The Australasian Society for Dermatology Research Inaugural Annual Scientific Meeting Sydney, Australia May 15, 2004
Committee Chairman Rodney Sinclair (Melbourne)
Local Organizing Committee Ross Barnetson (Sydney), Gary Halliday (Sydney), Peter Foley (Melbourne)
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Plenary Talks P1
Sensitising Human Melanoma Cells to Apoptosis Induced by The Immune System P. Hersey, X. D. Zhang, X. Y. Zhang, J. Borrow, and S. Gillespie Oncology & Immunology Unit, Newcastle Mater Hospital T lymphocytes kill melanoma cells by two different mechanisms. One is by release of perforin and granzymes from cytotoxic granules. The second by engagement of TNF family ligands on the lymphocytes with their receptors on melanoma cells. The TNF related apoptosis inducing ligand (TRAIL) and its receptors is the most important of the latter against melanoma. In killing by the (TRAIL-R1 and R2) is a frequent cause of resistance to killing by CD4 T cells. Complete loss of death receptor expression may occur due to loss of genes for receptors. More frequently, translation is inhibited by proteins binding to the 3UTR of the mRNA. Very little is yet known about this transitional control of death receptor expression. Resistance to apoptosis in melanoma cells with normal levels of TRAIL death receptor expression is associated with two groups of proteins. Those of the Bel-2 family appear to regulate release of pro-apoptotic proteins from mitochondria whereas inhibitor of apoptosis protein (IAP) family inhibit effector caspases, such as caspase 3. Smac/DIABLO from mitochondria binds to the IAP proteins and thereby allows the effector caspases to induce apoptosis. Several signal pathways appear important in regulating these 2 groups of proteins. These are the MAP kinase ERK1/2. Akt kinase and protein kinase C pathways. Agents that inhibit these pathways can sensitise melanoma cells to apoptosis and promise to be important new agents in treatment. Certain Histone deacerylase inhibitors also appear to target transcription factors that regulate these proteins and may be valuable adjuncts to immunotherapy. We propose that this dual approach offers much scope for improved treatment of melanoma.
Immunopathogenesis of Psoriasis: Role of Targeted Therapies Daniel N. Sauder Department of Dermatology, Johns Hopkins University Medical Institution The current theory on the immunopathogenesis of psoriasis is based on an interaction between antigen-presenting cells (APCs), such as Langerhans cells (LCs) in the skin and T lymphocytes. LCs first migrate to regional lymph nodes where they process antigens and present them on their surface through MHC class I or II molecules. The interaction between LCs and T cells triggers an immune response through the activation of T cells and subsequent release of cytokines that perpetuate the inflammation and the cell-mediated immune response. In particular, the cytokine tumor necrosis factor (TNF) is a key mediator of inflammation and joint destruction in the rheumatic diseases and has been implicated in the pathogenesis of psoriasis and psoriatic arthritis. During the past several years, a major focus in psoriasis research has been the development of novel biologic therapies for this disease. The aim of these therapies is to provide selective, immunologically directed intervention, with the hope that such specificity will result in fewer side effects than traditional therapies. Several different strategies have been developed for the biologic therapy of psoriasis:
Inhibition of initial cytokine release and LC migration Elimination of the pathologic T cells with therapies targeted against activated T cells Blockade of interactions leading to T-cell activation or migration to tissue Alteration of the balance of T-cell types (eg, by redirecting T-cell differentiation away from the T-1 type implicated in psoriasis toward the T-2 type) Inhibition of cytokines, such as TNF This presentation reviews the immunopathogenesis with particular emphasis on targeted immunotherapies.
Keratinocyte Stem Cell Biology Pritinder Kaur Epithelial Stem Cell Laboratory, Peter MacCallum Cancer Institute, Melbourne, Australia The immense regenerative capacity of the epidermis for cell renewal observed both in vivo and in vitro, has to date been attributed to the epidermal stem cell population. While there are many dogmas on the expected behaviour of keratinocyte stem cells, relatively little is known about the comparative tissue regenerative capacity of these cells versus their more committed progeny. Given our ability to prospectively isolate keratinocyte stem cells from their more committed progeny (Li et al, 1998), we have been able to test the hypothesis that extensive epithelial tissue regenerative capacity is indeed a hallmark of the keratinocyte stem cell population. Data generated in our lab demonstrate that current surrogate assays employed to define stem cell activity thus far (i.e. clonogenicity, long-term proliferative output, and short-term tissue reconstitution), believed to reflect the extensive capacity for self-renewal and superior proliferative potential of KSCs, do not in fact permit their unequivocal identification. Further, it appears that all basal keratinocytes possess significant proliferative potential (Li et al, 2004) throwing into doubt the need for stem cell purification for clinical applications. The insights gained from ongoing studies in our laboratory on the role of microenvironmental factors that promote skin tissue regeneration from various classes of keratinocyte progenitors including the stem cells will be presented. Li et al. (1998) PNAS 95:3902-3907. Li et al. (2004). J Clin Invest.113:390-400.
Keratin Associated Proteins: A One Hundred Year Perspective Rothnagel Joseph Biochemistry & Molecular Biology, University of Queensland, Brisbane Over the last hundred years we have travelled from the discovery of opaque granules in the outer layers of the epidermis and inner root sheath of the follicle to the molecular characterisation of the components of these granules. With the availability of electron microscopy in the 1950s and 60s, keratohyalin and trichohyalin granules were described as non-membrane bound dense deposits that were intimately involved with keratin filaments. In the early 70s the first biochemical studies were beginning to reveal the proteins that make up these granules. From these studies two proteins were identified, filaggrin and trichohyalin in skin and hair respectively, that became the archetypal keratin associated proteins. Through gene sequencing, these proteins have been shown to have common features, including a calcium binding domain and repetitive sequence motifs. Moreover, these genes lie in the same region on human chromosome 1 and are part of a large cohort of epidermal genes known as the epidermal differentiation cluster. Many of these genes share similarities with trichohyalin and filaggrin and their protein products are often found to be closely associated with keratin filaments. Although linkage studies have shown that certain genetic dermatoses map to this region of chromosome 1 including psoriasis, ichthyosis vulgaris, eczema and some cancers, there is no evidence for direct involvement of keratin associated proteins in the etiology of these disorders.
P5 The UVA Waveband in Sunlight Contributes to Immunosuppression and Gene Mutation in Humans. Gary M. Halliday,1 Nita S. Agar,1 Terence Poon,1 Ross StC. Barnetson,1 Honnavara N. Ananthaswamy,2 and Alexandra M. Jones1 1 Dermatology Research Laboratories, University of Sydney, Sydney, Australia; 2Department of Immunology, University of Texas, MD Anderson Cancer Centre, Houston, U.S.A Ultraviolet radiation (UV) induced immunosuppression and gene mutation are both important biological events which lead to skin cancer development. We developed procedures for determining effects of UV radiation on the human immune system using recall immunity to nickel. Ours studies show that both the UVA and UVB wavebands suppress immunity in humans. We hypothesized that a substantial portion of the mutagenic alterations produced in the basal layer of human skin cancers by sunlight are induced by wavelengths in the UVA range. Using laser capture microdissection (LCM) we examined separately basal and suprabasal keratinocytes from human skin squamous cell carcinomas (SCC) and premalignant solar keratosis (SK), for both UVA and UVB induced adduct formation and signature mutations. We found UVA fingerprint mutations were detectable in human skin SCC and SK, mostly in the basal germinative layer. This contrasted with a predominantly suprabasal localisation of UVB fingerprint mutations in these lesions. The epidermal layer bias was confirmed by immunohistochemical analyses with a superficial localisation of UVB-induced cyclobutane thymine dimers contrasting with the localisation of UVA-induced 8-hydroxy-2’deoxyguanine adducts to the basal epithelial layers. If unrepaired these adducts may lead to fixed genomic mutations. The basal location of UVA rather than UVB-induced DNA damage suggests that longer wavelength UVR is an important carcinogen in the stem cell compartment of the skin. Given the traditional emphasis on UVB, these results may have profound implications for future public health initiatives for skin cancer prevention. As UVA-induced genetic damage is present in the stem cell layer of human skin cancers, UVA is likely to be an important carcinogen for human skin cancer.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Oral Presentations O1
The Royal Prince Alfred Hospital Experience with alefacept – Preliminary Results Katherine Armour, and Ross Barnetson Department of Dermatology, Royal Prince Alfred Hospital, Missenden Rd, Camperdown, NSW 2050 Psoriasis is a T-cell-mediated immune disorder in which memory-effector T cells stimulate keratinocyte hyperproliferation. Alefacept (human LFA-3/IgG1) is a novel biologic agent that binds to CD2 on T cells to inhibit T cell activation and proliferation. It also binds to FcgRIII on natural killer cells and macrophages to cause apoptosis of CD4 þ CD45RO þ and CD8 þ CD45RO þ memory-effector T lymphocytes. Because CD2 is upregulated on memory T cells, alefacept has demonstrated selective effects on this T-cell population, which is implicated in the pathogenesis of psoriasis. We are involved in a phase 4, multicentre, openlabel study which aims to evaluate the duration of clinical response of one or two 12 week courses of once weekly 15 mg intramuscular administration of alefacept (AMEVIVE). There are 100 subjects in this study, and 25 of them are being treated in our institution. Several different measures have been used to evaluate the clinical severity of psoriasis. The Psoriasis Area and Severity Index (PASI) is one of the most commonly used tools. Use of the PASI was first reported in 1978. Whilst imperfect, with an important interobserver variability, the PASI score is currently considered to be the standard for assessing improvement in psoriasis in clinical trials. The PASI score assesses the extent of psoriasis on four body surface area (head, trunk, upper and lower limbs) and the degree of plaque erythema, scaling and thickness. The PASI score accounts for both the extent of body surface area affected by the erythema, scaling and thickness, and the severity of these measures. The score ranges from zero (indicating no disease) to 72 (indicating maximal disease). We performed PASI scores on our patients being treated with alefacept at weeks 1, 7, and 12 of treatment. Whilst finding the PASI score a useful measure of our patient’s progress, we also observed it to have some limitations. In particular, we felt that the PASI score at times underestimated the improvement in our patients’ psoriasis. Our early experience treating our patients with alefacept will be discussed.
Changes in Scalp Hair Follicle Density with Advancing Age Rodney Sinclair,a Adam Chapman,b and Jill Mageec Department of Dermatology, University of Melbourne; bSchool of Health Sciences, Deakin University; cMayne Health Dorovitch, Melbourne, Australia Background: Age-related reduction in hair is seen in the axillary and pubic regions as well as the scalp, however it has not been investigated qualitatively on the scalp. Horizontal sectioned scalp biopsy is an ideal tool to investigate the impact of advancing age on scalp hair follicle density and morphology. Objectives: To examine the effect of age and follicle miniaturization on total hair count in 1666 horizontally sectioned midscalp biopsies from 883 women aged between 13 and 84 with hair loss. Setting: Specialist hair loss referral clinic in a teaching hospital. Design: Analysis of data set. Methods: All scalp biopsies were taken from the crown. Miniaturization was assessed by calculating the ratio of terminal to vellus-like hairs (T:V) at the mid-isthmus level and considered significant if the ratio was p4:1. Fibrosis was documented when present. Linear regression was used to examine the association between total hair count, age and miniaturization. Results: The average number of hair follicles per biopsy was 39.6 (SD þ /10.8). A highly significant negative association (po0.0001) was found between age and total follicle number, although the predictive value of age in total hair count was found to be small (R2o2%). Controlling for T:Vp4:1, the association was weakened, but remained significant. The relationship unconfounded by T:Vp4:1 shows that for every additional year of ageing, 0.077 total hair follicles (0.22%) are lost per biopsy. Conclusions: Age and follicular miniaturization were found to be extremely weak predictors of total hair count in women with hair loss.
Anal Intraepithelial Neoplasia – Diagnosis and Differential Diagnosis R. Hillman,2 A. Herat,1 M. J. Whitfeld,1 and A. Meagher3 1 Skin & Cancer Foundation, Darlinghurst; 2Sexually Transmitted Infections Research Centre, Westmead Hospital, Sydney; 3Sydney, St Vincents Hospital, Sydney Background: ASIL is the term for anal squamous intraepithelial neoplasia, It is classified as either low grade or high grade atypia, depending on the depth of involvement of the atypia. High grade atypia includes Bowens disease, bowenoid papulosis, and in situ carcinoma. ASIL is the suspected precursor lesion of invasive anal carcinoma, the incidence of which is increasing. Invasive anal cancer without treatment has a high mortality. Its treatment also involves significant morbidity and mortality. ASIL is more common in some demographic groups, principally homosexual men, especially those who are HIV infected. It may be asymptomatic. Using the analogy of CIN & cervical cancer, it has been proposed that early diagnosis and treatment of ASIL may be an effective strategy in reducing the mortality from invasive anal carcinoma. Method: The aim of study was to investigate the presenting symptoms, signs, initial diagnoses, and pathology reports of patients presenting with anal complaints in whom a subsequent histological diagnosis of ASIL was made. The medical files of 100 patients with biopsy confirmed ASIL seen by a colorectal surgeon or dermatologist, were reviewed. The initial symptoms, signs and provisional diagnoses, and pathology reports were documented. Conclusions: The results highlight that the symptoms of ASIL may be minimal or involve itch only. ASIL is often diagnosed coincidentally when biopsies performed for other reasons. Multiple anal pathologies may make diagnosis more difficult. Pre-existing anal warts or anal warts and ASIL may make the diagnosis more difficult. A high index of suspicion should be maintained of the possibility of ASIL given its often-asymptomatic nature.
Buccal Cells as a Source of mRNA and Protein for Analysis of a Zinc Deﬁciency Disorder Agnes Michalczyk,1 Leigh Ackland,1 George Varigos,2 and Lance Smith3 Deakin University, Centre for Cellular & Molecular Biology, Melbourne, Vic; 2Department of Dermatology, Royal Children’s Hospital, Melbourne, Vic and 3ThermoTrace, Biosciences Division, Thermoelectron Corporation, Noble Park 3174, Melbourne, Vic Human tissue is essential for investigations to determine the cellular or molecular basis of disease. However, access to patient cells can be a limiting factor for these investigations. We developed a method for obtaining viable buccal cells from mouthwash samples, for use as a source of mRNA and protein. Our method has been used to investigate the role of a novel zinc transporter hZnT4, in a mammary disorder leading to reduced zinc secretion into milk. Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4 from mouthwash buccal cells were similar between control individuals and two unrelated mothers who produced zinc-deficient milk. Zinquin staining indicated the presence of labile pools of intracellular zinc that did not co-localise with hZnT4, suggesting that hZnT4 is not involved in transport of zinc into vesicles, which in the breast, may destined for secretion into milk. Using a keratinocyte-specific medium, buccal cells could be cultured on Matrigel-coated permeable filters for up to two weeks while maintaining expression of some epithelial-specific markers including cytokeratin 13, cytokeratin 10, transferrin receptor and b-integrin. The basal marker cytokeratin 14 was detected in a few cells. In summary, we show that buccal cells can be obtained from a non-invasive procedure, for use as a source of material for biochemical analyses and a population of these cells can be maintained in culture for several weeks.
Swimming Pool Flotation Devices: A Possible Source of Molluscum Contagiosum Infection A. Braue,w S. Bowden,w G. Varigos, and H. Kellyw Royal Melbourne Hospital, Melbourne, Australia; wVictorian Infectious Disease and Reference Laboratory, Melbourne, Australia The current research was designed to assess whether fomites (inanimate objects capable of transmitting infections) in swimming pool water could harbour and transmit molluscum contagiosum virus (MCV), a member of the poxvirus family. Molluscum contagiosum (MC) outbreaks have been linked to the use of public and school swimming pools, however the exact source of infection is unknown. Clinical observations at the Department of Dermatology, Royal Melbourne Hospital have noted patterns of MC infection consistent with the use of swimming pool flotation devices. Creation of in-house and quantitative real-time polymerase chain reaction (PCR) assays provided a diagnostic tool for detection of MCV. Probes were designed for specificity, and a portion of the MCV genome was cloned and used as a standard for quantification. Flotation devices were spiked and exposed to a variety of chlorine dilutions simulating swimming pool water. MCV DNA was recovered in each instance, however, this may not reflect infectious virus. As MCV grows poorly in cell culture, a surrogate virus – such as the related poxvirus, vaccinia – will be used to determine any effects of chlorine concentration on infectivity. Our results suggest that swimming pool flotation devices could be a common source of MCV spread. Given the popularity of swimming pool use among young people – and the fact that MCV is the most common poxvirus affecting humans – MC is a significant public health issue. A better understanding of its mode of transmission could guide the improvement of best practice in swimming pool management.
Regulation of Wheal and Flare by Tea Tree Oil: Complementary Human and Rodent Studies Prue H. Hart,# Zeinab Khalil,^ Annette L. Pearce, Narmatha Satkunanathan, Emma Storer, and John J. Finlay-Jones# Department of Microbiology & Infectious Diseases, School of Medicine, Flinders University, Adelaide, and #Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Perth, and ^National Ageing Research Institute, University of Melbourne, Parkville, Victoria, Australia When applied twenty minutes after injection of histamine into human forearm skin, tea tree oil (TTO), the essential oil steam-distilled from Melaleuca alternifolia, reduces the developing cutaneous vascular response. In this study, the effect of TTO on inflammatory microvascular changes were dissected at the base of an experimental blister on rat skin, and the active components of TTO identified. 1,8-Cineole, representing 2% of TTO, reduced vascular changes induced by sensory neuropeptides released when the distal portion of a cut sciatic nerve was electrically stimulated. Further, 1,8-cineole was without regulatory effect in a plasma extravasation response to substance p and its pre-terminal modulatory effect was confirmed in tests in sensory-denervated rats. In contrast, terpinene-4-ol (approx 40% TTO) reduced substance P-induced microvascular changes and protein extravasation by a direct effect on post-capillary venules, without sensory nerve involvement. a-Terpineol (3% of TTO) had regulatory properties on both pre- and post-sensory nerve terminals. In human skin, terpinene-4-ol applied 10 min after histamine injection, but not a-terpineol or 1,8-cineole, regulated the developing wheal and flare suggesting that the histamine-induced responses in humans (at the dose used in this study, 50 ml of 330 mM histamine) are in large part determined by histamine directly affecting the vasculature via post-terminal mediated events. The underlying strength of these studies is the use of a well established rat physiologic model to differentiate the mechanism of regulation of microvascular changes in human skin by modulatory agents.
123:6 DECEMBER 2004
Calcium Mobilisation does not Mediate Wound-Induced c-fos Expression in Fetal Skin Samantha Gangnuss,2 Allison J. Cowin,1,2 and Timothy E. Rayner1,2 1 Child Health Research Institute, Women’s and Children’s Hospital, North Adelaide and 2 University of Adelaide Department of Surgery, The Queen Elizabeth Hospital, Woodville, SA. Reepithelialisation is a key component of wound healing and is dependent on the activation and migration of epithelial cells. Clinically, enhanced re-epithelialisation is associated with improved healing and reduced scarring, particularly in burns. Embryonic day 17 (E17) fetal rat skin has a unique capacity to re-epithelialise a wound in explant culture and to heal wounds in a scar-free fashion in vivo. Our studies have shown re-epithelialisation in E17 skin explants is dependent on wound-induced c-fos expression mediated by activation of extra-cellular regulated kinase (ERK). To define the wound-induced events regulating ERK activation and c-fos expression, we investigated the role of calcium as a second messenger. Wounds were created on the dorsum of E17 fetuses which were incubated in media containing calcium agonists or antagonists and the effect on c-fos mRNA expression assessed by Northern blot. Pre-incubating fetuses with the calcium-binding fluorophore fura-2 showed wounding caused calcium mobilisation in the skin as increased fluorescence was observed at the wound margin after 30 s and remained at high levels for 5–10 min. This calcium mobilisation was temporally associated with wound-induced c-fos expression as mRNA transcripts were first detected after 10 min and reached a peak 30 min post-wounding. Incubating fetuses with inhibitors of voltage-gated channels (verapamil and nifedipine; 1 and 10 mM) or store-operated channels (SKF96365; 10 and 100 mM) had no effect on maximal wound-induced c-fos expression (2-3 fold increase) suggesting the influx of extra-cellular calcium was not required. Similarly, chelating cytoplasmic calcium with BAPTA-AM (20 and 40 mM) and blocking Ca2 þ mobilisation from the endoplasmic reticulum (ER) using the phospholipase C inhibitor U73122 (1 and 10 mM) also failed to suppress wound-induced c-fos expression. The calcium ionophore A23187 (10 mM) and thapsigargin (1 mM), which elevates intra-cellular calcium levels by preventing reuptake by the ER, both stimulated c-fos expression in unwounded skin (2-3 fold), with thapsigargin causing a synergistic increase in wound-induced expression (up to 8 fold). These results suggest that whilst calcium mobilisation in fetal skin can stimulate c-fos expression, wound-induced activation of c-fos occurs independently of calcium-mediated signalling events.
The Transcription Factors c-Rel and RelA Control Epidermal Development and Homeostasis in Embryonic and Adult Skin via Distinct Mechanisms R. Gugasyan, G. Varigos, A. Voss, T. Thomas, P. Kaur, R. Grumont, and S. Gerondakis The Walter & Eliza Hall Institute of Medical Research. Parkville, Melbourne. Australia Determining the roles of Rel/NF-kB transcription factors in mouse skin development using loss of function mutants has been limited by redundancy amongst these proteins and by embryonic lethality associated with the absence of RelA. Utilizing mice lacking RelA and cRel, which survive throughout embryogenesis on a TNF-a deficient background (rela/c-rel/ tnfa/), we show that c-Rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G1 progression and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed the mutant epidermis develops a TNF-a dependent hyper-proliferative condition. Collectively, our findings indicate that RelA and c-Rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a post-natal environment through the suppression of innate immune mediated inflammation.
Defective in vitro Epidermal Wound Healing in Mice Lacking the Tetraspanin CD151 Sean M. Geary, Allison Cowin,# Stephen Fitter,w Mark D. Wright,w and Leonie K. Ashman School of Biomedical Sciences, University of Newcastle, Callaghan NSW; #Institute for Child Health Research, Adelaide; +Austin Research Institute, Heidelberg VIC The tetraspanin CD151 forms complexes in cell membranes with integrins, especially alpha3 beta1, alpha6 beta 1 and alpha6 beta4 and modifies integrin-mediated cell migration in vitro. In both simple and stratified epithelia in human tissues, CD151 is localised mainly on the basolateral surface of cells in contact with basement membranes. It has been shown to be a component of hemidesmosomes. We recently generated CD151-null mice. In contrast to mice lacking alpha3, alpha6 or beta4 integrin chains which display skin detachment syndromes and neonatal mortality, CD151-null mice are viable, apparently healthy and have normal skin architecture. However, in vitro analysis indicates that the mice have defects in platelet integrin signalling, T lymphocyte function and, of particular relevance here, greatly reduced outgrowth of keratinocytes from skin biopsies in explant cultures. Isolated epidermal keratinocytes from null mice also showed defective migration into scratch ‘‘wounds’’ in monolayer cultures. Integrin expression in tissues and by isolated keratinocytes appeared normal, but reduced cell surface expression of another tetraspanin, CD9 was observed. Experiments on in vivo epidermal wound healing are about to be conducted and will be reported.
Novel Vitronectin-Growth Factor Complexes for Skin Repair D. Harkin, R. Dawson,1 P. Gillies,1 N. Johnson, C. Hyde, A. Noble, C. Towne, B. Hollier, L. Ainscough,1 R. Minchinton, and Z. Upton Tissue BioRegeneration and Integration Program, Science Research Centre, Queensland University of Technology, Brisbane, QLD, Australia and; 1Queensland Skin Bank, Australian Red Cross Blood Service, Brisbane, Brisbane, QLD, Australia Our research is focused on facilitating cellular therapies for tissue repair and regeneration. In particular, we are examining the potential of novel protein complexes comprised of growth factors and the extracellular matrix protein vitronectin. These complexes enhance the ability of cells to migrate and proliferate through coordinate activation of both the growth factor receptors as well as vitronectin-binding integrins. Thus far we have developed vitronectin complexes that can incorporate 3 different growth factors and we are examining the potential use of these agents for the treatment of diabetic ulcers and burns patients. For example, the potential of these complexes to replace serum and feeder cells during construction of cultivated epithelial autografts is being investigated. In addition, the technology is being examined in conjunction with methods for applying keratinocytes to wounds in the form of a sprayed aerosol of dissociated cells. Moreover, since vitronectin is a ‘‘sticky’’ protein we hypothesise that it can be used to incorporate growth factors into dermal tissue substitutes, thus encouraging the recruitment and survival of cells necessary for bio-integration. Data from our research projects addressing these concepts, as well as mechanistic studies examining the molecular basis underlying the responses of skin cells to these complexes, will be presented.
Human Growth Hormone (hGH) presented by K14hGH-transgenic Skin Grafts Induces a Strong Immune Response but no Graft Rejection Jie Zhong, Koji Matsumoto, Rachel De Kluyver, Germain J. P. Fernando, Graham R. Leggatt, and Ian H. Frazer Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102, Australia Although immune responses for the rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a minor transplantation antigen to investigate the consequences of antigen presentation from skin cells. Mice, transgenic for hGH driven from the keratin 14 promoter, express hGH in skin kerationcytes. This hGH transgenic skin is not rejected by syngeneic non-transgenic recipients, though a strong antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce significant immunity, and this response can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, such immune responses do not always lead to rejection despite induction of local inflammatory changes.
A Comparative Study Between Electron Microscopy and Immunoﬂuorescence Mapping For the Diagnosis of Epidermolysis Bullosa E. Yiasemides,1,4 J. Walton,2,4 L. Vonthethoff,3 P. Marr,3 and D. Murrell1,4 1 Departments of Dermatology; 2Orthopaedic Research Institute and; 3Anatomical Pathology, St George Hospital, Sydney and; 4The University of New South Wales, Sydney Introduction: Epidermolysis bullosa (EB) has been classified into 3 main subtypes based on electron microscopy (EM) which directly visualizes and quantifies specific ultrastructural features. Immunofluorescence antigenic mapping (IFM) is a technique that determines the precise level of skin cleavage by determining the site of binding of a series of antibodies. Methods: A prospective cohort of 31 patients thought to have EB on clinical grounds had both EM and IFM performed. The sensitivity and specificity of EM and IFM were calculated using the genetic results as the ‘‘gold standard’’. Statistical analysis involved the determination of positive and negative predictive values, accuracies, positive and negative likelihood ratios and post-test probabilities of the IFM and EM diagnosis being correct compared to the genetic diagnosis. Results: 28/31 had a positive EB diagnosis. The genetic results were available for 18 patients, including 7 cases where the EM and IFM results were discordant. EM subclassified EB into its 3 major forms in 23/28 cases (82%) and IFM in 28/28 cases (100%). Overall, EM sensitivities and specificities when compared to genetic results were 59% and 79%, respectively. IFM sensitivities and specificities when compared to genetic results were both 100%, giving likelihood ratios of having a particular type of EB of 3.5:1 for EM and 199:1 for IFM. Conclusion: These findings demonstrate that IFM is significantly more sensitive and specific as a diagnostic test than EM for the classification of the major types of EB.
O13 T-Cell Induced Keratinocyte Apoptosis Ilse S. Daehn,1,2 Antiopi Varelias,2 and Timothy E. Rayner1,2 1 The Child Health Research Institute, Women’s and Children’s Hospital, North Adelaide, SA and; 2The University of Adelaide Department of Surgery, The Queen Elizabeth Hospital, Woodville, SA Chronic skin inflammation is a central pathogenic process implicated in a number of conditions affecting the skin such as dermatitis, psoriasis and sun damage. The interaction of T cells with skin-resident keratinocytes plays an important role in the transition from acute resolving inflammation to the development of persistent chronic inflammation. In dermatitis, T cell mediated apoptosis of keratinocytes is proposed as a primary mechanism by which epidermal integrity is impaired resulting in skin lesions, increased invasion of allergens and amplification of the inflammatory process. The aim of this study was to establish an in vitro system suitable for investigating T cell mediated keratinocyte apoptosis using Jurkat T cells and a human keratinocyte cell line (HaCat). In order to differentiate between the effect of cytokines released by Jurkats following activation with mitogen (10 ng PMA) and direct interaction between Jurkats and HaCats involving both surface molecules and cytokines, HaCats were either incubated with Jurkat conditioned media or co-cultured with Jurkat cells (48 h). Apoptosis was determined by flow cytometry after staining HaCats with annexinV/ propidium iodide and caspase 3 activity was measured using an enzyme activity assay. The expression of the death receptor Fas (FasR) by HaCats was also assessed using flow cytometry. The conditioned media of mitogen activated Jurkats caused a 2.6-fold increase in HaCat apoptosis and a 2-fold increased in caspase 3 activity compared to control. Co-culture with activated Jurkats however resulted in a much greater increase in HaCat apoptosis (up to 8 fold) and a 2.5-fold increase in caspase 3. Caspase 3 activity was abolished by co-incubation with a specific caspase 3 inhibitor. HaCat FasR expression was upregulated by coculture with activated Jurkats with the mean fluorescent intensity increasing from 6.8 (control cells) to 10.6. Co-incubating HaCats with un-activated Jurkats did not change FasR expression on the keratinocytes. These results indicate that activated Jurkat T cells induce HaCat apoptosis by increasing FasR expression and inducing caspase 3 activity. Whilst secreted T cell cytokines induced HaCat apoptosis, the major effect appeared via direct interactions between the keratinocytes and T cell surface molecules. This model will assist in defining the mechanisms responsible for T cell induced keratinocyte apoptosis in inflammatory skin conditions like dermatitis.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
O14 T-Cell Surface Molecules Inhibit Skin Fibroblast Proliferation and Collagen Synthesis Antiopi Varelias,1,2 Guldana Ruzehaji,2 Ilse S. Daehn1,2, and Timothy E. Rayner1,2 Child Health Research Institute, Women’s and Children’s Hospital, North Adelaide, SA; 2 The University of Adelaide Department of Surgery, The Queen Elizabeth Hospital, Woodville, SA Introduction: The interaction of T cells with skin-resident cells plays an important role in the transition from acute resolving inflammation to the development of dysregulated chronic inflammatory conditions of the skin. Non-healing wounds, UV-induced photodamage and atopic dermatitis are all skin conditions characterised by persistent inflammation where the inappropriate accumulation of immune cells eventually leads to dermal degeneration and skin atrophy. This is compounded by the action of matrix-metalloproteinases (MMP) produced by skin-resident cells. This study aimed to characterise an in vitro model of skin inflammation suitable for investigating the effect T cells have on fibroblast function and how they may promote deterioration of the dermis. Methods: The effect of naive or activated Jurkat T cells on primary skin fibroblast proliferation, collagen synthesis and MMP activity was examined. The influence of T cell surface molecules and secreted cytokines on fibroblast function was determined by culturing fibroblasts with formaldehyde fixed T cells or T cell conditioned media, respectively. Dynamic interactions involving surface molecules and cytokines were also investigated by co-culturing live T cells with fibroblasts. Jurkats were activated with PMA (10 ng per mL) for 48 h prior to co-culture experiments. Results: Both fixed and live T cells adhered readily to fibroblasts although this was more pronounced with activated T cells. Fibroblast proliferation was significantly inhibited (po0.05) by co-culture with fixed T cells and T cell conditioned media. Both fixed and live T cell coculture, but not T cell conditioned media, significantly inhibited collagen synthesis by up to 90% (po0.05), indicating that direct interaction with the T cells was primarily responsible for mediating inhibition of collagen synthesis. No major changes in MMP production by fibroblasts were induced by T cell conditioned media or co-culture with fixed or live cells. Conclusions: These findings indicate that direct fibroblast-T cell interactions via T cell surface molecules reduce the proliferative and synthetic capacity of fibroblasts without greatly effecting the proteolytic environment.
Dose and Wavelength Dependence for Ultravoilet-Induced suppression of Contact Hypersensitivity Responses to a Recall Antigen in Humans T. A. Phan, G. M. Halliday, R. StC. Barnetson, and D. L. Damian Dermatology, Melanoma and Skin Cancer Research Institute, Sydney Cancer Centre, University of Sydney at Royal Prince Alfred Hospital, Camperdown, Sydney, NSW, Australia Ultraviolet (UV) radiation-induced immunosuppression plays a central role in cutaneous carcinogenesis. The relative contribution of different UV wavebands to this immunosuppression, is however still uncertain, as an action spectrum for UV immunosuppression has not yet been determined in vivo in humans. We investigated the effects of different UV wavebands on contact hypersensitivity (CHS) responses to nickel in healthy, nickel-allergic volunteers, using a xenon-arc lamp emitting solar-simulated (ss) UV, or fitted with different interference filters of 10 nm half-bandwidth used to produce 6 narrowband (NB) UV sources at 20 nm intervals. Volunteers were irradiated once on their backs with graded doses of NB UV and a known immunosuppressive dose of ssUV 48 h prior to the application of nickel patch tests to both the irradiated areas and adjacent, unirradiated areas serving as immunologically intact controls. The patch tests were removed after 48 h, and a reflectance spectrometer was used to measure the nickel-induced erythema index (NIEI) of each site. Immunosuppression of nickel CHS responses was calculated by subtracting the NIEI of test sites from the NIEI of unirradiated control sites. We found that UV at 290 and 310 nm caused dose dependent immunosuppression maximal at 35 and 400 mJ per cm2 respectively. UVA at 330 nm failed to cause significant immunosuppression, whereas 350 nm UV caused significant immunosuppression at a dose of 750 mJ per cm2. In conclusion, we have demonstrated dose-dependent suppression of recall CHS responses with both 290 nm and 310 nm NB UV sources, at doses equivalent to less than 5 min of midday Sydney sunlight. We also demonstrated immunosuppression by 350 nm UV at doses equivalent to less than 20 min of sunlight. With further studies in progress, the construction of a complete action spectrum for UV immunosuppression in humans will facilitate the development of sunscreens offering greater protection against UV immunosuppression, and hopefully greater efficacy in the prevention of premalignant and malignant skin lesions.
Melanin Protects from Erythema Induced by Solar-simulated and 310 nm Ultravoilet Radiation, but not 290 nm T. A. Phan, G. M. Halliday, R. StC. Barnetson, and D. L. Damian Dermatology, Melanoma and Skin Cancer Research Institute, Sydney Cancer Centre, University of Sydney at Royal Prince Alfred Hospital, Camperdown, Sydney, NSW, Australia We investigated the relationship between skin pigmentation and erythemal sensitivity to ultraviolet (UV) radiation using a solar simulated (ss) UV source and two narrowband sources centered at 290 nm and 310 nm. Skin pigmentation was measured in 139 healthy subjects of Fitzpatrick skin types I – IV as the ‘‘melanin index’’ (MI) with a reflectance spectrometer. A xenon-arc lamp was fitted with dichroic mirrors in combination with an atmospheric attenuation filter to produce an ssUV spectrum, or with interference filters of 12 – 13nm half maximal-intensity bandwidth to produce 290 and 310 nm spectra. Volunteers were irradiated on their mid to upper backs with a graded series of UV doses, and the minimal erythema dose (MED) was determined 24 h later as the minimum dose of UV that produced just perceptible erythema. Each subject had an ssUV MED and either a 290 nm or a 310 nm MED determined. The mean MEDs with the ssUV, 290 and 310 nm spectra were 3112, 24 and 203 mJ per cm2 respectively. There was a significant correlation between MI and both ssUV MED and 310 nm MED. However there was no significant correlation between MI and 290 nm MED. Our results thus confirm that MI is a useful predictor of an individuals’ sensitivity to ssUVinduced erythema. MI also predicts sensitivity to erythema caused by radiation at 310 nm, but is unable to predict sensitivity to short-wavelength UVB at 290 nm. This data is in keeping with in vitro absorption spectra which show that eumelanins absorb relatively more radiation at 310 nm and less at 290 nm than phaeomelanins. Other factors such as differences in stratum corneum thickness and genetic factors may also account for the differences in erythemal sensitivity to the different wavebands of UV. Measurements of MI could thus prove useful for MED estimation and planning of therapy in patients undergoing 311 nm narrowband UVB phototherapy.
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Evaluation of the Safety, Tolerability and Efﬁcacy of Melanotan in Healthy Caucasians Tkt Ooi,1 P. Walker,2 C. Reid,2 and R. StC. Barnetson1 Departments of Dermatology; 1Royal Prince Alfred Hospital, Sydney, NSW and; 2Royal Adelaide Hospital, Adelaide, SA, Australia There is good epidemiological evidence that melanin protects against sunburn and skin cancer resulting from excessive ultraviolet radiation (UVR) exposure. Melanotans is a synthetic analogue of a-MSH (melanocyte stimulating hormone) with potent melanogenic activity in animals and humans. Like UVR, Melanotan triggers the production of melanin by melanocytes, which acts as a shield to protect the epidermal keratinocytes from UV-induced DNA damage. This double-blind, randomised controlled study aimed to demonstrate that Melanotan is safe, tolerable, and effective in increasing skin melanin density (MD) compared to controls. Eighty-one healthy Caucasian adult subjects were randomised to either a fixed dose of Melanotan or placebo in a 3:1 ratio, given as 3 cycles of daily subcutaneous injections over 10 days each month for 3 consecutive months. Changes in skin pigmentation were quantified by serial skin reflectance measurements at 8 anatomical sites at specific time-points. The potential protective effect of Melanotan on UV-induced skin damage was measured by comparing the number of ‘‘sunburn cells’’ in pre- and post-treatment skin biopsies from UVirradiated sites. Clinically visible tanning was noted in a large proportion of Melanotan-treated subjects, with a highly significant increase in skin MD compared to controls (po0.0001, ANOVA) at all time-points for all sites, particularly in the fairer skin types. The largest changes in MD over baseline occurred in the abdomen, inner upper arm and forehead. Similarly, a highly significant reduction in the number of sunburn cells at post-treatment was observed in the active population (po0.001, ANOVA) compared to a non-significant change in the placebo group. The increase in MD in skin types I/II correlated with a 50% reduction in sunburn injury at the end of the study. As has been shown in previous studies, when Melanotan is delivered as a bolus aqueous injection, a number of subjects experienced some facial flushing, nausea, and loss of appetite. However, these adverse events were mostly mild and usually settled after the first few doses of each cycle. Melanotan had the greatest impact on those individuals with low constitutive melanin (skin types I/II). Therefore, given its ability to stimulate melanin production in the absence of UVinduced skin damage, Melanotan may prove a useful adjunct to current photoprotective strategies to stem the dramatic rise in skin cancer incidence in Australia and other parts of the world.
p14ARF Expression is a Predictor of Prognosis in SCC of the Anterior Tongue Rhonda A. Kwong, Larry H. Kalish, Ronaldo J. Bova, Ian E. Cole, Robert L. Sutherland, and Elizabeth A. Musgrove Cancer Research Program, Garvan Institute of Medical Research Purpose: The INK4A-ARF locus at chromosome 9p21 is frequently altered in head and neck squamous cell carcinoma (SCC) and encodes two distinct tumour suppressors, p16INK4A AND P14ARF. This study addressed the role of P14ARF, which has not previously been elucidated. Experimental Design: P14ARF protein expression was assessed by immunohistochemistry in a cohort of 145 patients with SCC of the anterior tongue. Using univariate and multivariate Cox’s proportional hazards models, the outcomes examined were time to diseases recurrence or death with or without clinicopathological covariates including: nodal status, diseases stage, treatment status and molecular markers with known functional or genetic relationships with P14ARF (p16INK4A, p53, pRb, p21CIP1/WAF1 and E2F-1). Results: On multivariate analysis, P14ARF positivity (nucleolar P14ARF staining and/or nuclear P14ARF staining in X30% of tumour cells) was an independent predictor of improved disease free survival (DFS; hazard ration [HR]: 2.9; p ¼ 0.003) and overall survival (OS; HR: 3.4; p ¼ 0.003). This was further enhanced when P14ARF positivity was co-segregated with positive (41%) p16INK4A staining (DFS; HR: 4.6; po0.0001; OS; HR: 4.3; p ¼ 0.0004). Patients with P14ARF negativity combined with high p53 staining (450%) had the poorest outcome (DFS; HR: 4.1 p ¼ 0.0002; OS; HR: 5.4 po0.0001) of any patient subgroup analysed. Conclusions: These data demonstrate that in patients with SCC of the tongue, overexpression of P14ARF protein predicts improved DFS and OS independent of established prognostic markers. Moreover, biological relationships and involvement of P14ARF in tumour inhibitory pathways, containing either p53 or p16INK4A, were verified in subgroup analyses of patients with variable relative expression of these proteins.
O19 melSIM: Software for the in-silico Simulation of Melanocytic Tumour Growth Stephen Gilmore Department of Dermatology, University of Melbourne In this talk I will describe the key features of melSim, a software package designed to simulate the stochastic growth of planar melanocytic tumours. The software for pattern generation has been designed to incorporate about ten key features of tumour cell biology, some of which are relatively specific for melanocytes. The algorithm for pattern analysis determines whether the pattern is fractal, and then calculates its fractal dimension. I will demonstrate the results of various simulations and compare these results with confocal images of real lesions. I will show how melSim has the potential to increase our understanding of the biology of melanocytic tumour growth, and I will discuss how analysing the results of the simulations has the potential to improve approaches to clinical diagnosis and management.
PLENARY TALKS P1 – Professor P. Hersey Sensitising Human Melanoma Cells to Apoptosis Induced by The Immune System Professor P. Hersey, X. D. Zhang, X. Y. Zhang, J. Borrow, and S. Gillespie P2 – Professor Daniel N. Sauder Immunopathogenesis of Psoriasis: Role of Targeted Therapies Daniel N. Sauder, M.D., FPCP(C), Department of Dermatology, Johns Hopkins University Medical Institution P3 – Dr Pritinder Kaur Keratinocyte Stem Cell Biology Pritinder Kaur. Epithelial Stem Cell Laboratory, Peter MacCallum Cancer Institute, Melbourne, Australia P4 – Dr Joseph Rothnagel Keratin Associated Proteins: A One Hundred Year Perspective Rothnagel, Joseph, Biochemistry & Molecular Biology, University of Queensland, Brisbane P5 – Professor Gary Halliday The UVA Waveband in Sunlight Contributes to Immunosuppression and Gene Mutation in Humans Gary M. Halliday1, Nita S. Agar1, Terence Poon1, Ross StC. Barnetson1, Honnavara N. Ananthaswamy2, Alexandra M. Jones1 SUBMITTED ABSTRACTS O1 – Dr Katherine Armour The Royal Prince Alfred Hospital Experience with alefacept – Preliminary Results K. Armour, R. Barnetson O2 – Associate Professor Rodney Sinclair Changes in Scalp Hair Follicle Density with Advancing Age Rodney Sinclaira, Adam Chapmanb, Jill Mageec O3 – Dr Asoka Herat Anal Intraepithelial Neoplasia – Diagnosis and Differential Diagnosis R. Hillman2, A. Herat1, M. J. Whitfeld1, and A. Meagher3 O4 – Agnes Michalczyk Buccal Cells as a Source of mRNA and Protein for Analysis of a Zinc Deficiency Disorder Agnes Michalczyk1, Leigh Ackland1, George Varigos2, and Lance Smith3
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
O6 – Associate Professor Prue Hart Regulation of Wheal and Flare by Tea Tree Oil: Complementary Human and Rodent Studies Prue H. Hart#, Zeinab Khalil^, Annette L. Pearce, Narmatha Satkunanathan, Emma Storer, and John J. Finlay-Jones# O7 – Dr Tim Rayner Calcium Mobilisation does not Mediate Wound-Induced c-fos Expression in Fetal Skin Samantha Gangnuss2, Allison J. Cowin1,2, and Timothy E. Rayner1,2 1
Child Health Research Institute, Women’s and Children’s Hospital, North Adelaide and 2University of Adelaide Department of Surgery, The Queen Elizabeth Hospital, Woodville, SA O8 – Dr Raffi Gugasyan The Transcription Factors c-Rel and RelA Control Epidermal Development and Homeostasis in Embryonic and Adult Skin via Distinct Mechanisms R. Gugasyan, G. Varigos, A. Voss, T. Thomas, P. Kaur, R. Grumont, and S. Gerondakis O9 – Professor Leonie Ashman Defective in vitro Epidermal Wound Healing in Mice Lacking the Tetraspanin CD151 Sean M. Geary, Allison Cowin#, Stephen Fitter, Mark D. Wright þ , and Leonie K. Ashman O10 – Dr Damien Harkin Novel Vitronectin-Growth Factor Complexes for Skin Repair D. Harkin, R. Dawson, 1P. Gillies, 1N. Johnson, C. Hyde, A. Noble, C. Towne, B. Hollier, L. Ainscough, 1 R. Minchinton, and Z. Upton O11 – Jie Zhong Human Growth Hormone (hGH) presented by K14hGHtransgenic Skin Grafts Induces a Strong Immune Response but no Graft Rejection Jie Zhong, Koji Matsumoto, Rachel De Kluyver, Germain J. P. Fernando, Graham R. Leggatt, and Ian H. Frazer O12 – Dr Eleni Yiasemides A Comparative Study Between Electron Microscopy and Immunofluorescence Mapping For the Diagnosis of Epidermolysis Bullosa E. Yiasemides1,4, J. Walton2,4, L. Vonthethoff3, P. Marr3, and D. Murrell1,4 O13 – Ilse Daehn T-Cell Induced Keratinocyte Apoptosis Ilse S. Daehn1,2, Antiopi Varelias2, and Timothy E. Rayner1,2 O14 – Dr Antiopi Varelias
O5 – Dr Anna Braue Swimming Pool Flotation Devices: A Possible Source of Molluscum Contagiosum Infection A. Brauew, S. Bowdenw, G. Varigos, and H. Kellyw
T-Cell Surface Molecules Inhibit Skin Fibroblast Proliferation and Collagen Synthesis Antiopi Varelias1,2, Guldana Ruzehaji2, Ilse S. Daehn1,2, and Timothy E. Rayner1,2
123:6 DECEMBER 2004
O15 – Dr Tai Anh Phan
O18 – Dr Rhonda-Anne Kwong
Dose and Wavelength Dependence for Ultravoilet-Induced suppression of Contact Hypersensitivity Responses to a Recall Antigen in Humans T. A. Phan, G. M. Halliday, R. StC. Barnetson, and D. L. Damian
p14ARF Expression is a Predictor of Prognosis in SCC of the Anterior Tongue Rhonda A. Kwong, Larry H. Kalish, Ronaldo J. Bova, Ian E. Cole, Robert L. Sutherland, and Elizabeth A. Musgrove O19 – Dr Stephen Gilmore
O16 – Dr Tai Anh Phan Melanin Protects from Erythema Induced by Solar-simulated and 310 nm Ultravoilet Radiation, but not 290 nm T. A. Phan, G. M. Halliday, R. StC. Barnetson, and D. L. Damian O17 – Dr Terry KT Ooi Evaluation of the Safety, Tolerability and Efficacy of Melanotan in Healthy Caucasians Tkt Ooi,1 P. Walker,2 C. Reid2, and R. StC. Barnetson1
melSIM: Software for the in-silico Simulation of Melanocytic Tumour Growth