Canadian Society for Investigative Dermatology Annual Meeting
Abstracts —July 1, 2000, Montreal , Quebec

President — Dr. Neil H. Shear

Actinic Prurigo: Clinical Feature and HLA Associations in a Manitoba First Nation and Northwest Territory Inuit Population

Marni C. Wiseman, Pamela H. Orr, Sharon M Mac-Donald, Marlis Schroeder, John W.P. Toole, University of Manitoba, Winnipeg, Manitoba; University of British Columbia, Vancouver, British Columbia

Actinic prurigo (AP) is an idiopathic familial photoder-matitis seen in First Nation and Inuit populations. The objective was to examine and compare the clinical feature s and HLA associations of AP in a Canadian Inuit and First Nation population. Thirty-seven Inuit subjects and 28 Cree First Nation subjects with AP were identified, administered a questionnaire, and underwent a cutaneous examination. Other causes of photosensitivity were excluded. HLA Class I and Class II typing and subtyping was perf o rmed by PCR and hybridization techniques. Of the Cree population, sub-jects were 75% female and 79% had a family history of pho-t o s e n s i t i v i t y. The mean age of onset was 21.5 years, with onset prior to age 21 in 57% of subjets. Eye involvement was re p o rted in 29% and nonexposed skin in 7%. Physi-cal examination revealed involvement of the face (50%), lip (17.8%), ear (21.4%), and dorsal aspect of the hand (50%). Class II HLA analysis revealed that 13 subjects (46.4%) were HLA DRB1 * 0407 positive compared with six contro l s (16.7%) (p = .01; odds ratio = 4.33). No other statistically significant HLA associations were identified. The Inuit pop-ulation consisted of 81% females and 68% had a family his-t o ry of photosensitivity. The average age of onset of photosensitivity was 29 years, with onset prior to age 21 in 24% of subjects. Involvement of the eyes was re p o rt e d in 62% and nonexposed skin in 19%. Physical examination revealed involvement of the face (64.9%), lip (32.4%), ear (13.5%), and dorsal aspect of the hand (24.3%). HLA D R B 1 * 14 was present in 51.2% of subjects and 26.2% of c o n t rols (p = .022, odds ratio = 2.975). Other significant HLA associations were not identified. Specifically, HLA DRB1 * 0 4 was present in 23/37 subjects (62.2%) and 25/42 contro l s (59.5%) (p = .81). Although AP in this First Nation and Inuit population is a clinically similar familial photoderm a t o s i s , age of onset is earlier in the First Nation population, and involvement of the eye, lip, and nonexposed areas is less com-mon, while involvement of the hand occurs more fre q u e n t l y than in Inuit. To our knowledge, this is the first Canadian study to confirm previous re p o rts of a strong association of HLA DRB1 * 0407 with AP. Intere s t i n g l y, this association was d i s c o v e red only in the First Nation population and the novel association of HLA DRB1 * 14 with AP was identified only in the Inuit population. Overall, the diff e rent HLA asso- ciation profiles and the features that clinically distinguish AP of the Inuit from AP of First Nation people support the concept that AP in these two populations may have a dis-tinct immunopathogenic basis that translates into a diff e r-ent phenotype.


Evidence for a Psoriasis Gene in the Psoriatic Popula-tion of Newfoundland and Labrador

Wayne Gulliver,3 Pat Charm l y,1 Eugene Farber,2 B . Fessenden,2 R. Leder,2 M. McEuen,2 Nall Lexie,2 and H. Weltman 2 1

Chiroscience R & D Inc, Bothell, Washington, USA, 2 Psoriasis Research Institute, Palo Alto, California, USA, and 3 NewLab Clinical Research Inc, St. John’s, New-foundland, Canada

The genetic basis of psoriasis is well known and has been c o n f i rmed by both population and genetic studies. Over the past 7 years we have identified, examined, and collected over 2,000 individual DNA samples from 650 psoriasis fami-lies (400 sibling pairs, 250 simplex families, and 40 skin biopsies). A Genome Scan (Weber screening set 9) with 387 markers (average spacing 9 cM, 1–19 cM) has indicated evi-dence for a linkage between psoriasis in this population and the HLA region. In an initial group of 103 families, con-taining approximately 200 affected sibling pairs, we o b s e rved a statistically interesting maximum multipoint NPL s c o re of 2.66 in the HLA region (chromosome 6p21). A sin-gle nucleotide polymorphism in the promoter region of the TNF – a gene was modestly significant (15% of affected vs. 8% of controls), in contrast to the HLACw6 allele (32% of affected vs. 12% of controls), which was statis-tically significant (p < .01). Other markers outside of this p o rtion of the HLA region do not show any significant asso-ciation, thereby defining the physical limits for containment of the susceptibility gene in this region. We conclude that the HLA Cw6-linked psoriasis allele is a major contribu-tor to the genetic component of psoriasis in the New-foundland and Labrador populations.


G e n o m e - Wide Screening for Genetic Alterations in Sezary Syndrome

Youwen Zhou, Gang Li, Vincent Ho, University of British Columbia, Vancouver, British Columbia

This study aims to develop a powerful genome-wide s c reening method for identification of genetic alterations using Sézary syndrome as a model. A novel, polymerase chain reaction (PCR)-based, molecular screening tool was developed from a recently described protocol, re p-resentational diff e rence analysis, with key modifications. Named “Selective Amplification of Unique Restriction Fragments” (SAURF), this method theoretically can detect genetic alterations resulting in unique or diff e re n t i a l restriction fragments in the genomic DNA, giving rise to unique DNA fragments after gel electro p h o resis. The mutations that can be detected may include gene deletions, gene amplifications, chromosomal re a rrangements, pre s-ence of exogenous genetic materials such as bacteria or v i ruses, and even some point mutations, all without prior knowledge of their presence. We tested the utility of this method by screening three re p resentative cell types fro m patients with Sézary syndrome for unique genetic diff e r-ences. Four unique DNA fragments from granulocytes, n o rmal lymphocytes, and Sézary cells were isolated using SAURF analysis. Two fragments were specifically deleted in Sézary cells. Sequence analysis showed they belong to the T-cell receptor and actin gene, re s p e c t i v e l y. Genomic S o u t h e rn blot analysis revealed that these two gene dele-tions were present in only one of the two Sézary patients studied, indicating that Sézary syndrome is not a homo-geneous disease. The other two fragments were due to non-specific amplification of genomic DNA, demonstrating that this powerful method is not erro r- f ree. Thus, SAURF is a powerful method that can detect novel genetic alterations. In addition, Sézary syndrome is a heterogenous disease, at least in terms of the genetic alterations present in their S é z a ry cells.


Intravenous Cyclosporine in Toxic Epidermal Necrol-ysis and Stevens-Johnson Syndrome

John R. Sullivan,1,2,3 Elizabeth Gow,2 Susan Freeman,3 Neil H. Shear,1 Christopher Commens,2 , 3 Divisions of Clin-ical Pharmacology and Derm a t o l o g y, Department of Med-icine, and Department of Pharm a c o l o g y, University of To ronto Medical School,1 Westmead Hospital, Division of D e rm a t o l o g y, Department of Medicine, University of Syd-ney, 2 Skin and Cancer Foundation of Australia,3 Sydney, New South Wales, Australia

Stevens-Johnson syndrome and toxic epidermal necro l-ysis are systemic reactions characterized by widespre a d epithelial cell death and cause significant morbidity and mor-tality. The objective was to assess safety and efficacy of cyclosporine as a disease-modifying intervention. Intra-venous cyclosporine was administered to six consecutive patients presenting with Stevens-Johnson syndrome and toxic epidermal necrolysis. All patients survived. Disease extension and high fever ceased within 24 hours of initi-ating therapy. Significant correlation was seen between length of hospital stay (Spearman r = .9747, p < .05), time to cutaneous re-epithelialization (Spearman r = .9474, p < .05), and the delay in initiating cyclosporine after the onset of blistering or mucosal ulceration. An additional forme f ruste presentation given cyclosporine had an abort i v e course. No significant adverse effect attributable to this inter-vention occurred. These observations suggest that early insti-tution of cyclosporine (within 24–48 h of onset) favourably alters patient outcome, whereas later administration (6 to 10 days) has less effect on the disease course. These actions may be additive to the postulated beneficial effects of intra-venous gamma globulin.


Photodynamic Therapy with Toluidine Blue Induces Apoptosis in Jurkat Cells

Jean-François Tre m b l a y, Gilles Viau, Michèle Boushira, R o b e rt Bissonnette, Division of Derm a t o l o g y, University of Montreal Hospital Centre, Montreal, Quebec

Toluidine blue O (tolonium chloride, TBO) belongs to the family of thiazine cationic dyes that are routinely used in histopathology. In order to explore the potential of TBO as a photodynamic therapy (PDT) photosensitizer, the cyto-toxicity of TBO-PDT was studied in Jurkat cells, a human leukemic T-cell line. The cells were incubated with various concentrations of TBO and exposed to bro a d - s p e c t rum vis-ible light from a slide projector under conditions where dark toxicity was absent. Cytotoxicity was assessed using the MTT colorimetric assay. DNA filtration elution assay as well as DNA electro p h o resis were perf o rmed to determ i n e if PDT with TBO induces apoptosis. TBO-PDT decreased Jurkat cell viability as measured with the MTT assay. This cytotoxic effect increased with increasing drug concentra-tion. DNA fragmentation was detected following TBO-PDT. DNA fragmentation increased according to time after PDT to reach 70% at 6 h post-PDT with 0.15 µg/mL of TBO and 11.2 J/cm 2 of broad-spectrum light. DNA fragmenta-tion was decreased following PDT with higher concentra-tions of TBO suggesting that necrosis rather than apoptosis may be predominant at higher drug-light combinations. In conclusion, TBO-PDT induces apoptosis in Jurkat cells. Fur-ther studies are needed to explore the potential of TBO-PDT in dermatology.


Photodynamic Therapy with 5-Aminolevulinic Acid Induces Rapid Apoptosis in Jurkat Cells

Faten Gad,1 Gilles Viau,1 Michèle Boushira,1 Richard B e rt r a n d , 2 R o b e rt Bissonnette.1 Division of Derm a t o l o g y,1 Department of Oncology,2 University of Montreal Hospi-tal Centre, Montreal, Quebec

Aminolevulinic acid (ALA) is converted by cells into pro-toporphyrin IX, which accumulates pre f e rentially in malig-nant or activated T lymphocytes. Pilot studies have suggested that photodynamic therapy (PDT) with ALA can improve diseases driven by either normal or malignant T lympho-cytes such as psoriasis or mycosis fungoides. Jurkat cells, a T-leukemic cell line, were used in this study to examine the induction of apoptosis by ALA-PDT. [ 14 C]-thymidine labelled Jurkat cells were incubated with ALA (.01–1 mM) in seru m - f ree medium for 2 h, followed by irradiation with 22.2 J/cm 2 of red light from a slide projector. After 24 h, DNA fragmentation was quantified by a filter elution a s s a y. To study the influence of total red light dose on DNA fragmentation, radiolabelled cells were incubated with 1 mM ALA for 2 h and exposed to doses of light fro m 2.22 J/cm 2 to 44.4 J/cm 2 . The same pro c e d u re was perf o rm e d after addition of ALA to Jurkat cells for periods ranging from 10 min to 6 h followed by exposure to 22.2 J/cm 2 of red light. The results showed that DNA fragmentation as high as 80% was observed 24 h after ALA-PDT with 1 mM ALA and 22.2 J/cm 2 of red light. The percentage of DNA fragmentation was found to increase with increasing doses of ALA, red light, as well as with longer incubation time with ALA. The induction of apoptosis after ALA-PDT was confirmed by the presence of a ladder pattern with DNA e l e c t ro p h o resis as early as 3 h after ALA-PDT. Fluore s c e n c e m i c roscopy revealed chromatin condensation after PDT in cells stained with Hoechst dye. In conclusion, ALA-PDT induces rapid apoptosis in Jurkat cells. This in vitro model could help define better parameters for ALA-PDT in situ-ations where induction of apoptosis in lesional T cells is an important endpoint.


Photodynamic Therapy with Topical Methylester Aminolevulinic Acid Delays the Appearance of UV- i n d u c e d Skin Tumours in Hairless Mice

S. Sharf a e i , 1 G. Vi a u , 1 D. Bouff a rd , 2 R. Bissonnette,1 1 Division of Derm a t o l o g y, and 2 Division of Pathology, Uni-versity of Montreal Hospital Centre, Montreal, Quebec

Photodynamic therapy (PDT) with topical aminole-vulinic acid (ALA) has recently been approved by the US Food and Drug Administration for the treatment of actinic keratoses. Methylester ALA (meALA) is an ALA-derivative that is also transformed into the photosensitizer pro t o-porphyrin IX after topical application. Recent studies have suggested that the selectivity of protoporphyrin IX synthesis for actinic keratosis compared to normal skin is enhanced with meALA as compared to ALA. In this project the influ-ence of weekly PDT with topical meALA on UV-induced skin cancer was studied in the hairless mouse. Groups of 20 mice were exposed 5 days/week to UV radiation (136 mJ/cm 2 ) generated by FS20 tubes. In addition, they were exposed weekly to bro a d - s p e c t rum visible light from a slide projector, 2 hours after application of 8% meALA in a cream base (5 mg/cm 2 ). Treatment of control gro u p s included daily UV exposure and weekly meALA only, daily UV and weekly visible light only, daily UV only, weekly meALA-PDT only, weekly meALA only, UV and weekly placebo cream and light, or no treatment. There was a sig-nificant diff e rence in tumour- f ree survival for mice exposed to UV and weekly to meALA-PDT as compared to all other groups. This was significant for tumours of 2 mm and more, larger tumours (> 4 mm), as well as for the presence of coalescing small tumours. Mice treated with meALA only or meALA and visible light did not develop tumours. In con-clusion, meALA- PDT delays the appearance of UV- i n d u c e d skin cancer in hairless mice. Further studies are required to determine if a similar approach could delay UV- i n d u c e d skin cancer in patients.


Evidence for Reactive Drug Metabolites in Lamotrigine-Associated Drug-Hypersensitivity Syndrome

N.H. Shear,1,2 J.R. Sullivan,1 , 2 S. Knowles, I. Malkiewicz, M. Neuman,2 Divisions of Clinical Pharmacology and Der-matology, Department of Medicine,1 Department of Phar-m a c o l o g y,2 University of To ronto Medical School, To ro n t o , Ontario

Reactive drug products are being increasingly re c o g n i z e d as important in the causation of many idiosyncratic drug reactions including the drug-hypersensitivity syndrome. A positive lymphocyte toxicity assay reflects a decre a s e d capacity to detoxify reactive drug metabolites or an incre a s e d susceptibility to these reactive drug products. The objec-tive was to investigate if lymphocytes from patients with lamotrigine-associated drug-hypersensitivity syndro m e show greater sensitivity to lamotrigine metabolites than cells f rom control subjects. Both patients and controls were recruited from the Drug Safety Clinic at a university hos-pital. Lymphocyte toxicity assays were performed on two patients suffering from the lamotrigine-associated drug-hypersensitivity syndrome. Fourteen control patients were similarly tested. Lymphocytes from hypersensitivity patients showed increased sensitivity to lamotrigine metabolites compared with controls. Sensitivity to phenytoin, carba-mazepine, and phenobarbitone metabolites was similar to controls. These findings add to growing evidence for the role of reactive drug metabolites and their impaired han-dling by patients suffering the lamotrigine-associated dru g -hypersensitivity syndrome.


E ffects of Pro i n f l a m m a t o ry Cytokines on the Follicular Keratinocytes in vitro: Implication in the Pathogenesis of Alopecia Areata

L. Tang, H. Lui, D.I. McLean, and J. Shapiro, Division of Derm a t o l o g y, The University of British Columbia, Va n-couver, British Columbia

We have previously observed that apoptosis in follicu-lar keratinocytes (FKC) is heavily involved in lesional alopecia areata (AA) follicles. At this time, there is no direct evidence to indicate what triggers the initiation of apoptosis in AA. Cytokines like IL-1a/b, TNF-a, IFN-g, and IL-2 have been linked to AA. However, neither the mechanisms nor the target HF cells for these cytokines are clear. We tested the effects of IL-1b, TNF-a, and IFN-g on the viability of diff e rent follicular cells in vitro by MTT assay. When FKC cells were treated alone with all these cytokines, cell viability was not much affected, except for the high doses of IFN-g. Pretreatment with IFN-g or IL-1b increased the susceptibility of FKCs to the cytotoxic effects of TNF-a. Neither the single treatment nor the combined cytokine tre a t-ment had any effects on survival rates of the follicular der-mal papilla (FDP) or follicular dermal sheath (FDS) cells. The decreased cell viability in the FKC was due to apop-totic cell death, which was determined by TUNEL assay. These observations clearly demonstrate that the FKC cells a re the targets for these pro i n f l a m m a t o ry cytokines and they may play an important role in the pathogenesis of AA. In addition, using Western blotting and immunohistochem-istry, we observed that Bcl-2 is expressed in the FDP and FDS, but hardly detected in the FKC. In contrast, Bcl-X was prominently expressed in the FKC cells. These apoptosis regulators might be correlated with the apoptosis in the dif-ferent components of AA-affected HF.


The Role of p33 ING1 in Cellular Stress Response and Its in vivo Relationship with p53

K-John Cheung, Jason Bush, Vincent Ho, Gang Li, Department of Medicine, Division of Dermatology, Van-couver Hospital and Health Sciences Centre, University of British Columbia, Vancouver, British Columbia

A recently identified tumour suppressor candidate, p33 I N G 1 , has been shown to participate in the negative control of cell cycle in the G 0 / G 1 phase, mediate anchorage-dependent g rowth, induce apoptosis in the absence of survival factors, and extend the cell’s replicative life span. It is found that g rowth inhibition of I N G 1 is dependent on the activity of p 5 3 t h rough transcriptional upregulation of the cyclin-dependent kinase (CDK) inhibitor, p 2 1 WA F 1 . Furt h e rm o re , i m m u n o p recipitation studies demonstrate that there is a physical association between p33 I N G 1 and p53. To furt h e r investigate the relationship between I N G 1 and p 5 3, we c o m p a red the expression level of I N G 1 in various murine o rgans differing in p53 status: p53 + / + and p53 - / - . We demon-strated that, at both the protein and RNA levels, p33 I N G 1 w a s e x p ressed independently of p53. In the course of studying the possible role of I N G 1 in cellular stress response, we found that p33 I N G 1 was elevated after UV irradiation in a dose-dependent manner, in both normal human keratinocytes and a keratinocyte cell line, HaCaT. This UV-induced ele-vation, according to our data, appeared to be involved in the repair of damaged DNA. Furt h e rm o re, we have shown that o v e re x p ression of I N G 1 enhances chemosensitivity of a melanoma cell line, MMRU, to camptothecin tre a t m e n t . These results, taken together, indicate that p33 I N G 1 in vivo e x p ression is independent of p53 and that p33 I N G 1 may play i m p o rtant roles in DNA repair and apoptosis.

JCMS 4(4) Contents