of Medicine, Princess Alexandra Hospital, The University of Queensland, Brisbane, Qld, Australia
In this presentation an overview on our specific naevus-orientated research program carried out in Southeast Queensland will be presented with special emphasis on the preliminary results of our NHMRC grant titled ‘Effects of naevogenesis susceptibility genes and phenotypic correlation with dermoscopic characteristics of naevi’. In addition, the results of a recent naevus surveillance study on a cohort of high-risk melanoma patients will be presented. And finally, a blueprint for staging of murine melanocytic lesions based on the Cdk4R24C/R24C::Tyr-NRASQ61K model will be discussed.
M. S. Stark1, G. M. Boyle1, J. M. Palmer1, J. Symmons1, C. Lanagan1, C. W. Schimdt1, H. P. Soyer2, P. Pollock3, A. Herington3, N. K. Hayward1
1Queensland Institute of Medical Research, Brisbane, Qld, Australia2School of Medicine, Princess Alexandra Hospital, The University of Queensland, Brisbane, Qld, Australia3Queensland University of Technology, Brisbane, Qld, Australia
We have recently discovered a profound upregulation (mean 204 fold) of the microRNA (miRNA) miR-514a-3p in 38/55 (69%) cutaneous melanoma cell lines compared to several other cancer types (3% or 1/34). This miRNA is a member of a cluster (miRNA-506–514) that has been shown previously to be involved in initiating melanocyte transformation along with promotion of melanoma growth. We have shown that miR-514a-3p specifically targets NF1, a tumour suppressor in melanoma. Generally, miRNAs negatively regulate target genes by binding to the transcript's 3′UTR however in this instance the NF1 mRNA has miR-514a-3p specific binding sites in its coding sequence (exons 9 and 23). Using a Dual-Luciferase Reporter assay, and following site-directed mutagenesis, we have shown a 4–5 fold recovery of luciferase signal compared to endogenous levels when both binding sites were mutated. NF1 negatively regulates H/KRAS and NF1 inactivation has been implicated in resistance to RAF/MEK-targeted therapies. Large-scale genome and exome sequencing projects have shown that NF1 loss of function can be frequently attributed to truncating mutations. Alternatively, we propose that binding of miR-514a-3p to NF1 transcripts provides another mechanism for the dysregulation of the MAPK pathway which may account for resistance to current therapies.
A. Taieb1,2,3, T. Jouary1,2,3, K. Boniface3, J. Seneschal1,2,3, M. Cario-Andre2,3, D. Mossalayi3, K. Ezzedine1,2,3
1Department of Dermatology and Pediatric Dermatology, Bordeaux, France2National Reference Center for Rare Skin Disorders, Bordeaux, France3Inserm U 1035, Université Bordeaux 2, Bordeaux, France
In the last 5 years we have studied prospectively a large cohort of children and adults with vitiligo. Clinical and pathological observations made in this group of patients have been of major importance to direct our bench research. In particular, the relation of segmental to nonsegmental vitiligo has been questioned, new forms of vitiligo have been delineated, and the importance of factors affecting immune responses (autoimmune and atopic diathesis) in patients and families have been studied. Recent data underline that vitiligo associated with thyroid autoantibodies has distinct clinical features from vitiligo without thyroid autoantibodies. In particular, patients with longer duration of disease and greater body surface involvement are more likely to present thyroid autoantibodies and should thus be monitored for thyroid function and anti-thyroid antibodies on a regular basis. Koebner's phenomenon and its role in primary melanocytorrhagy vs relation to inflammatory progression of disease is important for disease assessment and prognosis. A simple clinical score for Koebner's phenomenon was developed indicating that seven variables were independently associated with the presence of KP namely disease duration of more than 3 years, forehead + scalp areas, eyelids, wrists, genital + belt areas, knees and tibial crests. The score sustained adequate discrimination and calibration arguing that KP can be adequately predicted. Better nomenclature and outcome measures to perform muticenter randomized trials are future goals of our group within the Vitiligo European task force, now joined by other international colleagues since last year Vitiligo Global Issues Consensus Conferences held in Seoul and Bordeaux.
R. Tamagawa-Mineoka, H. Mizutani, N. Nakamura, N. Katoh
Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan
It has been widely known that platelets play an important role in inflammation, in addition to their function in hemostasis and thrombosis. On activation, platelets release various immune mediators including adhesion molecules which may induce and maintain inflammatory reactions. Recently we have reported that platelets are required for recruitment of leukocytes into murine inflamed skin in immediate hypersensitivity reaction and chronic dermatitis. However, the role of platelet-derived adhesion molecules in skin inflammation has not been understood yet.We investigated the role of platelet-derived adhesion molecules, P-selectin and junctional adhesion molecule (JAM)-A in a mouse model of contact hypersensitivity.
Mice were sensitized and hypersensitivity was elicited in ear skin with hapten, with or without platelet depletion by administration of antiplatelet antibody. Moreover, platelets from normal mice or platelets deficient with P-selectin or JAM-A were infused into platelet-depleted mice.
In platelet-depleted mice, the ear-swelling response and leukocyte infiltration into skin significantly decreased. Flow cytometry showed that the number of platelet-leukocyte aggregates were higher in blood of hapten-challenged mice compared with sham-challenged mice. Recruitment of leukocytes to skin was restored by infusing platelets from normal mice, and this was blocked by infusing platelets from P-selectin-deficient mice or platelets pretreated with anti-P-selectin antibody. Moreover, infusion of platelets pretreated with anti-JAM-A antibody also suppressed leukocyte recruitment. A combination of both antibodies showed further decrease in leukocyte recruitment.
These results suggest that platelets induce leukocyte recruitment to skin by forming platelet-leukocyte aggregates via distinct sets of adhesion molecules P-selectin and JAM-A.
K. Tashiro1, M. Shishido1, T. Gomi1, Y. Tanaka2
1Pola Chemical Industries, Kashio-cho, Totsuka-ku, Yokohama, Kanag, Japan2Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
Autophagy is fundamentally important in human health and disease. However, the contribution of autophagy to age-dependent changes in human skin has not been well described. In a past study, we found that autophagy is disrupted at the degradation step in dermal fibroblasts derived from aged women (aged fibroblasts), and this change could be involved in age-related alterations to extracellular matrix (ECM) components such as type I collagen, hyaluronan and elastin. The present study confirmed age-dependent changes in autophagy through in vivo experiments using transmission electron microscopy, and examined further contributions of autophagy to the function of dermal fibroblasts. To mimic disruption of autophagy in aged fibroblasts, young fibroblasts were treated with lysosomal enzyme inhibitors leupeptin and pepstatin. Treated fibroblasts showed decreased levels of nicotinamide adenine dinucleotide (NADH), which plays a key role in cellular energy metabolism. These findings suggest that age-dependent disruption of autophagy could be related to changes in cellular energy metabolism along with reduction of ECM components. In addition, we performed screening of plant extracts, and found that the extract of Hydrangea macrophylla var. thunbergii (Japanese name ‘Amacha’) significantly ameliorated disruption of autophagy in aged fibroblasts and increased NADH production. The extract of Hydrangea macrophylla var. thunbergii might thus improve skin conditions resulting from age-dependent changes in ECM and dermal fibroblasts.
M. K. D. Tissera1, J. K. K. Seneviratne1, B. D. W. Jayamanne2
1Lady Ridgeway Hospital for Children, Borella (Colombo 09), WP, Sri Lanka2National Dengue Control Unit, Ministry of Health, Narahenpita (Colombo 05), WP, Sri Lanka
Loss of pigment, either partial (hypopigmentation) or complete (depigmentation), can have a profound psychological impact, perhaps seemingly out of proportion for something that is almost exclusively benign. Atopic dermatitis (AD) is the commonest cause of hypopigmentation that appears to be increasing in frequency during recent decades. Most of the studies are based on the western population, few data available in the South Asian population. Other causes are pityriasis versicolor, photodermatitis, post inflammatory hypopigmentation, leprosy and vitiligo.
To determine the percentage of patients, could be diagnosed by the clinical examination alone, requiring additional procedures for diagnosis (Ex: Scraping, biopsy, etc) and percentage of patients having generalized disease.
A descriptive study of hypopigmented lesions at the skin clinic of Lady Ridgeway Hospital for Children Colombo, during 1st April–30th September 2011. All children had a complete clinical evaluation. Diagnosis of lesions done according to the current guidelines.
Out of presented patients, AD and AD related dermatological conditions were commonest (28.3%). Mean age was 2.94 years (SD ± 2.91), where 52.1% were females. 84.5% were diagnosed clinically, scrapings were performed in 11.4% of the remaining patients for diagnosis. Facial lesions (60.4%), facial and upper limbs (12.6%) and facial and truncal lesions (11.8%) were seen.
Commonest hypopigmented skin lesion related to atopic dermatitis. Most common anatomical sites were face, upper limbs and trunk. Most can be diagnosed clinically.
P. L. Tong1,2,3, J. Qin3, C. L. Cooper4,5, P. M. Lowe1,2, D. F. Murrell6,7, S. Kossard8, L. G. Ng9, B. Roediger3, W. Weninger1,2,3, N. K. Haass1,2,3,10
1Discipline of Dermatology, University of Sydney, Camperdown, NSW, Australia2Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia3Centenary Institute, Newtown, NSW, Australia4Sydney Medical School, University of Sydney, Camperdown, NSW, Australia5Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia6Faculty of Medicine, University of New South Wales, Randwick, NSW, Australia7Department of Dermatology, St. George Hospital, Kogarah, NSW, Australia8Skin and Cancer Foundation, Darlinghurst, NSW, Australia9Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore10University of Queensland Diamantina Institute, Woolloongabba, QLD, Australia
Multiphoton microscopy (MPM) is a novel imaging technology that has recently become applicable for diagnostic purposes. The use of (near) infrared light in MPM allows for deep tissue imaging. In addition, this modality exploits the autofluorescent nature of extracellular matrix fibres within the skin.
The aim of this study was to quantitate the structure and abundance of elastic fibres in human dermis in three dimensions utilizing autofluorescent signals generated by MPM for the objective examination of elastin-related skin disorders.
Cross-sections of skin samples from elastin-related disorders were analysed by MPM and correlated to histopathological examination. In situ visualisation of elastic fibres by MPM was conducted by en face imaging of ex vivo skin samples through the intact epidermis. Image analysis software was used to quantify elastic fibres in three dimensions.
Based on the MPM-detected autofluorescence specific for elastin, we developed the Dermal Elastin Morphology Index (DEMI), calculated as the ratio of elastic fibre surface area and volume. This enabled objective three-dimensional quantification of elastic fibres. Quantitative scoring of sun-damaged skin using the DEMI correlated with qualitative histopathological grading of the severity of solar elastosis. Furthermore, this approach was applied to changes in elastin in other elastin-related skin disorders, such as pseudoxanthoma elasticum (PXE), PXE-like syndrome, elastofibroma, focal dermal elastosis, anetoderma, mid-dermal elastolysis and striae distensae. We also imaged elastic fibres in intact ex vivo skin imaged en face through the epidermis, indicating that this approach could be used in patients in vivo.
MPM has the potential for non-invasive in vivo visualisation of elastic fibres in the dermis with near histological resolution. The DEMI is a novel approach to enable objective assessment of elastic fibres to support diagnosis as well as monitoring of disease progress or therapy of elastin-related skin disorders.
A. Q. T. Tran1, D. F. Murrell1, J. P. Lazo-Dizon1, J. Kim1, B. S. Daniels1, S. S. Venugopal1, L. M. Rhodes1, M. G. Laws2
1Dermatology, St George Hospital, Kogarah, NSW, Australia2Kirby Institute, Darlinghurst, NSW, Australia
It is not known which objective disease extent measure for atopic dermatitis is the most reliable. This study compared the inter-rater and intra-rater reliability of the four most commonly used outcome measures for atopic dermatitis: the objective SCORing Atopic Dermatitis (SCORAD), Eczema Area and Severity Index (EASI), Six Area, Six Sign Atopic Dermatitis (SASSAD) and Three Item Severity index (TIS), as well as analysing their correlation to three QOL instruments: the Patient-Orientated Eczema Measurement, Child/Dermatology Life Quality Index and SkinDex-29.
After power calculations, 12 atopic dermatitis patients having different degrees of severity were assessed on the same day by five independent trained dermatology assessors. Reliability was measured using the intra-class correlation coefficient calculated through one-way random effect ANOVA and Bland-Altman plots. Correlation between subjective and objective outcome measures was computed using the two-tailed Spearman's rho correlation with scatterplots.
EASI demonstrated a high intra-rater and moderate inter-rater reliability, ICC = 0.886 (95% CI: 0.744–0.952) and ICC = 0.73 (95% CI = 0.5–0.9), respectively. SASSAD showed moderate intra-rater and inter-rater reliabilities, ICC = 0.720 (95% CI = 0.424–0.878) and ICC = 0.68 (95% CI = 0.44–0.88). TIS showed high intra-rater reliability ICC = 0.886 (95% CI: 0.744–0.952) but low inter-rater reliability ICC = 0.497 (95% CI = 0.233–0.785). Objective SCORAD showed low intra-rater and inter-rater reliability, with ICC of 0.446 (95%CI = 0.037–0.730) and 0.498 (95% CI = 0.234–0.785), respectively. Only SASSAD demonstrated moderate correlation with SkinDex-29 ρ = 0.611 (P = 0.035).
In conclusion, this study found that the EASI score was the most reliable objective outcome measure and supports the use of EASI as a routine extent measure for atopic dermatitis studies and possibly for routine clinical use.
Telethon Institute for Child Health Research, West Perth, WA, Australia
Dendritic cells (DC(s)) are heterogeneous cells widely accepted as the key initiators of T cell immunity. To date, two major subsets of DCs are described: plasmacytoid DCs and conventional DCs. The latter consists of three lymphoid resident DC types found in all secondary lymphoid organs and three tissue-derived, or migratory, DC types that traffic from their tissue of origin to the lymph nodes. Subsets of these migratory DCs include epidermal-derived Langerhans cells and two dermal-derived DCs.
The aim of this study is to identify the DC subtypes responsible for presenting melanoma-derived antigens and to gain a mechanistic insight of such processes. To ascertain this information we utilize a mouse model of melanoma. Traditionally, such a model involves subcutaneous delivery of B16 cells. This results in tumour growth beneath the skin, a distal location to a host of distinct DC subsets resident in the skin, such as epidermal-derived LC.
This is in stark contrast to natural melanoma progression in humans. The etiology of this cancer clearly resides within the epidermal layer followed by vertical growth and dermal invasion. This certainly allows local epidermal- and dermal-derived DC to sample the malignant tissue before their migration to the tumour draining lymph node to initiate T cell immunity. Thus, to more closely mimic the human condition, we developed an orthotopic murine model of melanoma where tumour establishment and expansion is within the skin. We are using this novel model to dissect the complex immunosurveillance processes occurring during melanoma.
G. J. Walker1, R. Ram2, H. Handoko2, B. Ferguson1, H. K. Muller3, P. Soyer4, G. Morahan2
1Queensland Institute of Medical Research, Brisbane, Qld, Australia2Centre for Diabetes Research, Western Australia Institute of Medical Research, Perth, WA, Australia3School of Medicine, University of Tasmania, Hobart, Tas., Australia4Dermatology Research Centre, University of Queensland, Brisbane, Qld, Australia
Melanoma prevention and treatment strategies are transitioning to an individualized and mechanism-based approach, based on genetics. However inherited variation between individuals can manifest at any stage of neoplastic progression, and possibly the interaction of many genes is involved. Except for family history, the presence of above average numbers of naevi is the strongest melanoma risk factor. Naevus count is mostly genetically determined, although genes discovered to date explain only a small proportion of the heritability. As Dysplastic Naevus Syndrome (DNS) is at the upper extreme of naevus count, a powerful strategy to better understand melanoma progression is to look for genes that modify this phenotype. To do this we have combined our mouse model of DNS (Cdk4R24C::Tyr-NRAS) with the Collaborative Cross (CC), a large resource of inbred mouse strains. The CC facilitates not only rapid mapping of genes, but also provides a step-by-step way to move from quantitative trait locus, to gene, to pathway, to biology. We have mapped several melanoma-related phenotypes to very small chromosomal regions, containing only a very small number of genes. The identification of these genes, and the molecular pathways through which they act, will lead to a fuller understanding of naevus and melanoma development not only in DNS patients (who are at the extreme), but also in the general population. Especially attractive as potential therapeutic targets are genes that slow the development of lesions, as they are dominant over the oncogenic mutations known to generate melanoma in both our mice, and humans (CDK4R24C and NRASQ61K).
C. Q. Wang1,2, M. Ramirez1,2,3, C. G. Mendoza1,2,3, B. S. Daniel1,2,3, M. Law3, D. F. Murrell1,2,3
1Premier Specialists St George, Randwick, NSW, Australia2Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia3The Kirby Institute, University of New South Wales, Sydney, NSW, Australia
Objective outcome measures of disease activity and severity have been developed in AIBD, whilst treatment intensity has not been compared.
Two measures of therapeutic intensity for AIBD were developed, so that the amount of treatment needed to achieve similar levels of objective outcome could be compared. The Treatment Intensity Scoring System (TISS) assesses the number of dosage of therapy each patient is undertaking whilst the Treatment Adversity Scoring System (TASS) assesses the number and severity of adverse events that occurs during therapy.
Forty five patients with AIBD were retrospectively evaluated from May 2009 to September 2012 to calculate TISS and TASS scores for every visit, compared to their objective disease extent scores (ABSIS, PDAI, BPDAI) and quality of life scores (ABQOL, TABQOL). TISS scores ranged from 0 to 30 (8.7 mean), with higher scores attributed to relapses, severe disease and recalcitrant disease. TASS scores ranged from 0 to 10 (mean 0.7), with higher scores attributed to increasing treatments and respective dosages.
A positive statistically significant relationship was found between the TISS and the ABSIS, PDAI activity, ABQOL and TABQOL scoring systems.
E. Warrick1, C. Duval1, S. Nouveau1, C.-M. Benoit-Jay1, P. Bastien1, S. Deret2, A. Morita3, O. de Lacharrière1, F. Bernerd1
1L'oreal R&I, Clichy, France2Galderma R&D, Sophia-Antipolis, France3Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Actinic lentigines (AL) are benign skin hyperpigmented lesions associated with age and chronic sun exposure. AL develop earlier in Asian than in European skins. To better understand the aetiology of AL in both populations, the gene expression profile of AL from Japanese volunteers was analyzed and compared to previous results obtained on AL from European subjects. AL from the dorsal side of hands of 12 Japanese women were selected on the same epiluminescence criteria as in the European study. A whole genome transcriptomic study showed that 247 genes were differently expressed in AL vs adjacent non lesional skin (|Fold change| > 1.5 and P-value < 0.05). Interestingly, when comparing the Japanese and European AL profiles, we could show that AL lesions selected on the same clinical criteria from Japanese and European women share a common gene expression profile. In both populations, whereas the melanogenesis pathway was not altered, genes involved in multiple biological functions including epidermal proliferation and differentiation, inflammation, oxidative stress response and particularly extracellular matrix organization, were found modulated. In conclusion, European and Japanese AL share similar alterations of multiple actors involved in skin homeostasis, thus emphasizing the interest of a multi-targeted treatment for AL.
C. Duval1, S. Nouveau1, M. Monot1, P. Bastien1, E. Warrick1, C. Chagnoleau1, J.-P. Ortonne2, O. de Lacharrière1, F. Bernerd1
1L'oreal R&I, CLICHY, France2Dermatology Department, Hôpital l'Archet, Nice, France
Actinic lentigines (AL) are benign skin hyperpigmented lesions frequently found on sun-exposed sites in elderly people. However their histopathological features remain ill-defined. This study aimed at better describing morphological and biological features of AL expressing a similar clinical phenotype i.e. an elongated honeycomb pattern detected by epiluminescence and quantified through image analysis. Two biopsies were obtained from AL lesion and adjacent non lesional (NL) skin on the dorsal side of hands of 15 Caucasian women. In all lesions, HES staining revealed a drastic elongation of the dermal-epidermal junction associated with epidermal invaginations within the dermis. Morphometric analysis revealed a significant increase in the basal layer length and a higher undulation index in AL. The melanin content was found statistically higher and preferentially located within the epidermal basal layer in AL as compared to NL. However, melanocytes were found evenly distributed along the basement membrane, in both AL and NL, and the number of melanocytes per length of basal layer was similar. Furthermore, MITF and tyrosinase immunostainings showed that melanogenesis activity was not increased in AL. These findings indicate that both the higher melanin content and the disorganization of epidermal, dermal and junctional cutaneous structures should be taken into account in the follow-up of long-term treatments for age spots.
C. D. Weller1, S. M. Evans1, M. P. Staples1, P. Aldons2, J. J. McNeil1
1Monash University, Melbourne, Vic., Australia2Prince Charles Hospital, Brisbane, Qld, Australia
Multi-component compression is acknowledged as best practice treatment for venous leg ulcers, but many compression systems are not well tolerated, are unaffordable and are challenging to apply. Three layer tubular bandage system (3L) has been used for patients unable to tolerate conventional compression bandages, but clinical and cost effectiveness of 3L has not been evaluated despite frequent use in clinical settings. The aim of this study was to compare 3L with short stretch compression bandage (SS) for treatment of venous ulcers.
This multi-centre randomised controlled trial (RCT) recruited 45 participants with venous leg ulcers from wound clinics in Victoria and Queensland, Australia. Outcome measures included percentage wound reduction from baseline compared to week 12 following randomisation, proportion of ulcers healed, Quality of Life measures (SF 36 and Cardiff Wound Impact Schedule), patient-reported bandage adherence, recurrence rates at 3 months post healing and cost effectiveness. Outcome assessment was blinded.
The proportion of healed ulcers was higher for 3L bandage group [17/23 (74%) vs 10/22 (46%) (P = 0.05)]. Mean ulcer percentage reduction for 3L group was 82.4% vs70.1% The number of participants who reported tolerance at all treatment visits was 21 (91%) in 3L group vs 17 (73%). Self-reported compliance and study -related adverse events were similar in both groups. Health-related quality of life scores improved but differences between groups were not significant. Six of the 27 healed ulcers recurred within 3 months (P = 0.83). Cost per ulcer healed in 3L group was A$200 vs A$618 in SS group (P = 0.0001).
The 3L compression system applied weekly for up to 12 weeks increased healing rates when compared to SS bandage. The 3L compression system was well tolerated and more cost effective than the SS bandage group.
This RCT has demonstrated that 3L compression bandage improves healing rates in people with venous ulcers. Ease of application, less limb pain and greater comfort improved adherence to 3L compression system contributed to healing outcomes.
L. F. Xiang, M. Jiang, D. Dong, X. Xu
We isolated a population of adult stem cells with NCSC features from the bulge area of mouse whisker follicles. These bulge precursor cells, grown as three-dimensional spheroid structures, were capable of self-renewal through asymmetric cell division in vitro. They did not express squamous markers but expressed immature neural crest cell markers as well as the embryonic stem cell transcription factors. Furthermore, they exhibited multipotency that can give rise to neuronal, glia, myogenic, adipocyte, chondrocyte, and osteocyte lineage cells after targeted induction. This technology offered new opportunities for the use of these cells in regenerative medicine.
Next step, we developed an efficient method for differentiating hair NCSCs into melanocytes. The NCSCs-derived differentiated cells displayed a characteristic bipolar or tripolar morphology and expressed melanocytic markers with melanin pigment production. This could provide a novel in vitro system for studying the developmental biology and diseases of melanocytes.
Finally we shown an analysis by using NCSC isolated from mice with different age, in order to investigate whether the stem/progenitor cell pool with age or not. Hair NCSC abundance and function were found to decrease sharply with age, being extremely difficult to isolate, expand, differentiate and migrate when obtained from the elderly. Those changes represent novel insights into the aging process and could have implications regarding the potential for autologous stem cells therapy in older patients.
M. Yamada1, L. L. Lin1, K. Shakhbazov2, M. Dinger2, E. Payne1, I. H. Frazer2, P. Soyer1, T. Prow1
1Dermatology Research Centre, School of Medicine, Transitional Research Institute, Princess Alexandra Hospital, University of Queensland, Qld, Australia2Daimantina Institute, Transitional Research Institute, Princess Alexandra Hospital, University of Queensland, Qld, Australia
Actinic Keratosis (AK) is a very common precancerous lesion in photoaged caucasians. AK can progress to squamous cell-carcinoma, but the factors leading to transformation are debatable with the tumour suppressor gene p53 being a recognised candidate. When p53 is up-regulated, it promotes either growth arrest or apoptosis in response to cellular injury as well as is critical for genomic stability maintenance. UV exposure is a known cause of p53 mutations. There is a highly conserved and crucial p53 domain for signalling apoptosis located between codons 64 and 92. Particularly, codon 72 variant is most common coding-region polymorphism of TP53 gene.
We collected 25 clincially diagnosed AK lesions by shave biopsy. Half of the lesion was subjected to histopathology and the other half processed for whole transcriptome sequencing. All lesions were hitopathologically diagnosed within the AK to squamous cell carcinoma progression lineage, except for one that was diagnosed as solar elastosis. Whole transcriptome sequencing data was placed for p53 mutation profiles. We found that these lesions contained p53 mRNA with a wide variety of polymorphisms. Only one polymorphism was present at a high frequency in all but one patient, G to C mutation in codon 72 of p53.
The results have indicated that G/C polymorphism of p53 in codon72 is common in samples from AK patients. This may play a role in skin cancer risk, its progression and the efficacy of therapy. Finally, we are investigating the use of this polymorphism as a biomarker for squamous cell carcinoma.
K. Yin, K. Jagirdar, M. Harrison, R. Sturm, A. Smith
University of Queensland, St Lucia, Qld, Australia
Ultraviolet radiation (UVR) is one of the most common mutagens that humans are exposed to and the levels and frequency of exposure is a primary determinant of melanoma risk. In melanocytes, a number of signalling pathways have evolved to counter the damaging effects of UVR exposure. Amongst them is the MC1R G-protein coupled receptor that co-ordinates both melanin synthesis and induces post-UVR DNA repair when stimulated by its ligand α-MSH that signals via cAMP induction. Recently, the anti-microbial peptide β-defensin 3 (HBD3) has also been identified as one of the most highly induced genes in the skin following UVR exposure and has since emerged as an alternative agonist of the MC1R pathway that signals largely via MAPK. We are interested in exploring the mechanisms by which MC1R facilitates DNA repair and have previously identified the rapid and transient induction of the NR4A family of orphan nuclear receptors upon α-MSH stimulation in melanocytes. The induction of NR4A2 appears to be crucial for the ability of the MC1R pathway in enhancing DNA repair capacity that is independent of NR4A2 transcriptional function. Using fluorescence imaging, we have revealed that upon UVR irradiation, NR4A2 translocates to distinct nuclear foci by a mechanism requiring both p38-mediated phosphorylation and the involvement of poly-(ADP-ribose)-polymerase activity. Furthermore, our recent works show that NR4A2 co-localise and interact with proteins involved in the recognition and repair of DNA. Our findings indicate that NR4A2 represent a novel component of DNA damage response, placing it as an interesting candidate for understanding the mechanisms that underpin genomic maintenance and cyto-protection following UVR-induced carcinogenesis that have direct relevance to melanoma formation.