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March 2007 - Vol. 156 Issue 3 Page 411-612
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RESEARCH SNIPPETS
pages xv–xv
Editorial
Has teledermatology in the U.K. finally failed?
J.S.C. English and D.J. Eedy pages 411–411
Review article
Maturity of teledermatology evaluation research: a systematic literature review
N. Eminović, N.F. de Keizer, P.J.E. Bindels and A. Hasman pages 412–419
Cutaneous biology
Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed
A.-A. Raymond, M.-C. Méchin, R. Nachat, E. Toulza, R. Tazi-Ahnini, G. Serre and M. Simon pages 420–427
Testosterone metabolism to 5α-dihydrotestosterone and synthesis of sebaceous lipids is regulated by the peroxisome proliferator-activated receptor ligand linoleic acid in human sebocytes
E. Makrantonaki and C.C. Zouboulis pages 428–432
The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology
G. Morissette, L. Germain and F. Marceau pages 433–439
Clinical and laboratory investigations
Expression of p16, CD95, CD95L and Helix pomatia agglutinin in relapsing and nonrelapsing very thin melanoma
L.A. Fearfield, J.M.G. Larkin, A. Rowe, R. A'Hern, C. Fisher, N. Francis, R. MacKie, B. McCann, M.E. Gore and C.B. Bunker pages 440–447
Inactivation of the CDKN2A and the p53 tumour suppressor genes in external genital carcinomas and their precursors
N. Soufir, S. Queille, M. Liboutet, O. Thibaudeau, F. Bachelier, G. Delestaing, B.C. Balloy, J. Breuer, A. Janin, L. Dubertret, C. Vilmer and N. Basset-Seguin pages 448–453
Serum chemokine profile in patients with bullous pemphigoid
H. Nakashima, M. Fujimoto, N. Asashima, R. Watanabe, Y. Kuwano, N. Yazawa, N. Maruyama, H. Okochi, A. Kumanogoh and K. Tamaki pages 454–459
Resolution of inflammatory acne vulgaris may involve regulation of CD4+ T-cell responses to Propionibacterium acnes
H.E. Wilcox, M.D. Farrar, W.J. Cunliffe, K.T. Holland and E. Ingham pages 460–465
Tight junction components occludin, ZO-1, and claudin-1, -4 and -5 in active and healing psoriasis
S. Peltonen, J. Riehokainen, K. Pummi and J. Peltonen pages 466–472
Cutaneous mucinosis associated with dermatomyositis and nephrogenic fibrosing dermopathy: fibroblast hyaluronan synthesis and the effect of patient serum
M. Edward, L. Fitzgerald, C. Thind, J. Leman and A.D. Burden pages 473–479
Sweet's syndrome: a spectrum of unusual clinical presentations and associations
C.Y. Neoh, A.W.H. Tan and S.K. Ng pages 480–485
Cutaneous side-effects in patients with rheumatic diseases during application of tumour necrosis factor-α antagonists
H-H. Lee, I-H. Song, M. Friedrich, A. Gauliard, J. Detert, J. Röwert, H. Audring, S. Kary, G.-R. Burmester, W. Sterry and M. Worm pages 486–491
Changes in skin barrier function following long-term treatment with moisturizers, a randomized controlled trial
I. Buraczewska, B. Berne, M. Lindberg, H. Törmä and M. Lodén pages 492–498
Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga
R. Inoue, A. Nishio, Y. Fukushima and K. Ushida pages 499–509
Contact dermatitis and allergy
Compositae dermatitis from airborne parthenolide
E. Paulsen, L.P. Christensen and K.E. Andersen pages 510–515
Dermatological surgery and lasers
Comparative study of photodynamic therapy vs. CO2 laser vaporization in treatment of condylomata acuminata, a randomized clinical trial
K. Chen, B.Z. Chang, M. Ju, X.H. Zhang and H. Gu pages 516–520
Epidemiology and health services research
Teledermatology in the U.K.: lessons in service innovation
T.L. Finch, F.S. Mair and C.R. May pages 521–527
The Family Dermatology Life Quality Index: measuring the secondary impact of skin disease
M.K.A. Basra, R. Sue-Ho and A.Y. Finlay pages 528–538 Erratum article printed in: British Journal of Dermatology 156:4 p. 791 doi: 10.1111/j.1365-2133.2007.07825.x
Photobiology
Repeated low-dose ultraviolet (UV) B exposures of humans induce limited photoprotection against the immune effects of erythemal UVB radiation
J. Narbutt, A. Lesiak, A. Sysa-Jedrzejowska, A. Wozniacka, A. Cierniewska-Cieslak, J. Boncela, C. Jochymski, W. Kozlowski, A. Zalewska, M. Skibinska and M. Norval pages 539–547
Therapeutics
A pilot study of the safety and efficacy of picolinic acid gel in the treatment of acne vulgaris
M.P. Heffernan, M.M. Nelson and M.J. Anadkat pages 548–552
Treatment of refractory erosive oral lichen planus with extracorporeal photochemotherapy: 12 cases
A.D. Guyot, D. Farhi, S. Ingen-Housz-Oro, A. Bussel, N. Parquet, C. Rabian, H. Bachelez and C. Francès pages 553–556
Concise communication
Reporting of study design in titles and abstracts of articles published in clinically oriented dermatology journals
R. Ubriani, N. Smith and K.A. Katz pages 557–559
Case reports
Solitary plaque mycosis fungoides on the penis responding to topical imiquimod therapy
L.Y.T Chiam and Y.C. Chan pages 560–562
Paraneoplastic pemphigus without an underlying neoplasm
G.T. Park, J.H. Lee, S.J. Yun, S.C. Lee and J.B. Lee pages 563–566
Allogeneic bone marrow transplantation in a 7-year-old girl with congenital erythropoietic porphyria: a treatment dilemma
S.M. Taibjee, O.E. Stevenson, A. Abdullah, C.Y. Tan, P. Darbyshire, C. Moss, H. Goodyear, A. Heagerty, S. Whatley and M.N. Badminton pages 567–571
Gene corner
Galli–Galli disease is an acantholytic variant of Dowling–Degos disease
E. Sprecher, M. Indelman, Z. Khamaysi, J. Lugassy, D. Petronius and R. Bergman pages 572–574
Correspondence
A survey of approach to wound healing by secondary intention among British Society for Dermatological Surgery members
A. Lally and R. Turner pages 575–576
Acute necrotic pancreatitis induced by severe hypercalcaemia due to tacalcitol ointment
C. Knackstedt R. Winograd A. Koch F. Abuzahra C. Trautwein and H.E. Wasmuth pages 576–577
Raised limb bands in infancy: a post-traumatic aetiology?
M.M. Marque B. Guillot G. Le Gallic and D. Bessis pages 578–579
Human papillomavirus type 5 infection in a patient with Hailey–Hailey disease successfully treated with imiquimod
C-C. Chan H-Y. Thong Y-C. Chan and Y-H. Liao pages 579–581
Perioral dermatitis in a patient with myasthenia gravis following systemic corticosteroid treatment
J.M. Goss K.M. Nord M.R. Olarte M.E. Grossman pages 582–582
Confluent and reticulated papillomatosis successfully treated with amoxicillin
R.F. Davis and K.E. Harman pages 583–584
Folliculotropic mycosis fungoides with CD30+ large-cell transformation in a young woman: beneficial effect of bexarotene
C. Mitteldorf R. Stadler H.P. Bertsch and C. Neumann pages 584–586
Lichen scrofulosorum caused by Mycobacterium szulgai: a new cause of a tuberculide reaction
G.L. Ross, H. Chong, T. Collyns, D.M. Gascoyne-Binzi and R.P.E. Sarkany pages 586–587
Long-term efficacy of topical tacrolimus on oral lesions of chronic graft-versus-host disease
J.-C. Fricain, V. Sibaud, N. Swetyenga, R. Tabrizi, F. Campana and A. Taïeb pages 588–590
Minocycline-induced pigmentation of pre-existing capillaritis
V. Madan and J.T. Lear pages 590–591
Anti-desmoglein-1 antibodies are prevalent in Tunisian patients with hydatidosis and leishmaniasis
M. Kallel Sellami, M. Zitouni, W. Tombari, M. Ben Ayed, O. Abida, L. Laadhar, M. Mokni, B. Fezza, H. Turki, I. Mokhtar, A. Ben Osman, R. Kamoun Mohamed, P. Joly, F. Tron, D. Gilbert, H. Masmoudi, S. Makni and (members of the 'Franco-Tunisian Group of Survey and Research on Tunisian endemic pemphigus') pages 591–593
Needle-free anaesthesia prior to botulinum toxin type A injection treatment of palmar and plantar hyperhidrosis
A. Benohanian pages 593–596
Increased human papillomavirus type 31 DNA load in a verrucous high-grade intraepithelial neoplasia of a human immunodeficiency virus-infected patient with extensive bowenoid papulosis
A. Kreuter, N.H. Brockmeyer, H. Pfister, P. Altmeyer, U. Wieland and For the German Competence Network HIV/AIDS pages 596–598
Telangiectatic cutaneous metastasis from carcinoma of the prostate
S. Reddy, R.H. Bang and M.E. Contreras pages 598–600
Isolated recurrence of vesicobullous incontinentia pigmenti in a schoolgirl
S. Darné and A.J. Carmichael pages 600–602
Infantile systemic hyalinosis: a case report and mutation analysis in a Chinese infant
Y-C. Huang, Y-Y. Xiao, Y-H. Zheng, W. Jang, Y-L. Yang and X-J. Zhu pages 602–604
Scytalidium infection associated with dyskeratosis congenita
J. Garg, R. Tilak, A.K. Gulati, S. Singh, P. Prakash and A. Garg pages 604–606
Pityriasis rubra pilaris and hypothyroidism. Efficacy of thyroid hormone replacement therapy in skin recovery
V. Orlandini, O. Cogrel, M.S. Doutre, C. Beylot and M. Beylot-Barry pages 606–607
Nicorandil-induced peristomal ulcers: is nicorandil also associated with gastrointestinal fistula formation?
S. Ogden, Y. Mukasa, C.C. Lyon and I.H. Coulson pages 608–609
Variability in the clinical pattern of cutaneous side-effects of drugs with systemic symptoms: does a DRESS syndrome really exist?
S.H. Kardaun, A. Sidoroff, L. Valeyrie-Allanore, S. Halevy, B.B. Davidovici, M. Mockenhaupt and J.C. Roujeau pages 609–611
Book review
Textbook of Pediatric Dermatology 2nd edition
D.K.B. Armstrong pages 611–611
News and Notices
News and Notices
pages 611–611
Corrigenda
Corrigenda
pages 612–612
Corrigenda
pages 612–612
Corrigenda
pages 612–612
RESEARCH SNIPPETS DOI 10.1111/j .1365-2133.2007.07787.x
Testosterone metabolism to 5a-dihydrotestosterone and synthesis of sebaceouslipids is regulated by the peroxisome proliferator-activated receptor ligandlinoleic acid in human sebocytesDespite clinical evidence that androgens stimulate sebaceous lipids, experiments in vitrohave shown ineffectiveness. This contradiction led to the assumption that cofactorsmay be required for lipid regulation. In this study, the combination of testosteroneand the natural peroxisome proliferator-activated receptor (PPAR)-a/d ligand linoleicacid exhibited a synergistic effect on lipid synthesis and on 5a-dihydrotestosteronelevels in cultured sebocytes. Furthermore, the 5a-reductase type I inhibitor LY191704only unfolded its action under concurrent cell exposure to both compounds. Thesedata suggest a catalytic PPAR effect on cellular testosterone activation and pointtowards a new mode of androgen involvement in sebum production and probably ofthe pathogenesis of sebaceous gland-associated diseases such as acne.Makrantonaki E, Zouboulis CC. Testosterone metabolism to 5a-dihydrotestosterone and synthesis of sebaceous lipids is regulatedby the peroxisome proliferator-activated receptor ligand linoleic acid in human sebocytes. Br J Dermatol 2007; 156:428–32.
Serum chemokine profile in patients with bullous pemphigoidIn bullous pemphigoid (BP), autoantibodies initiate a cascade of inflammatory responsethat causes subepidermal blisters. Nakashima et al. report serum levels of chemokines,including IP-10, MIG, MIP-1a, MIP-1b, RANTES, eotaxin, MCP-1, MCP-2, MCP-3 andGRO-a, in patients with BP. Serum levels of IP-10 and MCP-1 were significantlyincreased in patients with BP compared with healthy controls. Furthermore, serumlevels of IP-10, MIG, MCP-1 and eotaxin in patients with BP significantly increasedwith the affected area. This study suggests that, while various chemokines are involvedin the pathomechanism of BP, MCP-1 and IP-10 have a significant contribution inleucocyte recruitment.Nakashima H, Fujimoto M, Asashima N et al. Serum chemokine profile in patients withbullous pemphigoid. Br J Dermatol 2007; 156:454–59.
Resolution of inflammatory acne vulgaris may involve regulation of CD4+ T-cellresponses to Propionibacterium acnesPropionibacterium acnes is strongly implicated in the pathogenesis of inflammatory acne butits role remains unclear. Wilcox et al. have analysed the CD4+ T-cell response to P. acnesand bacterial heat shock proteins in patients with acne, individuals with acne that hadpreviously resolved (‘resolved individuals’) and nonacne controls by lymphocytetransformation and limiting dilution analysis (LDA). A significantly lower proportionof resolved individuals demonstrated a single-hit LDA response to P. acnes. This mayrepresent negative regulation of the CD4+ T-cell response to P. acnes in these subjects andsuggests that such regulation may be important in the spontaneous resolution of acne.Wilcox HE, Farrar MD, Cunliffe WJ et al. Resolution of inflammatory acne vulgaris may involve regulation of CD4+ T-cellresponses to Propionibacterium acnes. Br J Dermatol 2007; 156:460–65.
Compositae dermatitis from airborne parthenolideAnecdotal clinical and plant chemical reports suggest Compositae dermatitis to be trulyairborne. Feverfew (Tanacetum parthenium) and its main allergen parthenolide areEuropean sensitizers suspected of causing airborne contact allergy. Among 12individuals allergic to feverfew/parthenolide, eight had positive patch-test reactionsto a high-volume air sampler filter extract, collected around feverfew plants.Subsequently, parthenolide was detected in the extract at a concentration of510 ng mL)1, and the most sensitive person tested positive to 8Æ1 ng parthenolide.Thus, the nonvolatile parthenolide is carried on airborne plant particles in allergenicamounts and thereby induces dermatitis on exposed skin in some feverfew-allergicpatients.Paulsen E, Christensen LP, Andersen KE. Compositae dermatitis from airborneparthenolide. Br J Dermatol 2007; 156:510–15.
(a) (b)
(c)
(e)
(d)
EDITORIAL DOI 10.1111/j .1365-2133.2007.07750.x
Has teledermatology in the U.K. finally failed?
Although two papers published in this issue provide us with
additional information about the current state of teledermatol-
ogy services, we probably remain some way off a final evalua-
tion of the current role of this technology in the provision of
dermatology services in the NHS. Over the past decade many
clinicians in the U.K. have expressed concerns over the devel-
opment of teledermatology and this was probably reflected by
the fact that the Action on Dermatology in 2001 had only three
pilot sites out of 15 devoted to looking at teledermatology as a
way of improving access for patients to secondary care. One of
these never started and the status of the other two is unknown.
It was thought by the Department of Health and Government
policy advisers that the use of digital photography and
improvements in electronic communications such as store-
and-forward technology would provide a potential panacea for
specialties like dermatology in an effort to decrease outpatient
waiting times. The reason for this seems simple: the images
obtained from digital photography are of such high quality
that why couldn’t images of skin conditions be e-mailed to a
dermatologist and save the patient a hospital visit? This would
reduce costs and decrease waiting times, but clinical effective-
ness and cost-effectiveness of such services remain largely
untested. If only dermatology were that simple! In an article
on palpation of the skin, Dr Cox recently summarized some of
the reasons, obvious to dermatologists, why this approach is
simplistic.1 They include the fact that it is often necessary to
palpate the skin in order to help make the diagnosis, one can-
not judge the patient’s concerns and anxieties just from a series
of digital images, other areas of pathology are missed that are
not photographed and often the images are of poor quality,
thus making a diagnosis difficult.
In a recent NHS R&D Technology Assessment, patients
referred to a 2-week wait clinic were invited to have a series
of digital photographs, with and without dermoscopy, imme-
diately before their face-to-face consultation.2 An unexpectedly
high proportion (33%) of referrals proved to have a malig-
nancy or a severely dysplastic lesion, with almost 22% having
a malignant melanoma or squamous cell carcinoma, possibly
reflecting the rise in incidence of skin cancers reported else-
where. If the highest level of clinician confidence had been
applied, no cancers would have been missed, but only 20% of
patients would have avoided an outpatient appointment. It
was concluded that it is unlikely that this approach can dra-
matically reduce the need for conventional clinical consulta-
tions while still maintaining clinical safety.2
The first paper in this issue is a literature review of teleder-
matology evaluation and research.3 The authors carried out a
systematic review of the literature over the last 40 years and
concluded that most of the studies published are feasibility
studies rather than phase III or phase IV studies. They found
that compared with other specialties in telemedicine, teleder-
matology seems to be a ‘mature application’. However, more
research on clinical outcomes such as preventable referrals and
time to recovery would be needed to prove that teledermatol-
ogy is indeed a promising and cost-saving technology.
The second paper reports qualitative research into telederma-
tology in the U.K. and is an extremely thorough study invol-
ving interviewing various stakeholders in teledermatology
development within the NHS.4 One of the authors’ conclusions
was that the original policy vision of how teledermatology
would be used as a technological answer for long waiting lists
and consultant shortages has failed to be realized. They also
found that teledermatology services were initially viewed as a
diagnostic service but then increasingly gained acknowledge-
ment as a triage and management service.
The successful implementation of teledermatology as a rout-
ine service requires understanding of and paying great atten-
tion to the interplay between social and technical aspects of
teledermatology. This means it is not a quick, simple fix for
dermatology. However, it is uncertain that the politicians and
policy makers who lead developments in the Health Service
will listen.
J . S .C . ENGL I SH
D . J . E EDY*
Department of Dermatology,
Queen’s Medical Centre,
Nottingham University Hospitals
NHS Trust, Nottingham
NG7 2UH, U.K.
*Department of Dermatology,
Craigavon Area Hospital Group,
Portadown BT63 5QQ, U.K.
E-mail: John.English@nuh.nhs.uk
References
1 Cox NH. Palpation of the skin – an important issue. J R Soc Med2006; 99:598–600.
2 Bowns IR, Collins K, Walters SJ, McDonagh AJG. Telemedicine indermatology: a randomised controlled trial. Health Technol Assess 2006;
10(43):1–58.3 Eminovic N, de Keizer NF, Bindels PJE, Hasman A. Maturity of tele-
dermatology evaluation research: a systematic literature review. Br J
Dermatol 2007; 156:412–19.4 Finch TL, Mair FS, May CR. Teledermatology in the U.K.: lessons in
service innovation. Br J Dermatol 2007; 156:521–7.
� 2007 The Authors
Journal compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp411 411
REVIEW ARTICLE DOI 10.1111/j .1365-2133.2006.07627.x
Maturity of teledermatology evaluation research:a systematic literature reviewN. Eminovic, N.F. de Keizer, P.J.E. Bindels* and A. Hasman
Departments of Medical Informatics and *General Practice, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
CorrespondenceN. Eminovic.
E-mail: n.eminovic@amc.uva.nl
Accepted for publication15 August 2006
Key wordsevaluation, maturity, phases, systematic review,
teledermatology, telemedicine
Conflicts of interestNone declared.
Summary
Background There is a growing interest in teledermatology in today’s clinical prac-tice, but the maturity of the evaluation research of this technology is still unclear.Objectives This systematic review describes the maturity of teledermatology evalu-ation research over time and explores what kind of teledermatology outcomemeasures have been evaluated.Methods Systematic review of literature found in Medline database (1966 up toApril 2006). A telemedicine evaluation strategy consisting of four consecutiveresearch phases (parallel to drug and diagnostics evaluation research) extendedwith a fifth postimplementation phase was used to classify all included studies bytwo independent reviewers. In addition, main characteristics (store-and-forwardor real-time, study design, outcome measures) were registered.Results Three hundred and forty-five papers were systematically selected from Med-line, and 244 papers were excluded. For two randomized controlled trials (RCTs),multiple papers in phase III were found. After correcting for this, 99 studiesremained included (11 phase I, 72 phase II, two phase III, six phase IV, eightpostimplementation phase). The number of phase II studies is the largest and stillgrowing, while other phases are much less represented. Diagnostic accuracy wasthe most often used outcome measure and was found in phase I, II and IV. Store-and-forward teledermatology has been evaluated more since 2001, but mostphase IV studies (RCTs, including cost aspects) are on real-time teledermatology.Conclusions Most teledermatology evaluation studies are classified as feasibility stud-ies (phase II). The number of phase III and IV studies remains low through theyears. Compared with other specialties in telemedicine (i.e. telesurgery, telepaedi-atrics), teledermatology seems to be a mature application. However, more evalu-ation studies with a focus on clinical outcomes such as preventable referrals ortime to recovery are needed to prove that teledermatology indeed is a promisingand cost-saving technology.
Teledermatology can be defined as the use of imaging and
telecommunication technologies to provide skin services by a
dermatologist to another health professional (general practi-
tioner, nurse, other specialist) or directly to a patient. Tele-
medicine in general enables health professionals to
communicate with each other and with their patients over a
geographical or physical distance. Together with other visually
oriented specialties, dermatology is one of the clinical special-
ties in which telemedicine is most applied.
Two types of teledermatology can be distinguished: store-
and-forward (SAF) and real-time (RT) teledermatology.1 The
SAF variant uses asynchronous data transfer technology (e.g.
e-mail) while RT teledermatology is based on synchronous
data transfer technologies (e.g. videoconferencing software).
RT teledermatology requires both communicating parties (e.g.
physician, dermatologist and patients) to be available at the
same time, which is not necessary when using an SAF variant.
This variation in teledermatology is not only technology
based, but also has impact on organizational and clinical char-
acteristics of teledermatology.
Although telemedicine and teledermatology are increasingly
becoming integrated in local regular healthcare systems, there
Additional information added after online publication 5th January2007: Preliminary results for this study were presented at the conference
Medical Informatics in Europe (MIE), August 2006, Maastricht, theNetherlands.
� 2006 The Authors
412 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp412–419
is lack of valid scientific evidence showing positive effects,
which is mainly due to the low quality of the studies.2–5
Implementation and evaluation of teledermatology seem to be
performed in parallel while medical interventions are usually
first fully investigated through different research phases before
being implemented.
The primary aim of this review is to describe the maturity
status of teledermatology evaluation research over time, for
both SAF and RT, as a function of the four consecutive tele-
medicine evaluation phases defined by Holle and Zahlmann.6
These four phases are comparable with the four phases of
drug research. The secondary aim of the study is to explore
the outcome measures used in the different evaluation phases.
Materials and methods
Search strategy
We identified English language published teledermatology
studies by searching Medline database (1966 up to April
2006) using various search queries combining the key words
‘teledermatology’,‘dermatology’,‘telemedicine’,‘skin’,‘e-health’
and ‘electronic mail’. As we were only interested in ori-
ginal full papers reporting on the evaluation of a specific
teledermatology service, we excluded literature reviews, com-
ments, abstracts, letters and editorials. The second step inclu-
ded the manual screening of the selected full papers by two
independent reviewers. Papers which were not about derma-
tology but about another specialty (e.g. radiology, pathology)
and papers where the evaluation of a specific teledermatology
service was not the primary aim were excluded. Papers on a
telemedicine application for several specialties were included
only if the results on dermatology were separately reported.
Literature reviews1,7 were screened to retrieve possibly missed
references. Papers from conference proceedings were excluded
if a full journal paper on the same study was obtained in the
selection procedure.
Classification of evaluation studies
Included studies were assigned independently to one of the
four phases of the Holle and Zahlmann strategy.6 With per-
mission of the authors, we added an extra phase: the postim-
plementation phase. This fifth phase should be considered as
an extra category and not specifically as a continuation of the
four phases. Table 1 shows the five phases which are deter-
mined by the study design, the number of participants in the
study and the setting of the study.
We distinguished two main study designs: observational
and intervention studies. Intervention studies were further
divided into: uncontrolled trial; nonrandomized controlled
trial with the same patients as control group (e.g. a patient
has been seen through teledermatology first and face-to-face
afterwards); nonrandomized controlled trial with other
patients as control group; and randomized controlled trial
(RCT).
In cases where multiple papers on one study had been pub-
lished, the study was classified once in one phase. However, if
multiple papers on one study were classified in different
phases (for example, clinical outcomes in phase III and costs
in phase IV), than the study was classified in each phase separ-
ately. When different phases of a study were described in one
paper, the paper was classified only once, in the highest
phase.
We predefined the following outcome measures: diagnos-
tic accuracy, diagnostic reliability, patient satisfaction with
provided care, patient and doctor satisfaction with the sys-
tem, costs, preventable referrals, quality of images, learning
effect in doctors and delay in treatment. When measuring
diagnostic accuracy the telediagnosis is compared with a
Table 1 Four phases of Holle and Zahlmann,6 and added fifth phase (‘postimplementation’) with their characteristics and main criteria used inthis review to classify teledermatology studies
Phase Study design Usual participants Main criteria used by the reviewers
I Exploratory, small interventionstudies
Researchers and project members,simulated patients
Experimental setting, i.e. teleconsultationdid not result in a diagnosis or
treatment planII Feasibility studies, can be controlled
intervention studies, but rarelyinvolve a separate control group
Specially trained and highly
motivated potential users,real or simulated patients
Field or experimental studies with
potential users
III RCT Unselected sample of users withminimal training, real patients
RCT on clinical outcomes
IV (Simulation) cost studies usingthe benefits results from an RCT
Not always applicable,see also phase III
Costs are compared betweenconventional dermatological care and
teledermatology and/or with any kindof benefits resulting from an RCT
Postimplementation Telemedicine part of the regularcare, observational study
Actual users of telemedicine Teledermatology is fully integratedin the regular care
RCT, randomized controlled trial.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp412–419
Maturity of teledermatology evaluation research, N. Eminovic et al. 413
gold standard diagnosis (face-to-face diagnosis, biopsy etc.).
Diagnostic reliability is diagnostic agreement between differ-
ent examiners who determined a diagnosis using the same
or a different, but not gold standard, diagnostic method.
Patient satisfaction with the teledermatology system refers to
how the patients experience a new technology such as tele-
dermatology, while patient satisfaction with care refers to
how the patients feel about the health care delivered in
general.
All discrepancies in classifying the selected papers between
the reviewers were solved by consensus. Interobserver reliabil-
ity regarding the classification into phases was calculated to
determine the validity of the classification procedure.
Results
The Medline search resulted in 345 unique references. Of
these studies, 181 studies were included and 164 excluded
(see Fig. 1). Forty-five papers were about another specialty
(radiology, pathology) or about all kinds of medical condi-
tions including dermatology as a subset but without report-
ing separate results. After reading the full papers, 80 papers
were excluded. Most of these papers did not report on an
evaluation of a specific teledermatology service (n ¼ 46).
Five studies needed to be excluded as they were in some
way duplicate publications: conference proceedings and a full
version paper. Finally, 101 papers were included for the clas-
sification into five phases. It appeared that in two situations
two related papers on the same RCTs were classified in the
same phase, phase III. Following the rules, these studies were
classified once, making the total number of classified studies
as 99.
The two reviewers agreed about the classification of the
studies into phases in 84% of cases (j ¼ 0Æ7).
Phase trends
In 1996 two teledermatology evaluation studies were pub-
lished. These first teledermatology studies were a phase I8 and
phase II study9 (Fig. 2). Phase I studies have been published
sporadically with a total of 11 studies up to 2006.
Seventy-two studies were assigned to phase II. In 1998, 10
phase II studies were published while only one phase I and
one postimplementation study were published. The same high
number of phase II studies was found again in 2003 and
2005.
The number of phase III studies was limited to two, pub-
lished in 2002 and 2004.10,11 Phase IV and the postimple-
mentation phase were represented by six and eight studies,
respectively. In 2000 three cost studies were published12–14
and another two were published in 2001.15,16 The last pub-
lished phase IV study dates from 2003.17
Two large groups of trials were found: U.K. Multicentre
Teledermatology Trials14,18–21 and Health Waikato Tele-
dermatology Trials.13,22,23 These trials investigated RT
Fig 1. Two steps of the literature selection
procedure. The first step of selection
procedure was based on title, abstract and
paper characteristics, while the second step
was based on the full paper. RCT, randomized
controlled trial; td, teledermatology; conf.,
conference.
Fig 2. Cumulative number of studies per phase and over the years.
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414 Maturity of teledermatology evaluation research, N. Eminovic et al.
teledermatology and covered different phases. The U.K. trials
covered phases I–IV, while the Waikato trials covered phases
II–IV.
Outcome measures and study design
The diagnostic accuracy was the most common outcome
measure used in 53 studies (Table 2). In seven of 11 phase
I studies, the quality of images was investigated. The two
phase III RCTs investigated delay in treatment and prevent-
able referrals as clinical outcome measures, but also inclu-
ded patient and doctor satisfaction with the system. Central
themes of the eight postimplementation phase studies were
patient and doctor satisfaction with the teledermatology
system.
In total, there were 43 intervention studies controlled by
the same group of patients. An uncontrolled study design was
applied in 30 studies, mainly in phase II (21 of 72), but also
in phase I (six of 11). We found four RCTs in phase II which
were small and not focusing on clinical outcomes and could
therefore not be classified as phase III or IV.24–27
Store-and-forward vs. real-time teledermatology
The majority of the included studies (63%) investigated the
SAF variant of teledermatology (Fig. 3). The RT variant was
investigated in 29% of the studies, with the majority per-
formed before 2001. Since 2001 more SAF applications were
the subject of evaluation compared with RT teledermatology
applications. The combination of the two technologies was
evaluated twice.14,28 In six studies it was not clear what kind
of technology was used. Three of these six studies were phase
I studies in which technical aspects and quality of images
were evaluated.29–31 The other three studies were two recent
phase II32,33 and one postimplementation phase study.34
SAF technology was investigated in all phase III studies,
while four of six phase IV studies evaluated RT12,13,15,16 and
one SAF teledermatology.17
Table 2 Outcome measures and study designs in five phases
Phase n (SAF, RT, both) Outcome measures (number of studies) Study design (number of studies)
I8,21,29–31,38–43 11 (5, 3, 0)
3 unknownSAF or RT
Diagnostic accuracy (5)
Diagnostic reliability (3)Quality of images (7)
Doctor satisfaction with the system (3)Preventable referrals (1)
Confidence in the findings (1)Technical aspects (2)
Observational (2)
Intervention controlled bythe same patients (3)
Uncontrolled (6)
II9,18–20,22–28,32,33,44–102 72 (47, 18, 1)2 unknown
SAF or RT
Diagnostic accuracy (47)Diagnostic reliability (17)
Quality of images (12)Patient satisfaction with the system (16)
Doctor satisfaction with the system (11)Patient satisfaction with care (5)
Costs (5)Preventable referrals (12)
Learning effect GPs (3)
Confidence in the findings (1)Time needed for consultation (8)
Technical aspects (3)
RCT (4)Intervention controlled by
other patients (7)Intervention controlled by the
same patients (40)Uncontrolled (21)
III10,11 2 (2, 0, 0) Patient satisfaction with the system (2)
Doctor satisfaction with the system (2)Patient satisfaction with care (2)
Preventable referrals (1)Delay in treatment (2)
RCT (2)
IV12–17 6 (1, 4, 1) Diagnostic accuracy (1)Costs (6)
Preventable referrals (3)Time needed for consultation (1)
RCT (6)
Postimplementation34,103–109 8 (4, 3, 0)1 unknown
SAF or RT
Patient satisfaction with the system (2)Doctor satisfaction with the system (2)
Costs (2)Delay in treatment (1)
Time needed for consultation (1)
Observational (5)Uncontrolled (3)
SAF, store-and-forward; RT, real-time; GP, general practitioner; RCT, randomized controlled trial.
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Maturity of teledermatology evaluation research, N. Eminovic et al. 415
Discussion
Teledermatology has been the subject of evaluation studies for
10 years now. The results of our review show that evaluation
of teledermatology takes place in different research phases.
Despite a growing implementation of teledermatology in
today’s clinical practice, there are no clear trends towards
phase III and IV studies in more recent years in comparison
with the first years of teledermatology evaluation.
The number of phase II studies grew continuously over the
past 10 years and is still growing. Phase III, IV and V studies
are much less represented. There are different possible expla-
nations for this. Phase I studies, usually reporting on technical
issues such as, for example, quality of images, are often not
published separately as scientific journal papers (possible pub-
lication bias). Technical evaluations are also more likely to be
published in technical journals which are not covered by the
Medline database.
Only two RCTs could be classified in phase III and six in
phase IV. Performing high-level evidence studies such as RCTs
in telemedicine is often seen as difficult as telemedicine is a
complex intervention depending on local variations in admin-
istration and organisation.35,36 Characteristics of teledermatol-
ogy such as its relatively low risk, low investment costs and
the possibility of using it in addition to regular care are likely
to speed up its implementation despite the lack of scientific
research on the pros and cons. However, detailed and sophis-
ticated evaluation of a new technology requires many
resources and should be performed only after some basic but
important evaluation steps have been carried out in phase I
and II. Furthermore, for certain nonclinical outcome measures
of teledermatology (e.g. diagnostic accuracy), a phase II study
should provide sufficiently strong evidence and a phase III or
IV study is not expected. These issues are likely to reduce the
push to perform RCTs in teledermatology.
Postimplementation studies are, like phase I studies, not
often published in scientific journals. Commercial telederma-
tology suppliers probably perform this kind of evaluation fre-
quently, but mainly for their own use without academic
ambitions. In our review we obtained only Medline indexed
literature. Further in-depth analysis of phase I or postimple-
mentation phase studies would require obtaining literature
databases of technical journals and nonpublicly available
reports.
More detailed and sophisticated evaluation of any new tech-
nology is expected to be carried out after some basic evalu-
ation steps have been performed, such as in phase I and II. As
no or limited scientific evidence about the impact of teleder-
matology on clinical outcome has been demonstrated,4 more
detailed evaluation studies (phase III and IV) are needed.
Diagnostic accuracy is the most often evaluated outcome
measure in the teledermatology studies included in our
review. This result was not surprising as this outcome measure
is strongly related to the visual aspect of teledermatology.
With the changing technology (i.e. different cameras with dif-
ferent possibilities and settings) and large variations in teleder-
matology settings, this outcome is likely to be studied
repeatedly in most teledermatology evaluations. However, it is
regrettable that important clinical outcomes such as prevent-
able referrals and delay in treatment are not more often inves-
tigated, especially in phase III and IV studies.
Earlier studies showed that RT teledermatology was more
clinically efficient than the SAF variant.14 However, SAF tele-
dermatology is less expensive and under certain circum-
stances more cost effective, making it more attractive than
the RT variant. This increasing interest in SAF has also been
shown in our review as RT was especially popular in the
early evaluations and SAF has been much more popular over
the past 5 years. However, most phase IV studies that we
found were about RT. Therefore we support Whited’s con-
clusion that there is a lack of economic evaluation of SAF
teledermatology.7
An earlier study concluded that teledermatology was
maturing because of the reasonably good quality and quan-
tity of the published literature when compared with other
specialties (i.e. telesurgery, telepaediatrics).37 Hailey et al.4
found for teledermatology the strongest indication for bene-
fits of telemedicine applications. Although these conclusions
might be true when compared with other telemedicine
applications, our review shows that there is no clear trend in
maturity of teledermatology evaluation research over the past
10 years as there is a lack of phase III and IV studies. At this
moment, we found no indications for the maturation of tele-
dermatology evaluation research or that the number of phase
III and phase IV studies is likely to increase in the near
future. The timing of our study might be too early to meas-
ure an increase of phase III and phase IV studies as these
Fig 3. Cumulative number of store-and-forward (SAF) vs. real time
(RT) teledermatology evaluation studies in the past 10 years.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp412–419
416 Maturity of teledermatology evaluation research, N. Eminovic et al.
studies are often time consuming in both performing and
reporting. Although teledermatology seems to be a valuable
application, we believe that there is a need for more phase
III and IV studies to provide a high level of evidence on
positive clinical outcomes and cost effectiveness of telederma-
tology prior to its implementation and wide use in clinical
practice.
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CUTANEOUS BIOLOGY DOI 10.1111/j .1365-2133.2006.07656.x
Nine procaspases are expressed in normal humanepidermis, but only caspase-14 is fully processedA.-A. Raymond, M.-C. Mechin, R. Nachat, E. Toulza, R. Tazi-Ahnini,* G. Serre and M. Simon
UMR 5165 CNRS – University of Toulouse III, Institut Federatif de Recherche Claude de Preval, IFR30 (INSERM-CNRS-Universite Paul Sabatier-Centre
Hospitalier Universitaire de Toulouse), Faculte de Medecine, 37 allees Jules Guesde, 31073 Toulouse, France
*Division of Genomic Medicine, University of Sheffield, Medical School, Sheffield, U.K.
CorrespondenceMichel Simon.
E-mail: msimon@udear.cnrs.fr
Accepted for publication17 August 2006
Key wordscaspase, differentiation, epidermis, protease,
psoriasis
Conflicts of interestNone declared.
M.-C.M. and R.N. participated equally in this
work.
Summary
Background During normal stratification of the epidermis, keratinocytes undergo acomplex programme of terminal differentiation. This programmed cell deathresults in corneocyte accumulation to form the stratum corneum (SC). Terminaldifferentiation and apoptosis share numerous features such as elimination of nu-clei and organelles, changes in cell shape, and activation of transglutaminases andproteases. Caspases are cysteine proteases that play a central role in apoptosis.Therefore they may also be involved in the terminal differentiation of keratino-cytes.Objectives To identify the caspases expressed in normal human epidermis and todefine their pattern of expression and activation.Methods We analysed mRNAs from human epidermis by reverse transcription–polymerase chain reaction (RT–PCR), skin cryosections by immunohistologicalmethods, and epidermal protein extracts by Western blotting.Results The mRNAs encoding caspase-1, -2, -3, -4, -6, -7, -8, -9, -10 and -14were detected by RT–PCR. Accordingly, the immunohistological analyses showedclear expression in the epidermis of the corresponding proteins except caspase-2and caspase-8, with only a weak expression of caspase-9. Moreover, procaspase-1, -2, -3, -4, -6, -7, -9, -10 and -14, and the fully processed caspase-14, wereimmunodetected in total epidermis extracts. However, only procaspase-1 and theprocessed caspase-14 were detected in extracts of superficial SC. In addition tothese two proteins, procaspase-4 was detected in extracts of superficial SCobtained from lesional psoriatic skin.Conclusions This study, the first exhaustive description of caspase expression andprocessing in normal human epidermis, indicates that in vivo granular keratino-cytes express nine procaspases, and in addition the activated form of caspase-14.This confirms that only caspase-14 is involved in keratinocyte differentiation, andsuggests that keratinocytes are ready to induce apoptosis in response to cutaneousdamage.
Apoptosis involves a signalling pathway in which cysteinyl
aspartate-specific proteinases, called caspases, play a central
role.1 These cytoplasmic enzymes are synthesized as zymogens
(procaspases), comprising a prodomain, as well as a large
(p20) and a small (p10) subunit. The proforms are activated
by proteolytic cleavage between their subdomains (by other
proteases or by themselves thanks to their autocatalytic activ-
ity), which leads to the formation of a mature caspase, a tetra-
mer consisting of two p20/p10 heterodimers. Most of the 15
caspases reported until now can be divided into initiator (-1,
-2, -4, -5, -8, -9, -10, -11, -12 and -13) and effector caspases
(-3, -6, -7 and -14). The former display a long prodomain
whereas the prodomain of the latter is made up of fewer than
40 amino acids. Caspase-11 (murine), -13 (bovine) and -15
(described in various other mammalian species) do not exist in
humans.2–4 Upon binding to signal-transducing molecules, ini-
tiator caspases activate downstream effector caspases which
finally cleave various cellular substrates (structural proteins
such as actin, laminin or components of cellular junctions, and
regulatory proteins such as a DNAse inhibitor),5,6 thereby
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420 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp420–427
inducing the demise of the cell. Caspase-14 is peculiar as it has
a restricted pattern of expression, as opposed to all other cas-
pases identified, and is not activated during apoptosis.7
The skin is one of the first lines of defence against the envi-
ronment. The epidermal protective barrier is established by
keratinocytes that undergo a complex programme of terminal
differentiation, from the stratum basale towards the surface of
the skin. During the differentiation programme, keratinocytes
stop bcl-2 expression, show transglutaminase and protease
activation, rearrange their cytoskeleton, change their shape
and lose their nucleus and organelles. This leads to epidermal
terminal differentiation being considered as a ‘planned cell
death pathway’.8 However, epidermal differentiation differs
from classical apoptosis in that it does not result in the frag-
mentation of cells into apoptotic bodies and their subsequent
phagocytosis, but induces the formation of dead cornified cells
called corneocytes. Stacking of the corneocytes in the upper
epidermis forms a resistant barrier, the stratum corneum (SC).
We nevertheless assumed that epidermal keratinocytes use
some apoptosis elements during their differentiation, in par-
ticular one or several caspases. This seems to be the case for
the terminal differentiation of rodent lens epithelial cells,9
erythrocytes10 and T cells,11,12 or during postnatal develop-
ment of rat cerebellum.13
There are few reports on any relationships that might exist
between caspases and keratinocyte terminal differentiation.
When keratinocytes, cultured in conditions that induce differ-
entiation, are treated with the broad-spectrum caspase inhibi-
tor Z-VAD-FMK, SC development has been shown to be
perturbed.14 Some studies have been carried out on caspase
expression in the epidermis, but the results are as yet incom-
plete and sometimes in contradiction. Caspase-1, -2, -3, -4
and -7 mRNAs have been detected by reverse transcription–
polymerase chain reaction (RT–PCR) in cultured human kera-
tinocytes but those of caspase-5 and -6 have not.15 In mouse
epidermis, caspase-2, -3, -6, -8, -9 and -14 mRNAs have been
detected but those of caspase-1, -7, -11 and -12 have not.16
Caspase-1 and -3 have been partially purified from human
corneocyte extracts.15 It has been suggested that procaspase-3
is activated during late keratinocyte differentiation.14 How-
ever, processed forms of caspase-3, -6 and -7 have not been
detected in human epidermis.7 More data are available about
caspase-14 which is only expressed in the skin,17 choroid
plexus, retinal pigment epithelium and thymic Hassall’s bod-
ies.18 Its expression, conserved among mammals,19 was repor-
ted in adult and embryonic human epidermis to be restricted
to the differentiated keratinocytes, where the processed forms
(p17 and p11) were detected.7 In particular, active caspase-14
was observed in SC extracts.20,21 Immunogold cytochemistry
localized caspase-14 to the amorphous material of keratohy-
aline granules and to the keratin bundles. It is associated with
nuclear remnants, corneodesmosomes and intracellular matrix
of corneocytes.19,22 Caspase-14 is also expressed in hair folli-
cles and sebaceous glands of human epidermis.20
Our objective was to analyse by RT–PCR the caspases
expressed in normal human epidermis, to define their pattern
of expression using immunohistochemistry and indirect
immunofluorescence, and to determine by Western blotting
which caspases are processed.
Materials and methods
Human tissues
All described studies concerning human tissues/subjects were
conducted in accordance with the Declaration of Helsinki Prin-
ciples, and with the ethical guidelines of the University of
Toulouse III, the University of Sheffield and the French Minis-
try of Research and Technology. Each patient provided
informed consent to testing. Plastic surgery specimens of nor-
mal human breast or abdominal skin, obtained from patients
undergoing plastic surgery without any history of skin dis-
eases, were kindly provided by Professor J.-P. Chavoin (Plastic
Surgery Department, Rangueil Teaching Hospital, Toulouse,
France). Samples of SC were obtained from volunteers with
normal skin and without any history of genetic skin disorders
or atopy, and from lesional and nonlesional skin of three
patients with psoriasis recruited in Sheffield.
Analysis of mRNA expression by reverse
transcription–polymerase chain reaction
Human epidermis cDNA, prepared as previously described23
and tested for the absence of genomic contamination, was
used as a template for PCR analysis with specific caspase pri-
mer pairs, chosen in reference to previously published
works.24,25 The specificity of the primers was confirmed using
the BLAST program.26 The sequence of primers and their posi-
tion on caspase cDNA are shown in Table 1. Positive control
reactions with Jurkat cell cDNA (Biochain Institute Inc.,
Hayward, CA, U.S.A.) were carried out in parallel. Glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) primers were
used to confirm normalization of the cDNAs. PCR reactions
were performed in 25-lL volumes using 0.75 U of Goldstar
Taq DNA polymerase (Eurogentec, Seraing, Belgium) with the
supplied buffer, 1.5 mmol L)1 MgCl2, 5 mmol L)1 of each
deoxyribonucleoside triphosphate, 5 pmol of each primer and
1 lL of each cDNA sample. The PCR conditions were as fol-
lows: 94 �C for 2 min, followed by 35 or 45 cycles at 94 �Cfor 1 min, between 52 �C and 62 �C (52 �C for caspase-3, -5
and -6, 56 �C for caspase-4, -7, -10, -12, -14 and GAPDH,
58 �C for caspase-8, 60 �C for caspase-9, 62 �C for caspase-1
and -2) for 1 min, and 72 �C for 1 min, and a final elonga-
tion at 72 �C for 1 min.
Extraction of proteins from human epidermis and
superficial stratum corneum
Dermoepidermal cleavage was performed by heat treatment,
and the epidermis extracted in a Tris–ethylenediamine tetra-
acetic acid (EDTA) buffer containing Nonidet P-40 as previ-
ously reported.27
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Expression of caspases in human epidermis, A.-A. Raymond et al. 421
Superficial SC samples were obtained by a tape-stripping
procedure.28 Proteins were dissolved in a Tris–EDTA buffer
supplemented with 2% sodium dodecyl sulphate (SDS) and
50 mmol L)1 dithiothreitol as previously reported.29
Protein concentrations were measured using the Bio-
Rad protein assayTM (Bio-Rad Laboratories GmbH, Munich,
Germany).
Antibodies
The characteristics of the antibodies used in this study are
summarized in Table 2.
Protein electrophoresis and immunoblotting
Proteins were separated by SDS–polyacrylamide gel electro-
phoresis (PAGE) on 12Æ5% acrylamide gels and electrotrans-
ferred to nitrocellulose membranes. The membranes were
stained with Ponceau Red, then heated at 70 �C for 45 min in
order to cross-link the small proteins, incubated for 30 min in
blocking buffer [Tris-buffered saline (40 mmol L)1 Tris-HCl
pH 8, 0Æ9% NaCl) supplemented with 2Æ5% low-fat dried milk
or fish gelatin, and 0Æ05% Tween-20], and probed with anti-
bodies as previously described.27 Immunoreactivities were
revealed with the ECLTM Western blotting kit as described by
the manufacturer (Amersham Biosciences, Little Chalfont,
U.K.).
Immunohistology
Immunohistochemistry was performed on unfixed 5–7-lm
thick cryosections of breast or abdominal skin samples
obtained from several donors. The primary antibodies diluted
in phosphate-buffered saline (PBS) were applied for 1 h at
37 �C or overnight at 4 �C (Table 2). For detection of cas-
pase-2, -3 and -14 we used the Histostain-Plus kit, as
described by Zymed Laboratories Invitrogen (Cergy Pontoise,
France), the manufacturer. Anticaspase-4, -6, -7 and -10 anti-
bodies were revealed with a horseradish peroxidase-conju-
gated rabbit antigoat IgG secondary antibody (Tebu-bio, Le
Perray en Yvelines, France) and 3-amino-9-ethylcarbazole as
the substrate.
For indirect immunofluorescence, cryosections were
blocked with PBS containing 2% bovine serum albumin
(BSA) and 0Æ05% Tween-20 and then incubated with pri-
mary antibodies diluted in PBS containing 1% BSA and
Table 1 Primer sequences
Caspase-1 Upstream 5¢-AAC CCA GCT ATG CCC ACA TCC-3¢ (96–116)Downstream 5¢-TTA ATG TCC TGG GAA GAG GTA-3¢ (953–933)
Caspase-2 Upstream 5¢-GTT ACC TGC ACA CCG AGT CAC G-3¢ (557–578)Downstream 5¢-GCG TGG TTC TTT CCA TCT TGT TGG TCA-3¢ (790–764)
Caspase-3 Upstream 5¢-GAA TAT CCC TGG ACA ACA-3¢ (157–174)Downstream 5¢-ACG CCA TGT CAT CAT CAA-3¢ (618–601)
Caspase-4 Upstream 5¢-GGT CAT CAT TGT CCA GGC-3¢ (792–809)
Downstream 5¢-CCA TTG TGC TGT CTC TCC-3¢ (994–977)Caspase-5 Upstream 5¢-ACC ACA TGC TAA AGA ACA-3¢ (81–98)
Downstream 5¢-CGA TTT GCA GAA GAG GTT-3¢ (434–417)Caspase-6 Upstream 5¢-ACC CGC AGG TTT TCA GA-3¢ (249–265)
Downstream 5¢-CAT GAG CCG TTC ACA GT-3¢ (715–699)Caspase-7 Upstream 5¢-AGC CTG GGT TTT GAC GTG-3¢ (385–402)
Downstream 5¢-ACC GTG GAA TAG GCG AAG-3¢ (755–738)Caspase-8 Upstream 5¢-TAT GGC ACT GAT GGA CAG GA-3¢ (1323–1342)
Downstream 5¢-AGA TCA CGC CAC TGC ACT C-3¢ (1882–1864)Caspase-9 Upstream 5¢-AAG ACC ATG GGT TTG AGG TG-3¢ (970–989)
Downstream 5¢-TGT CGT CAA TCT GGA AGC TG-3¢ (1519–1500)Caspase-10 Upstream 5¢-AGA AGT CCA GCT CAG CCT-3¢ (352–369)
Downstream 5¢-ACT CGG CTT CCT TGT CTA-3¢ (813–796)Caspase-12 Upstream 5¢-GGA CCA AGC ACT GGG ATC A-3¢ (795–813)
Downstream 5¢-TAC CTG AGT TGC TTC TTA TGA GT-3¢ (1168–1146)Caspase-14 Upstream 5¢-ATA TGA TAT GTC AGG TGC CCG-3¢ (83–102)
Downstream 5¢-CCG AAA CAT GTG TTC CAG AG-3¢ (170–151)GAPDH Upstream 5¢-TAG CCC AGG CAC TTG GAG CC-3¢ (299–319)
Downstream 5¢-CGG TTT AAG TAA CAG TAT GG-3¢ (1121–1101)
Primer location on cDNA sequences (in bp) is written in brackets. GenBank references ofcDNAs used: NM033293 (caspase-1), BC002427 (caspase-2), NM032991 (caspase-3),
BC017839 (caspase-4), NM004347 (caspase-5), BC004460 (caspase-6), BC015799(caspase-7), NM033355 (caspase-8), NM001229 (caspase-9), NM032977 (caspase-10),
AF464193 (caspase-12) and NM012004 (caspase-14). GAPDH, glyceraldehyde-3-phos-phate dehydrogenase.
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422 Expression of caspases in human epidermis, A.-A. Raymond et al.
0Æ05% Tween-20 for 1 h at 37 �C or overnight at 4 �C(Table 2). The sections were washed twice in PBS contain-
ing 0Æ05% Tween-20 and twice in PBS and then incubated
for 1 h at room temperature with the secondary antibody
[tetramethylrhodamine isothiocyanate-conjugated goat anti-
mouse IgG (Zymed), fluorescein isothiocyanate (FITC)-con-
jugated goat antirabbit IgG (Zymed) or FITC-conjugated
rabbit antigoat IgG (Tebu-bio)] diluted in PBS containing
1% BSA and 0Æ05% Tween-20. After two washes in PBS
containing 0Æ05% Tween-20 and two washes in PBS, the
sections were finally mounted in an antifading solution
(Fluoroprep; BioMerieux, Marcy-l’Etoile, France). For nega-
tive controls, sections were incubated with nonimmune
sera, with irrelevant rabbit antipeptide antibodies or without
primary antibodies.
Results
At least nine caspase genes are expressed in normal
human epidermis
To test for the expression of caspase genes, RT–PCR analysis
was performed on cDNA from human epidermis with primer
pairs specific for each human caspase. In a first 35-cycle
amplification, the mRNAs encoding caspase-1 (858 bp), -2
(234 bp), -3 (462 bp), -4 (203 bp), -6 (467 bp), -7
(371 bp), -9 (550 bp), -10 (462 bp) and -14 (88 bp) were
detected. However, only weak signals were observed for cas-
pase-1, -2, -6, -7 and -10. Therefore a 45-cycle amplification
was performed. The results obtained (Fig. 1) confirmed the
previous data; in particular the mRNAs encoding caspase-5
and -12 were not amplified. Moreover, an amplicon of
560 bp corresponding to caspase-8 was barely detected in
only two of four cDNA samples. Parallel negative and positive
PCR controls were performed without any cDNA and with
cDNA from Jurkat cells, respectively.
Caspase-14 is the only fully processed caspase in normal
human epidermis
To explore the expression of caspases at the protein level,
extracts of epidermis obtained from either abdominal or breast
skin were separated by SDS–PAGE and immunodetected using
anticaspase antibodies (Table 2). The same results were
Table 2 Anticaspase antibodies
Caspase Type Specificity Dilution for WB Dilution for IHE Dilution for IIF
1 SA101, rabbit polyclonal p20 and precursor 1 : 1000 Not carried out 1 : 320
2 F-7, mouse monoclonal p10 and precursor 1 : 500 1 : 10–1 : 160 1 : 10–1 : 1603 H-277, rabbit polyclonal p20 and precursor 1 : 1000 1 : 160 1 : 160
4 N-15, goat polyclonal p20 and precursor 1 : 500 1 : 40 1 : 206 A-16, goat polyclonal p10 and precursor 1 : 500 1 : 20 1 : 10
7 C7724, rabbit polyclonal p20 and precursor 1 : 500 1 : 40 1 : 808 C-20, goat polyclonal p20 and precursor 1 : 500 Not carried out 1 : 10
9 F-7, mouse monoclonal p10 and precursor 1 : 250 Not carried out 1 : 1010 C8351, rabbit polyclonal p20 and precursor 1 : 1000 Not carried out 1 : 40
14 H-99, rabbit polyclonal p20 and precursor 1 : 5000 1 : 80 1 : 80
WB, Western blot; IHE, immunohistoenzymology; IIF, indirect immunofluorescence. All anticaspase antibodies were purchased from SantaCruz Biotechnology (Santa Cruz, CA, U.S.A.) (200 lg mL)1) except anticaspase-1 which came from Biomol International (Plymouth Meet-
ing, PA, U.S.A.) (100 lg mL)1), and anticaspase-10 and anticaspase-7 which came from Sigma-Aldrich (L’Isle d’Abeau, France). The letterand number correspond to the catalogue references.
Fig 1. The expression of 10 caspase genes was detected by reverse
transcription–polymerase chain reaction (PCR) in normal human
epidermis. PCR reactions were performed with human epidermis
cDNA (lane 1), without any cDNAs (lane 2) and with Jurkat cell
cDNA (lane 3), using primers specific to each caspase as indicated.
The PCR results obtained using primers specific to caspase-8 gene and
cDNA from two different individuals are shown. The size of PCR
products, analysed on agarose gels, is indicated on the left.
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Expression of caspases in human epidermis, A.-A. Raymond et al. 423
obtained with both types of extracts. We detected the pro-
forms of the caspases corresponding to the cDNAs detected in
RT–PCR [procaspase-1 (45 kDa), -2 (48 kDa), -3 (33 kDa),
-4 (43 kDa), -6 (34 kDa), -7 (32 kDa), -9 (46 kDa), -10
(59 kDa) and -14 (28 kDa)] except caspase-8. Under the con-
ditions used, only weak expression of procaspase-9 was im-
munodetected, in comparison with the others. However,
strong expression of procaspase-8 and -9 was detected in sim-
ilar extracts of cultured human keratinocytes (data not
shown). Caspase-1, -2, -3, -6, -7, -9 and -10 were present
only as unprocessed proenzymes. The anticaspase-4 antibody
recognized both the proenzyme and a 35-kDa protein prob-
ably corresponding to a processing intermediate as proposed
by Kamada et al.30 or resulting from alternative splicing,
whereas the fully processed 20-kDa form was not detected. In
the case of caspase-14, we detected both the proenzyme and
the p17 fragment, indicating full processing of this caspase
(Fig. 2).
We then analysed by immunoblotting the presence of
caspases in SC extracts obtained by tape-stripping from five
healthy individuals. Only procaspase-1 and the p17 fragment
of caspase-14 were detected in all SC extracts (Fig. 3).
Caspases are detected in all layers of the normal
epidermis
To identify the sites of caspase expression in normal human
epidermis, breast and abdominal skin cryosections were ana-
lysed by immunohistoenzymology and/or indirect immuno-
fluorescence using the same antibodies. Identical results were
obtained with both techniques, and on sections from both
anatomical sites. Control experiments were always negative.
No labelling was observed with the anticaspase-2 and anticas-
pase-8 antibodies. The anticaspase-1, -4, -6 and -10 antibodies
displayed the same pattern of staining. They labelled the entire
epidermis except the SC with an increased staining intensity at
the stratum granulosum level. Moreover, sweat glands were
labelled by anticaspase-1, -4 and -10 antibodies and muscular
fibres, only observed on sections of breast skin, were stained
by the anticaspase-1 antibody (Fig. 4 and data not shown).
The anticaspase-3 antibody labelled the entire epidermis and
Fig 2. The only fully processed caspase in normal human epidermis is
caspase-14. Twenty micrograms of proteins extracted from normal
human epidermis of breast skin were immunoblotted with anticaspase
antibodies (lane 1) or without primary antibodies (lane 2). Arrows
and arrowheads point to proteins specifically recognized by each
antibody. The position of molecular mass standards (kDa) is indicated
on the right. Note that the antigoat immunoglobulin secondary
antibody reacts nonspecifically with the heavy chains of human
immunoglobulins (50-kDa bands; panels Casp-4, -6 and -8), and that
the antimouse immunoglobulin secondary antibody reacts
nonspecifically with the light chains of human immunoglobulins
present in the extracts as monomers or heterodimers (25- and 75-kDa
bands; panel Casp-2).
Fig 3. Caspase-1 and -14 are immunodetected in extracts of normal
superficial stratum corneum. Equal amounts of proteins (20 lg) of
samples obtained from five different normal individuals using a tape-
stripping procedure were used for immunoblotting with anticaspase-1
and anticaspase-14 antibodies, as indicated. The p17 fragment of
caspase-14 is indicated on the right, and the position of molecular
mass standards (kDa) on the left.
Fig 4. Immunofluorescence staining of caspases in human epidermis.
Normal human skin cryosections were analysed by indirect
immunofluorescence using the anticaspase antibodies, as indicated,
and an irrelevant affinity purified antipeptide rabbit antibody (negative
control). Note that sweat gland myoepithelial cells [caspase-1 (b)]
and arrector pili muscle cells [caspase-1 (c)] are labelled by the
anticaspase-1 antibody. The dotted lines indicate the dermoepidermal
junction. Scale bars ¼ 75 lm.
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp420–427
424 Expression of caspases in human epidermis, A.-A. Raymond et al.
muscular fibres (data not shown). The anticaspase-7 antibody
stained with the same intensity the entire epidermis except the
SC (Fig. 4). Only a labelling of some, usually granular, kera-
tinocytes was observed with the anticaspase-9 antibody (data
not shown). When using the anticaspase-14 antibody, we
observed labelling of the entire epidermis, including the SC as
described previously.7,31
Caspase-14 is also the only fully processed caspase
detected in psoriatic stratum corneum
To evaluate a potential role for caspases in psoriasis, we ana-
lysed their presence in SC samples obtained from lesional and
nonlesional psoriatic skin (three patients) using the same anti-
bodies. As observed in normal skin, procaspase-1 (data not
shown) and the p17 processed fragment of caspase-14
(Fig. 5) were detected. Additionally, the proform (45 kDa)
and smaller forms of caspase-4 (including the previously
observed 35-kDa band) were observed in the extracts of le-
sional psoriatic SC. However, the fully processed 20-kDa form
of caspase-4 was not detected (Fig. 5).
Discussion
We analysed the expression of caspases in human skin using
different techniques including PCR on cDNA from human epi-
dermis, Western blotting of epidermis extracts and immuno-
histological analysis of skin cryosections.
We detected the mRNAs encoding caspase-1, -2, -3, -4, -6,
-7, -9, -10 and -14. In agreement, all the corresponding pro-
forms of the proteases were immunodetected. In some cases
(two of four different cDNAs), weak expression of caspase-8
mRNA was detected but we were unable to immunodetect the
corresponding protein. In agreement with these results, cul-
tured human keratinocytes have been shown to express the
same mRNAs except those encoding caspase-6.15,17,32–35 The
expression in mouse keratinocytes and epidermis of caspase-2,
-3, -6, -8 and -14 mRNA but not -7 and -12 was also
observed.16 Our results confirm and extend a previous study
showing the presence of procaspase-3, -6, -7 and -14 in
mouse epidermis.7
Most of the caspases were localized in all the epidermal
layers, with a more intense immunostaining of the granular
layer for caspase-1, -4, -6 and -10. Caspase-9 was only detec-
ted in some, usually granular, keratinocytes. This explains the
low detection of this caspase using Western blotting. The
anticaspase-8 antibody did not detect any proteins on skin
sections, confirming the Western blotting results. This strongly
suggests that caspase-8 expression is controlled at the post-
transcriptional level. Concerning caspase-2, although the
proform was detected in epidermal extracts, we did not
successfully obtain staining of human skin using immuno-
histological techniques with two different antibodies, one
from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.;
Table 2) and one from Sigma-Aldrich (L’Isle d’Abeau, France;
C7349), both specific for the precursor and p10 fragment.
This could be due to a low sensitivity of the method or more
probably to inaccessibility of the antigen.
Surprisingly, the only activated caspase seems to be caspase-
14, as detected by Western blotting of epidermis extracts. This
indicates that the amount of fully processed caspases is too
low to be immunodetected or more probably that most cas-
pases are not activated at all. This result raises the following
question: why do keratinocytes express unprocessed procas-
pases? We could speculate about an anticipation of a cutane-
ous stress harmful for the survival of keratinocytes or for the
Fig 5. Caspase-14 and procaspase-4 are detected in extracts of
psoriatic superficial stratum corneum (SC). Equal amounts of proteins
(20 lg) of samples obtained from the SC of three normal individuals
and from both lesional (L) and nonlesional (NL) SC of three patients
with psoriasis were separated by sodium dodecyl sulphate–
polyacrylamide gel electrophoresis, and transferred to membranes.
The membranes were stained with Ponceau Red and used for
immunoblotting using antibodies to caspase-14 and caspase-4, and an
isotype control antibody (anticaspase-6), as indicated. The position of
molecular mass standards (kDa) is indicated on the left. The p17
fragment of caspase-14, the proform (Casp-4) and the p35 fragment
of caspase-4 (arrowhead) are indicated on the right.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp420–427
Expression of caspases in human epidermis, A.-A. Raymond et al. 425
integrity of the skin barrier, e.g. ultraviolet (UV) irradiation.
Indeed, caspase-1, -3, -8 and -9 have been shown to be acti-
vated in human keratinocytes during UV-induced apopto-
sis,32–34,36,37 this induction being considered as a protective
function against skin cancer. In agreement, the intense immuno-
staining of the granular layer observed for most caspases
might be related to the fact that cutaneous stresses first affect
the most superficial layers of the epidermis. All classes of cas-
pases seem to be ready to be activated: inflammatory caspase-1
and -4, initiator caspase-2, -9 and -10, and effector caspase-3,
-6 and -7.
Caspase-14 was suggested to play a role in keratinocyte ter-
minal differentiation. In the same way, different caspases were
shown to be involved in cell differentiation, e.g. caspase-3 in
lens epithelium differentiation,9,38 and caspase-1 in neuronal
differentiation of PC12 cells.39 Here, we show that fully proc-
essed caspase-14 is detected at the SC surface. This suggests
that the enzyme might be important for the late steps of
terminal differentiation, e.g. during desquamation. Indeed,
immunoelectron microscopy showed that it was localized near
corneodesmosomes.22 For desquamation to occur, corneodes-
mosome components must be proteolysed. We previously
demonstrated the involvement of proteases of the kallikrein
family in the degradation of the extracellular components.40
We also suggested that intracellular components (plakoglobin
and the intracellular part of desmoglein 1) must also be degra-
ded, but the involved proteases have not been identified. In
agreement with this hypothesis, intracellular and transmem-
branous components of desmosomes are cleaved by caspases
after induction of apoptosis in the keratinocyte cell lines
HaCaT and A431.6,41 To test for a role of caspase-14, we
activated human recombinant procaspase-14 by granzyme B
in vitro and incubated an extract of epidermis containing plako-
globin and desmogleins with the active caspase-14. However,
we did not observe cleavage of these proteins (data not
shown).
Finally, we looked for a potential disruption of caspase
expression in psoriatic epidermis. Western blot analysis of SC
samples revealed, as in normal epidermis, the presence of
procaspase-1 and caspase-14. It is surprising that the level of
caspase-14 in the superficial SC of psoriatic skin (both lesional
and nonlesional skin) seems to be not so different from its
level in the SC of normal skin21 (this work). Indeed, a reduc-
tion in the expression of the caspase is generally observed
using immunohistochemistry in the parakeratotic SC of psori-
atic skin.7,42,43 This could be explained in part by the well-
known masking of antigens/epitopes in the hyperkeratotic SC.
This result also suggests that despite a reduction in the level of
procaspase-14 in the noncornified keratinocytes of psoriatic
epidermis, a peculiar mechanism of regulation ensures the
same level of processing and accumulation of the active
caspase-14 in both normal and diseased tissue. In addition to
these two caspases, the expression of procaspase-4 was also
noticed in the SC of lesional psoriatic skin. This could be linked
to the aberrant expression of other apoptosis-related molecules
in the psoriatic skin such as ICAD (inhibitor of caspase-3-
related DNase), Bcl-2 or Bax.44 Alternatively, this result sug-
gests a possible involvement of caspase-4 in psoriasis physio-
pathology. In particular, the caspase-4 gene is one of the
numerous interferon-induced genes,45–47 interferon-c being an
important cytokine in psoriasis. Moreover, the caspase-4 gene
was recently shown to be upregulated in psoriasis.47
In conclusion, keratinocytes express in the epidermis the
proforms of the initiator caspase-1, -2, -4, -9 and -10, and of
the effector caspase-3, -6 and -7. Therefore, they are probably
ready to respond to major stresses and to induce apoptosis.
Moreover, they express an active caspase-14, which is prob-
ably necessary for the last steps of terminal differentiation but
the exact function of which remains to be discovered.
Acknowledgments
We thank Professor A.-P. Arrigo for the gift of anticaspase-3
antibodies, Professor J.-P. Chavoin for providing us with
human skin and Dr M. Guerrin for reading the manuscript.
This study was supported by grants from the Centre National
de la Recherche Scientifique, the European Regional Develop-
ment Funds, the European Social Funds, the Institut National
de la Sante et de la Recherche Medicale, and from the Societe
de Recherche Dermatologique.
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Expression of caspases in human epidermis, A.-A. Raymond et al. 427
CUTANEOUS BIOLOGY DOI 10.1111/j .1365-2133.2006.07671.x
Testosterone metabolism to 5a-dihydrotestosterone andsynthesis of sebaceous lipids is regulated by theperoxisome proliferator-activated receptor ligandlinoleic acid in human sebocytesE. Makrantonaki* and C.C. Zouboulis*�
*Laboratory of Biogerontology, Dermatopharmacology and Dermatoendocrinology, Institute of Clinical Pharmacology and Toxicology, Charite University Medicine
Berlin, Campus Benjamin Franklin, Garystrasse 5, 14195 Berlin, Germany
�Departments of Dermatology and Immunology, Dessau Medical Center, Auenweg 38, 06847 Dessau, Germany
CorrespondenceChristos C. Zouboulis.
E-mail: christos.zouboulis@klinikum-dessau.de
Accepted for publication17 August 2006
Key words5a-reductase inhibitor, androgens,
lipids, peroxisome proliferator-activated
receptors, sebocytes
Conflicts of interestNone declared.
Summary
Background Despite the clinical evidence that androgens stimulate sebaceous lipids,androgens in vitro have shown no similar effects. This contradiction led to theassumption that cofactors may be required for lipid regulation and peroxisomeproliferator-activated receptor (PPAR) ligands were suggested to be adequatecandidates.Objectives The influence of testosterone and linoleic acid, a PPAR ligand, as singleagents and in combination with of LY191704, a 5a-reductase type I inhibitor,was examined on 5a-dihydrotestosterone (5a-DHT) synthesis and lipid contentin human SZ95 sebocytes.Methods Cell proliferation and viability were measured by the 4-methylumbelliferylheptanoate fluorescence assay and by the Boehringer Lactate Dehydrogenase Assaykit, respectively. 5a-DHT enzyme-linked immunosorbent assay was used for thedetection of 5a-DHT synthesis in cell supernatants after treatment, whereas lipidproduction was documented by means of the Nile red lipid microassay and fluor-escence microscopy.Results Testosterone promoted 5a-DHT synthesis (P < 0Æ001), whereas linoleicacid increased sebaceous lipids (P < 0Æ001). The combination of testosterone andlinoleic acid exhibited a synergistic effect on the synthesis of 5a-DHT (P < 0Æ01vs. testosterone) and sebaceous lipids (P < 0Æ01 vs. linoleic acid). Furthermore,LY191704 reduced 5a-DHT and sebaceous lipid levels (P < 0Æ01 and P < 0Æ001in comparison with testosterone/linoleic acid, respectively). Cell proliferationand viability remained unchanged under treatment with all compounds tested.Conclusions These data suggest a catalytic effect of PPAR ligands on cellular testo-sterone activation by 5a-reduction and the importance of the latter for theregulation of sebaceous lipids.
Among the numerous endocrine signals that affect skin, those
of androgens play a predominant role. High testosterone levels
have been implicated in enhanced sebaceous gland activity in
humans1,2 and consequently in diseases marked by seborrhoea,
such as acne vulgaris. Enhanced sebaceous gland activity is
attributed to the potent androgen 5a-dihydrotestosterone
(5a-DHT)3 as sebaceous gland cells possess all necessary
enzymes for conversion of testosterone to 5a-DHT.4
The effects of testosterone and 5a-DHT are mediated by
binding to the nuclear androgen receptor (AR), also expressed
in human sebaceous gland cells.5 The AR is a member of the
steroid superfamily of ligand-dependent transcriptional factors.
5a-DHT binds to the AR with greater affinity than testosterone
and the 5a-DHT/AR complex appears to be more stable6 and,
therefore, more effective.
In contrast to the in vivo observations, in vitro experiments
with human sebocytes have shown that testosterone affects
proliferation in a dose-dependent manner7,8 but not lipid syn-
thesis.9,10 This contradiction led to the assumption that cofac-
tors may be required for the induction of the entire so-called
androgenic influence of the sebaceous gland.11 Current
research has indicated that peroxisome proliferator-activated
� 2007 The Authors
428 Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp428–432
receptors (PPARs) and their ligands could be the primary can-
didates.9,10 PPARs regulate multiple lipid metabolism genes in
mitochondria, peroxisomes and microsomes, all prominent in
sebocyte cytoplasm.9,10
In the experiments presented here we examined the influ-
ence of testosterone and linoleic acid (LA), a PPAR-a/d lig-
and12 previously shown to induce lipid synthesis of human
sebocytes in vitro,9,10 as single agents and in combination on
5a-DHT synthesis and lipid content in SZ95 immortalized
human sebaceous gland cells.8 Furthermore, the mechanism of
testosterone 5a-reduction and its influence on lipid synthesis
was examined by adding the 5a-reductase type I inhibitor
LY19170413 to the experimental setting.
Materials and methods
Cell culture
Immortalized human SZ95 sebocytes8 were maintained in
Sebomed� medium (Biochrom, Berlin, Germany) supplemen-
ted with 10% fetal calf serum, 5 ng mL)1 recombinant
human epidermal growth factor, 1 mmol L)1 Ca2+ and
50 lg mL)1 gentamicin. The cells were incubated with testo-
sterone (2 · 10)8 mol L)1) (Sigma, Munich, Germany), LA
(10)4 mol L)1) (Sigma) and with the 5a-reductase inhibitor
LY191704 (10)8 mol L)1), provided by Schering AG (Berlin,
Germany). All cells used for the experiments were between
passages 26 and 28.
Cell proliferation
Cell proliferation was assessed after incubation of 1500 SZ95
sebocytes/well in 96-well plates for 48 and 96 h by the
4-methylumbelliferyl heptanoate fluorescence assay, measured
automatically as previously described.14
Detection of cell viability
SZ95 sebocytes were incubated at a density of 20 000 cells/
well in 96-well plates for 48 h and then treated with the
active compounds. After 24 h the supernatants were collected
and centrifuged to remove cell detritus. Finally, 100 lL of the
supernatant was used to measure lactate dehydrogenase release
employing the Boehringer Lactate Dehydrogenase Assay kit
(Mannheim, Germany) and adhering to the manufacturer’s
instructions.
5a-dihydrotestosterone enzyme-linked immunosorbent
assay
After a 48-h adhesion period and a 24-h incubation of SZ95
sebocytes with the active compounds in 24-well plates, the
supernatants were collected, centrifuged in order to remove
cell debris and then the 5a-DHT released was measured by a
5a-DHT enzyme-linked immunosorbent assay (IBL, Hamburg,
Germany) according to the manufacturer’s instructions.
Nile red microassay
After a 48-h adhesion period and a 24-h treatment of 20 000
SZ95 sebocytes/well in 96-well plates with the active com-
pounds, the Nile red microassay for evaluation of lipid content
was performed as previously described:15 485 nm excitation
and 565 nm emission filters were used for neutral lipids
detection and 540 nm excitation and 620 nm emission filters
for polar lipids.
Fluorescence microscopy
SZ95 sebocytes were seeded on 18-mm coverslips in culture
dishes at an approximately 40% confluence. After a 48-h ad-
hesion period, SZ95 sebocytes were treated for 24 h, whereas
control cells received no treatment. Then, the cells were
washed twice with phosphate-buffered saline (PBS), and
100 lL of a 100 lg mL)1 Nile red solution in PBS was added.
The plates were then incubated at 37 �C for 10 min, and the
fluorescence released was determined by Axiophot SIP 31182
fluorescence microscope (Carl Zeiss, Gottingen, Germany).
Statistical analysis
Values represent the mean values ± SD of at least three experi-
ments. Statistical significance was calculated by the two-sample
independent-groups t-test. Mean differences were considered
to be significant when P < 0Æ05.
Results
Preliminary experiments
Testosterone (2 · 10)8 mol L)1), LA (10)4 mol L)1) and
LY191704 (10)8 mol L)1) concentrations used and the treat-
ment duration (up to 96 h) were chosen in preliminary
experiments in order not to influence SZ95 sebocyte prolifer-
ation and viability. They were tested as single compounds and
in combination (data not shown). The testosterone levels
administered were similar to those circulating in healthy
young men.
Regulation of 5a-dihydrotestosterone synthesis
in SZ95 sebocytes
As expected, 5a-DHT synthesis was markedly increased by
treatment with testosterone (P < 0Æ001), while it was not
influenced by LA treatment (Fig. 1). Interestingly, 5a-DHT
synthesis was further increased in SZ95 sebocytes by treatment
with testosterone and LA in comparison with testosterone
(P < 0Æ05) or LA (P < 0Æ001) alone (Fig. 1).
Amplification of lipid synthesis in SZ95 sebocytes
Testosterone alone had no effect on sebocyte lipid content,
while LA increased significantly neutral (sebocyte) lipids
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Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp428–432
Androgens and PPAR ligands, E. Makrantonaki and C.C. Zouboulis 429
(+ 523%, P < 0Æ001 vs. control) and polar (membrane)
lipids (+ 50%, P < 0Æ001 vs. control) (Fig. 2). The com-
bination of testosterone and LA exhibited a synergistic effect
on the amplification of neutral lipids (+ 599%, P < 0Æ001
vs. control and + 14Æ5%, P < 0Æ01 vs. LA) but not polar
lipids (+ 59%, P < 0Æ001 vs. control and nonsignificant vs.
LA) (Fig. 2).
The lipid content of the treated SZ95 sebocytes was com-
pared after staining with the Nile red dye and visualization of
the fluorescence by fluorescence microscopy (Fig. 3). In con-
trast to untreated SZ95 sebocytes (Fig. 3a), lipid accumulation
was enhanced in LA-treated cells (Fig. 3c), while testosterone
alone showed no effect (Fig. 3b). Treatment with testosterone
and LA induced synergistically the lipid synthesis (Fig. 3d),
corresponding to the results of the Nile red microassay.
LY191704 reduces 5a-dihydrotestosterone and lipid
synthesis in SZ95 sebocytes treated with testosterone
and linoleic acid
The 5a-reductase type I inhibitor LY191704 reduced 5a-DHT
synthesis in SZ95 sebocytes treated with testosterone and LA
() 29%, P < 0Æ01 vs. testosterone/LA treatment) (Fig. 4c) but
did not affect 5a-DHT synthesis in SZ95 sebocytes treated
with either testosterone or LA (Fig. 4a,b). In addition, neutral
lipids were decreased after treatment of SZ95 sebocytes with
all three compounds () 30%, P < 0Æ001 in comparison with
testosterone and LA) (Fig. 3e, 5c) but were not affected after
incubation with LY191704 and either testosterone or LA
(Fig. 5a,b).
Discussion
Several studies have demonstrated that there is an association
between local overproduction of active androgens and skin
disorders such as acne. Patients with acne produce higher rates
of testosterone and 5a-DHT in their skin than healthy individ-
uals.16 In addition, only a few patients with androgenic dis-
orders exhibit hyperandrogenaemia, an observation which
indicates the predominance of peripheral tissue events for the
occurrence of clinical signs.17
In order to gain greater insights into the peripheral andro-
gen activation and activity we examined the effects of testos-
terone at physiological levels and of LA, as single agents and
in combination, on 5a-DHT production and lipid synthesis
in SZ95 sebocytes. Interestingly, the combination exhibited
Fig 1. 5a-dihydrotestosterone (5a-DHT) synthesis in SZ95 sebocytes
after a 24-h treatment in 24-well plates with testosterone (T) and/or
linoleic acid (LA), determined by enzyme-linked immunosorbent
assay. SZ95 sebocytes treated with T + LA produced significantly
higher levels of 5a-DHT in contrast to cells treated only with T
(P < 0Æ05). Control cells without treatment were set at 0% and the
production of 5a-DHT in the treated cells was calculated as percentage
of control. Values represent the mean of three experiments ± SD
(***P < 0Æ001).
Fig 2. Lipid synthesis in SZ95 sebocytes after a 24-h treatment in
96-well plates with testosterone (T) and/or linoleic acid (LA),
determined by Nile red microassay. The production of neutral (light
columns) and polar lipids (dark columns) is illustrated. T showed no
effect on lipid synthesis, whereas LA increased significantly the
production of neutral (P < 0Æ001 vs. control) and polar lipids
(P < 0Æ001 vs. control). The combination of T and LA exhibited a
synergistic effect on neutral lipids (P < 0Æ001 vs. control and P < 0Æ01
vs. LA). Lipid synthesis in control cells without treatment was set at
0% and the lipid production in the treated cells was calculated as
percentage of control. Values represent the mean of three
experiments ± SD with 10 wells evaluated for each data point in each
experiment (***P < 0Æ001).
(a) (b)
(c)
(e)
(d)
Fig 3. Lipid synthesis in SZ95 sebocytes without treatment (a), after a
24-h treatment with testosterone (T) (b) and/or linoleic acid (LA)
(c,d) and LY191704 (e), shown by means of fluorescence
microscopy. The production of sebaceous lipids is illustrated after
staining of SZ95 sebocytes with 100 lg mL)1 Nile red dye. Treatment
only with T showed no effect (b), while LA enhanced lipid
accumulation (c). The combination of T + LA acted synergistically on
lipid synthesis (d). In SZ95 sebocytes treated with
T + LA + LY191704 (e), lipid synthesis was inhibited in contrast to
SZ95 sebocytes treated with T + LA (d).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp428–432
430 Androgens and PPAR ligands, E. Makrantonaki and C.C. Zouboulis
an unexpected synergistic effect on 5a-DHT production and
synthesis of sebaceous lipids. This is, to our knowledge, the
first work indicating that LA ‘activates’ the stimulatory effect
of testosterone on sebaceous lipogenesis that would be
expected according to in vivo data. Androgens alone have
been found ineffective on sebaceous lipid synthesis in previ-
ous in vitro experiments.9,10 This illustrates that cofactors such
as LA are required for regulation of sebaceous lipids by
androgens and may be responsible for the pathogenesis of
sebaceous gland-associated diseases with excessive sebum
production, such as acne.18 Indeed, Rosenfield et al.9 have
previously demonstrated the interaction of 5a-DHT with
PPAR ligands in inducing differentiation of sebocyte-like rat
preputial cells and lipid synthesis. However, in this study,9
PPAR-c agonists, such as BRL, were shown to be more
effective in stimulating lipid synthesis in the presence of
androgens, whereas no effect was seen with the PPAR-a/dagonist LA. This discrepancy is probably due to the fact that
in our experiments human sebaceous cells (SZ95 sebocytes)
were used, for which recent results have also demonstrated
that PPAR-a is the most important PPAR that regulates lipid
synthesis and inflammation.11,19 In addition, PPAR-a, d, c1and c2 have been shown to be expressed at mRNA and pro-
tein levels in SZ95 sebocytes,10 whereas LA has been docu-
mented to stimulate intracellular accumulation of sebaceous
lipids.9,10
Further to corroborate the importance of testosterone
5a-reduction in the stimulation of sebaceous lipid synthesis, we
performed similar experiments adding LY191704 [8-chloro-
4-methyl-1,2,3,4,4a,5,6,10b-octaahydro-benzo(f)quinolin-3(2H)-
one], a potent, selective, nonsteroidal inhibitor of human
steroid 5a-reductase type I,13 which catalyses the conversion
(a)
(c)
(b)
Fig 4. 5a-dihydrotestosterone (5a-DHT) synthesis in human sebocytes incubated with LY191704 (LY) and testosterone (T) (a) and/or linoleic
acid (LA) (b,c), determined by enzyme-linked immunosorbent assay. LY showed a significant inhibitory effect on 5a-DHT synthesis (P < 0Æ01)
only in the presence of T + LA (c). LY + T (a) and LY + LA (b) showed no effect on 5a-DHT production. As controls were used cells treated
only with T (a), LA (b) and T + LA (c), respectively, and the production of 5a-DHT was calculated as percentage of control. Values represent the
mean of three experiments ± SD (**P < 0Æ01).
(a)
(c)
(b)
Fig 5. Lipid synthesis in SZ95 sebocytes after a 24-h treatment in 96-well plates with LY191704 (LY) and testosterone (T) (a) and/or linoleic
acid (LA) (b,c), determined by Nile red microassay. The production of neutral (light columns) and polar lipids (dark columns) is illustrated. LY
inhibited lipid synthesis in SZ95 sebocytes (P < 0Æ001) only in the presence of T + LA (c). LY + T (a) and LY + LA (b) showed no inhibitory
effect on lipid production. As control were used cells treated only with T (a), LA (b) and T + LA (c), respectively, and the production of lipids
was calculated as percentage of control. Values represent the mean of three experiments ± SD with three wells evaluated for each data point in
each experiment (***P < 0Æ001).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp428–432
Androgens and PPAR ligands, E. Makrantonaki and C.C. Zouboulis 431
from testosterone to 5a-DHT in peripheral tissues by an
NADPH-dependent reaction.
The isozyme 5a-reductase type I is expressed predominantly
in skin. It is present in the cytoplasm and cell membrane com-
partment in skin cells20 and particularly in facial sebocytes,4
illustrating the key role of sebaceous gland cells in androgen
metabolism.
In accordance with in vivo studies, treatment with LY191704
and either testosterone or LA did not show any inhibitory
effect on 5a-DHT and sebaceous lipid synthesis.21 However,
LY191704 showed its inhibitory action when cells were
exposed concurrently to testosterone and LA. Hence, we hypo-
thesize that the 5a-reductase type I can be inhibited only after
being activated by LA. The simultaneous presence of the pre-
cursor molecule testosterone is of great importance for this
action. Further studies are needed to clarify this hypothesis.
In conclusion, our results suggest that lipid production is
regulated by the PPAR-a/d ligand LA in human sebocytes and
that PPAR ligand-induced 5a-reduction of testosterone is,
indeed, a mechanism for amplifying sebaceous lipid synthesis.
Acknowledgments
This work was supported by research grants of the Deutscher
Dermatologen Kongreß 1995 – Verein zur Forderung der Der-
matologie eV and of Schering AG, Berlin, Germany.
References
1 Pochi PE, Strauss JS. Sebaceous gland response in man to the
administration of testosterone, delta-4-androstenedione, and dehy-droisoandrosterone. J Invest Dermatol 1969; 52:32–6.
2 Giltay EJ, Gooren LJ. Effects of sex steroid deprivation/administra-tion on hair growth and skin sebum production in transsexual
males and females. J Clin Endocrinol Metab 2000; 85:2913–21.3 Zouboulis CC, Degitz K. Androgen action on human skin – from
basic research to clinical significance. Exp Dermatol 2004; 13 (Suppl. 4):5–10.
4 Fritsch M, Orfanos CE, Zouboulis CC. Sebocytes are the key regula-tors of androgen homeostasis in human skin. J Invest Dermatol 2001;
116:793–800.5 Fimmel S, Saborowski A, Orfanos CE et al. Development of efficient
transient transfection systems for introducing antisense oligonucleo-tides into human epithelial skin cells. Horm Res 2000; 54:306–11.
6 Anderson KM, Liao S. Selective retention of dihydrotestosterone byprostatic nuclei. Nature 1968; 219:277–9.
7 Akamatsu H, Zouboulis CC, Orfanos CE. Control of human sebo-cyte proliferation in vitro by testosterone and 5-alpha-dihydrotesto-
sterone is dependent on the localization of the sebaceous glands.J Invest Dermatol 1992; 99:509–11.
8 Zouboulis CC, Seltmann H, Neitzel H et al. Establishment and char-acterization of an immortalized human sebaceous gland cell line
(SZ95). J Invest Dermatol 1999; 113:1011–20.9 Rosenfield RL, Deplewski D, Kentsis A, Ciletti N. Mechanisms of
androgen induction of sebocyte differentiation. Dermatology 1998;
196:43–6.10 Chen W, Yang CC, Sheu HM et al. Expression of peroxisome prolif-
erator-activated receptor and CCAAT/enhancer binding proteintranscription factors in cultured human sebocytes. J Invest Dermatol
2003; 121:441–7.11 Zouboulis CC, Eady A, Philpott M et al. What is the pathogenesis of
acne? Exp Dermatol 2005; 14:143–52.12 Kliewer SA, Forman BM, Blumberg B et al. Differential expression
and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 1994; 91:7355–9.
13 Hirsch KS, Jones CD, Audia JE et al. LY191704: a selective, nonste-roidal inhibitor of human steroid 5 alpha-reductase type 1. Proc Natl
Acad Sci USA 1993; 90:5277–81.14 Zouboulis CC, Garbe C, Krasagakis K et al. A fluorometric rapid
microassay to identify anti-proliferative compounds for humanmelanoma cells in vitro. Melanoma Res 1991; 1:91–5.
15 Zouboulis CC, Seltmann H, Hiroi N et al. Corticotropin-releasinghormone: an autocrine hormone that promotes lipogenesis in
human sebocytes. Proc Natl Acad Sci USA 2002; 99:7148–53.16 Sansone G, Reisner RM. Differential rates of conversion of testo-
sterone to dihydrotestosterone in acne and in normal humanskin – a possible pathogenic factor in acne. J Invest Dermatol 1971;
56:366–72.17 Orfanos CE, Adler YD, Zouboulis CC. The SAHA syndrome. Horm
Res 2000; 54:251–8.18 Kligman AM. An overview of acne. J Invest Dermatol 1974; 62:268–
87.19 Alestas T, Ganceviciene R, Fimmel S et al. Enzymes involved in the
biosynthesis of leukotriene B(4) and prostaglandin E(2) are activein sebaceous glands. J Mol Med 2006; 84:75–87.
20 Chen W, Zouboulis CC, Fritsch M et al. Heterogeneity and quantita-tive difference of type 1 5 alpha-reductase expression in cultured
skin epithelial cells. Dermatology 1998: 196 51–2.
21 Leyden J, Bergfeld W, Drake L et al. A systemic type I 5 alpha-reductase inhibitor is ineffective in the treatment of acne vulgaris.
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432 Androgens and PPAR ligands, E. Makrantonaki and C.C. Zouboulis
CUTANEOUS BIOLOGY DOI 10.1111/j .1365-2133.2007.07681.x
The antiwrinkle effect of topical concentrated2-dimethylaminoethanol involves a vacuolar cytopathologyG. Morissette, L. Germain* and F. Marceau
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Quebec, Quebec QC, G1V 4G2, Canada
*Laboratoire d’Organogenese Experimentale, Hopital du Saint-Sacrement du CHA, Quebec QC, G1S 4L8, Canada
CorrespondenceFrancois Marceau.
E-mail: francois.marceau@crchul.ulaval.ca
Accepted for publication21 September 2006
Key words2-dimethylaminoethanol, triethanolamine, vacuolar
adenosine triphosphatase, vacuolar cytopathology
Conflicts of interestNone declared.
Summary
Background The ‘cosmeceutical’ agent 2-dimethylaminoethanol (DMAE) is a tertiaryamine found in high concentration in numerous topical antiwrinkle preparations.Objectives We hypothesized that a 337 mmol L)1 (3%) DMAE reservoir applied tothe skin could reproduce the cytopathology induced by other amines by main-taining a millimolar drug concentration within a certain depth of the skin layers,and that vacuolar cell expansion could account for the very rapid effect on theapparent skin fullness.Methods Morphological and functional assays were applied to cultured rabbit der-mal fibroblasts treated with tertiary amines in vitro. A morphological verificationof the vacuolization caused by topical DMAE was also attempted in vivo using theinner skin of the rabbit ear and in vitro using primary cultures of human cutane-ous epithelial cells.Results Fibroblasts responded to DMAE (2Æ5–10 mmol L)1) by massive vacuoliza-tion (0Æ5–4 h; phase contrast observations). Triethanolamine, another chemicalfrequently used topically, was also active in this respect (10 mmol L)1). The vac-uolar adenosine triphosphatase inhibitor bafilomycin A1 prevented DMAE- ortriethanolamine-induced vacuolization; adding bafilomycin A1 or cell washoutslowly reversed the established vacuolization induced by DMAE. Further effectsof DMAE in cultured fibroblasts included a moderate cytotoxicity (10 mmol L)1)that was abated by bafilomycin A1 cotreatment, a concentration-dependent mito-tic arrest (2Æ5 mmol L)1) and transient and mild effects on cell ploidy. The epi-dermis of the rabbit external ear was significantly thickened and exhibited clearperinuclear swelling indicative of vacuolization in response to 3% DMAE (1 h;paraffin tissue sections). Cultured human cutaneous epithelial cells responded toDMAE by vacuolization (inhibited by bafilomycin A1 cotreatment).Conclusions The vacuolar cytopathology induced by concentrated organic aminesmay be the cellular basis of the antiwrinkle effect of DMAE.
Despite their wide use, the pharmacology and toxicology of
agents used in cosmetology are rarely documented in a
detailed manner in the scientific literature. The ‘cosmeceutical’
agent 2-dimethylaminoethanol (DMAE) is an ingredient of
numerous antiwrinkle creams; in the lay press, it has been
dubbed ‘an instant anti-aging face-lift.’1 Some objective clin-
ical information is available about DMAE: a placebo-controlled
trial consisting of a 337 mmol L)1 (3%) DMAE facial gel
applied for 16 weeks showed efficacy in attenuating wrinkles
of various sizes and improving lip shape and the fullness of
skin.2 The agent was well tolerated during the trial and its
1-year extension. Another randomized, double-blind, split-face
study using 3% DMAE showed a nonsignificant trend towards
increased skin firmness.3 Other related aminoethanol deriva-
tives (di- and triethanolamine) are also widely exploited in
topical formulations.4
The mode of action of DMEA in dermatology is unknown.
There have been speculations about possible interference with
cholinergic neurotransmission (perhaps by analogy with the
antiwrinkle effect of botulinum toxin) and about a mild anti-
inflammatory effect.2 DMEA is a tertiary amine (pKa 8Æ88) and
many cultured cell types respond to a millimolar concentration
of such compounds by a massive vacuolization. This is a form
of cytopathology that is not fully understood but is explained
by ion trapping of the charged organic amines in acidic com-
partments of the cells and secondary osmotic swelling of the
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp433–439 433
organelles.5,6 The reaction is relatively rapid (1–2 h for most
amines) and is driven by vacuolar adenosine triphosphatase
(V-ATPase), as shown by it being fully prevented by treatment
with the specific inhibitor bafilomycin A1; morphological ana-
lyses have shown that it involves at least the trans-Golgi.5,6
Massive vacuolization induced by amine drugs such as pro-
cainamide often involved supratherapeutic concentrations and
is of toxicological interest. Notable exceptions are the local
anaesthetics (lidocaine, etc.) that, used as supramillimolar
solutions applied in confined anatomical areas, cause such
reactions in vivo at sites of injection and in cultured cells of
various types.5,7–9 For some amines, such as triethylamine and
procainamide, the sustained vacuolization response of cultured
cells is associated with only a low frequency of cytotoxicity
that is abated by concomitant bafilomycin A1 treatment, a
mitotic arrest, an inhibition of protein translocation along the
cell secretory pathway and some inhibition of cell migra-
tion.5,6 Other agents that could be considered as substituted
triethylamines (e.g. metoclopramide, chloroquine) also caused
vacuolization, but were more toxic at millimolar concentra-
tions probably as a result of additional actions at other bio-
chemical and/or subcellular levels.5,6,10
We hypothesized that a 337 mmol L)1 (3%) DMAE reser-
voir applied to the skin could reproduce the cytopathology
induced by other amines by maintaining a millimolar drug
concentration within a certain depth of the skin layers, and
that vacuolar cell expansion could account for the very rapid
effect on the apparent skin fullness. This was tested in cultured
rabbit dermal fibroblasts and human epidermal cells. Another
tertiary amine widely used in cosmetics, triethanolamine,11
has been compared with DMAE. A histological verification of
the vacuolization caused by 3% DMAE was also attempted
using the inner skin of the rabbit ear.
Materials and methods
Drugs
All drugs were obtained from Sigma-Aldrich (St Louis, MO,
U.S.A.).
Cells and microscopic techniques
The institutional Review Board approved the animal studies
and the animals were cared for in accordance with the Guide
to the Care and Use of Experimental Animals (Canadian Coun-
cil on Animal Care). Rabbit dermal fibroblasts were grown
from explants of fat-free subcutaneous tissue removed from
male New Zealand white rabbits (1Æ5–2 kg). The culture med-
ium was Dulbecco’s modified Eagle’s medium (Invitrogen,
San Diego, CA, U.S.A.) supplemented with 10% fetal bovine
serum and antibiotics.12 An institutional research committee
approved the anonymous use of primary cultures of human
epidermis, harvested and maintained as described.13 Six-well
plates were seeded with 20 000 irradiated 3T3 feeder
cells cm)2 and 10 000 keratinocytes cm)2 at passage 2, as
described.13 Drugs were added to the complete culture med-
ium for each cell type; bafilomycin A1 was initially dissolved
in dimethyl sulphoxide (final concentration of solvent
< 0Æ1%).
The vacuolization of cultured cells, photographed in phase
contrast such that clear vacuoles could be observed on a dark
background, was quantified as the percentage of individual
cell pixels above a set threshold, after increasing the contrast
of the picture in a standardized manner (Adobe Photoshop,
version 6.0; Adobe Systems Inc., San Jose, CA, U.S.A.). These
numerical values were averaged and compared using nonpara-
metric statistical tests.
Cytotoxicity assay
Subconfluent fibroblasts plated in 35-mm Petri dishes were
maintained in their regular culture medium; test drugs or
combinations were added for 4 or 24 h. After this time, the
medium was removed and centrifuged at 200 g for 5 min.
The pellet containing some detached cells was resuspended in
reaction medium comprising 1 mL of fresh 12Æ5 lg mL)1 flu-
orescein diacetate (Sigma-Aldrich) in phosphate-buffered sal-
ine diluted from a 5 mg mL)1 stock in filtered acetone. The
cells were reintroduced into the Petri dish and incubated for
30 min at 37 �C in the dark; propidium iodide (100 lL of
0Æ5 mg mL)1) was then added, the dishes were further incu-
bated for 3–5 min at room temperature and then observed
(epifluorescence 100·) without rinsing, using two sets of
excitation–emission filters (the red fluorescence indicates pro-
pidium iodide uptake by nonviable cells, the green fluores-
cence shows the hydrolysis of the fluorescein ester by viable
cells). The proportion of nonviable cells (adherent or not)
was established by counting.
Other cell-based assays
A fibroblast proliferation assay was applied as previously des-
cribed for smooth muscle cells.5 For the ploidy assay, fibro-
blasts maintained in complete culture medium were treated
with drugs for various time periods (up to 48 h), detached
using trypsin–ethylenediamine tetraacetic acid, fixed with 95%
ethanol, stained with propidium iodide and analysed using the
EPICS XL cytofluorometry apparatus (Beckman Coulter, Fuller-
ton, CA, U.S.A.; excitation 488 nm, emission 620 nm); results
were analysed using the System II software (version 3.0; Beck-
man Coulter).
Effect of 2-dimethylaminoethanol application on rabbit
ear skin
Freshly prepared emulsions of DMAE (3%) in heavy mineral
oil (Fisher Scientific, Nepean, ON, Canada) were applied to
the inner bare surface of the external ear of conscious rabbits.
The contralateral ear was the control (treated with the oil
vehicle). Animals were killed by CO2 inhalation after 1 h.
The procedure has no observable impact on the behaviour of
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Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp433–439
434 2-Dimethylaminoethanol-induced vacuolization, G. Morissette et al.
animals and no consistent macroscopic effect on the skin. The
treated areas were dissected and multiple tissue samples were
fixed and processed for paraffin sections (haematoxylin and
eosin stain).
Results
Massive fibroblast vacuolization induced by
2-dimethylaminoethanol
Rabbit dermal fibroblasts are spindle-shaped cells that respond
to HCl-buffered DMAE (pH 7Æ4; structure in Fig. 1c) by a
massive vacuolization over a period of 4 h (phase contrast
observations at 4 h, Fig. 1a; graphical representation of digit-
ized images, Fig. 1b). A period of 4 h was chosen for stan-
dardized observations, but a robust morphological response
was observed as early as 30 min (DMAE 10 mmol L)1;
data not shown). A concentration response study showed that
the 1 mmol L)1 level was not usually active to induce vacuoli-
zation, but that a concentration-dependent response occurred
in the 2Æ5–10 mmol L)1 range. At 10 mmol L)1, the cells
usually exhibited a more or less retracted morphology, some
being rounded and detached. In massively vacuolated cells,
nuclei were often located at the periphery and exhibited a
condensed appearance, as previously observed with smooth
muscle cells treated with other amines.5,6 Triethanolamine
(structure, Fig. 1c; pKa 7Æ8) is related to DMEA but is more
polar; it was less active than DMEA at inducing vacuolization
of the fibroblasts (Fig. 1a,b). However, the response to
10 mmol L)1 of each agent was of comparable magnitude.
Not only did bafilomycin A1 prevent DMAE- or triethanol-
amine-induced vacuolization (P < 0Æ001, Mann–Whitney test
for the 10 mmol L)1 concentration of each amine; Fig. 1b),
but it also slowly reversed the established morphological alter-
(a) (b)
(c)
(d)
Fig 1. Massive vacuolization of rabbit dermal fibroblasts induced by 2-dimethylaminoethanol (DMAE) or triethanolamine [(EtOH)3N]. (a) Phase
contrast observation of cells treated for 4 h with buffered (pH 7Æ4) DMAE or (EtOH)3N introduced into the regular culture medium (original
magnification · 100). (b) Quantification of the vacuolization as a percentage of individual cell-surface pixels above a set threshold following
image treatment (see Materials and methods). Values are means ± SEM of 106–236 determinations. (c) Structure of DMAE and (EtOH)3N.
(d) Bafilomycin A1 (300 nmol L)1) reverses the vacuolized appearance of cells pretreated with DMEA (10 mmol L)1; treatment schedules as
indicated). The control at the left of each data set has received the dimethyl sulphoxide vehicle of bafilomycin A1 for the same period.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp433–439
2-Dimethylaminoethanol-induced vacuolization, G. Morissette et al. 435
ations induced by DMAE (10 mmol L)1; Fig. 1d), suggesting
that V-ATPase is continuously functioning to maintain a pro-
ton gradient that attracts the amine and water in specific
organelles. Cell washout with fresh culture medium also
reversed the vacuolization induced by DMAE (pretreatment for
8 h) in a manner dependent both on time and on DMAE con-
centration (Fig. 2). Thus, the morphological effect of the
amine was not significant at 4 h after washout in cells pre-
treated with 2Æ5 mmol L)1, whereas those exposed to the
higher concentration level (10 mmol L)1) were still vacuolar;
at 16 h post-washout, vacuolization was not significant for the
10 mmol L)1 DMAE concentration (digitized morphological
analysis, Fig. 2b).
Treatment with DMAE (5 or 10 mmol L)1 only) for 24 h
was associated with a significant cytotoxicity that was also pre-
vented by bafilomycin A1 cotreatment (Fig. 3). Treatment with
DMAE for 4 h was not cytotoxic despite extensive vacuolization
at some concentrations (Fig. 3). For comparison, the DNA-
binding antimitotic agent actinomycin D showed no cyto-
toxicity at 4 h, but induced an extensive mortality at 24 h that
was not abated by bafilomycin A1 (Fig. 3). Similar findings
were made using rabbit vascular smooth muscle cells, except
that DMAE-induced cytotoxicity was more extensive (74% of
cells). Cotreatment of smooth muscle cells with bafilomycin
(a)
(b)
Fig 2. Regression of 2-dimethylaminoethanol (DMAE)-induced vacuolization following cell washout with fresh culture medium. Rabbit dermal
fibroblasts were pretreated for 8 h with DMAE or its saline vehicle, then were observed, washed and observed again after further incubation at
37 �C, as indicated. (a) Sample photographic record. (b) Quantification of the vacuolization, as in Figure 1b. Values are means ± SEM of 67–141
determinations.
Fig 3. Cytotoxicity assay applied to dermal fibroblasts maintained in
the presence of 2-dimethylaminoethanol (DMAE) (4 or 24 h): effect
of drugs or drug combinations on fibroblast mortality. The controls
are common for DMAE and actinomycin D (indicated as the ‘zero’
concentration of DMAE). Proportion of nonviable cells established
using 177–474 cells per experimental point.
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436 2-Dimethylaminoethanol-induced vacuolization, G. Morissette et al.
A1 was again very effective in preventing cell death (reduced
to 3%, data not shown).
Organic amines at concentrations that elicit vacuolization,
such as procainamide (2Æ5 mmol L)1) and bafilomycin A1,
exert an antiproliferative effect on vascular smooth muscle.5,6
The same applied to DMAE when used at a noncytotoxic con-
centration (2Æ5 mmol L)1; Fig. 4a). After treatment for 48 h
with the cytotoxic agent actinomycin D, the cell number in
Petri dishes was lower than the number of seeded cells
(50 000), but this was not the case for the noncytotoxic con-
centrations of DMAE or procainamide.
In a previous study, procaine at a concentration that pro-
duced vacuolization inhibited the mitosis of hepatoma cells or
fibroblasts by retarding the late S and G2 phases of the cell
cycle.14 DMAE (2Æ5 mmol L)1) was only transiently active in
this respect in rabbit fibroblasts (Fig. 4b), as the effect of this
amine on ploidy was neutral for treatments longer than 6 h.
Sub-G1 peaks, indicative of apoptosis, were small and uninflu-
enced by DMAE in these experiments (data not shown).
In other cell cultures, bafilomycin A1 treatment (24 h,
300 nmol L)1) had a more sustained effect on fibroblast
ploidy: the proportion of cells in S phase was 55% (33% in
controls); in G1/G0 phase was 41% (54% in controls); and in
G2/M phase was 4% (12Æ8% in controls; n ¼ 2). Under the
same experimental conditions, colchicine exerted a profound
reduction in the proportion of cells in G1/G0 phase with an
increase in the proportion of cells in G2/M phase (Fig. 4c;
a control for cell reactivity).
In vivo 2-dimethylaminoethanol application
Measurements were performed using a photographic record
(paraffin tissue sections, 1000·) of the inner bare skin of the
rabbit external ear treated for 1 h with 3% DMAE or vehicle
(Fig. 5; n ¼ 2). The epidermis was significantly thickened by
DMAE treatment (Fig. 5a), and recognizable cellular structures
located in the suprabasal layers of the epidermis were more
frequent (arrowheads in Fig. 5a). Furthermore, DMAE-treated
skin exhibited numerous examples of clear perinuclear areas
indicative of vacuolization (koilocytosis, arrowheads; statistical
analysis, Fig. 5b) in the epidermal cells as a response to 3%
DMAE. In the affected cells, nuclei appeared somewhat con-
densed and the cell volume was enlarged. The tissues were
otherwise unremarkable (no signs of inflammation).
Effect of 2-dimethylaminoethanol on human epidermal
cells
As the in vivo experiments indicated the epidermis as a major
target for topically administered DMAE, the effect of a
2Æ5 mmol L)1 concentration of the amine was verified in pri-
mary cultures of human skin epithelial cells (4 h treatments,
Fig. 6). DMAE induced intracellular vacuoles of various sizes,
many of which were very small and difficult to photograph.
Bafilomycin A1 cotreatment prevented this morphological
response yet had no effect alone (Fig. 6).
Discussion
Virtually all cultured cell types respond within hours to a
wide array of organic amines by massive cell vacuolization
explained by ion trapping in acidic organelles and subse-
quent osmotic swelling. DMAE and triethanolamine, agents
used in cosmetics, are active in the millimolar concentration
range, as demonstrated with rabbit dermal fibroblasts and
human keratinocytes from the epidermis and hair follicles.
(a)
(b) (c)
Fig 4. (a) Effect of 2-dimethylaminoethanol
(DMAE), actinomycin D (actino D) or
procainamide (PA) on the proliferation of
rabbit dermal fibroblasts. Approximately
50 000 cells were seeded in Petri dishes
3 days before the counts and the drugs were
introduced for the last 48 h. The controls are
common for all drugs (indicated as the ‘zero’
concentration of DMAE). Values are
means ± SEM of triplicate determinations.
Ploidy in rabbit dermal fibroblasts treated
with (b) DMAE (2Æ5 mmol L)1) or
(c) colchicine (pooled results of experiments
conducted with 10 or 100 lmol L)1) for
various time periods. Values are means ± SEM
of three different determinations based on
different primary cell lines.
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2-Dimethylaminoethanol-induced vacuolization, G. Morissette et al. 437
The central role of V-ATPase in this phenomenon, previ-
ously established for triethylamine and procainamide-related
drugs,5,6 also applies to DMAE and triethylamine, which are
more hydrosoluble and exhibit higher concentration thresh-
olds for the morphological response. However, the active
DMAE concentration (2Æ5 mmol L)1) is considerably lower
than that found in topical preparations (as described above),
supporting the relevance of this cellular response. The mild
cytotoxicity of DMAE in fibroblasts might derive from an
extreme vacuolization reaction, perhaps with vacuole rupture,
as both activities are abated by the V-ATPase inhibitor bafilo-
mycin A1. However, the correlation between vacuolization
and cytotoxicity varies for each amine and is clear only for
the less toxic drugs.6 Another common response to amines
that induce vacuolization is mitotic arrest with no persistent
effect on ploidy; it has previously been observed that mitotic
arrest occurs only at a concentration of procainamide that
induces vacuolization, and the same holds true for DMAE
(Fig. 4a). Bafilomycin A1 itself induces a complete mitotic
arrest in cultured smooth muscle cells,6 as well as in several
tumour-derived cell lines in a recent study.15 The latter
report documents that this effect was not apoptotic; it was
parallel to an increase in S and G2/M phases that was not
sustained over a continuous treatment period of 48 h. DMAE
only transiently exhibited this type of effect on ploidy,
although the agent effectively blocked cell division. Thus, the
Golgi-disrupting action of concentrated amine drugs may be
the basis of the mitotic arrest without overt cytotoxicity, as
discussed elsewhere.6
(a)
(b)
Fig 5. Histology of the inner bare skin of the rabbit external ear (paraffin sections). The skin was treated in vivo with either 3% 2-dimethyl-
aminoethanol (DMAE) for 1 h or the oil vehicle. (a) Epidermal thickness. Left: sample photographic record (haematoxylin and eosin; original
magnification · 1000). Arrowheads point to recognizable cellular structures in the suprabasal cell layers; these structures were more common in
the photographic record from DMAE-treated skin. Right: comparison of epidermal thickness in 19 control and 28 DMAE-treated fields. Values are
means ± SEM; *P < 0Æ001 (Student’s t-test). (b) Koilocytosis in the epidermal cells in DMAE-treated tissues. Left: photographic record.
Arrowheads point to koilocytic cells. Right: average proportions of koilocytic cells in the whole photographic record as a function of treatment.
Values are means ± SEM of proportions established by two independent observers. Both sets of data indicate a highly significant effect of DMAE
(P < 0Æ001, v2 test).
Fig 6. Morphological effect of 2-dimethylaminoethanol (DMAE)
(2Æ5 mmol L)1, 4 h) on primary cultures of human cutaneous
epithelial cells (phase contrast, original magnification · 100). The
vacuolization response was prevented by cotreatment with bafilomycin
A1 (300 nmol L)1). DMSO, dimethyl sulphoxide.
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438 2-Dimethylaminoethanol-induced vacuolization, G. Morissette et al.
Rabbit skin has a thin stratum corneum and epidermis, and
the rate of transdermal drug absorption is generally faster than
for human skin.16 Within the limitations of the model, topical
DMAE (3%) was found to increase the epidermal thickness in
1 h in rabbit ears. The relationship between the histological
observations and the cellular cytopathology characterized in
cultured cells is indicated by the increased frequency of appar-
ently vacuolar and dilated cells in the DMAE-treated epidermis.
Thus, it is plausible that a reservoir of concentrated DMAE
applied to the skin elicits cellular expansion through massive
vacuolization for an extended period of time, and this could
lead to the improved appearance of wrinkled skin. It is of
great interest that the application of EMLA cream, a topical
local anaesthetic formulation containing 2Æ5% lidocaine and
2Æ5% prilocaine, has recently been found to increase the thick-
ness of the human epidermis.17 Indeed, these local anaesthet-
ics can be considered as substituted triethylamine derivatives,
several of which induce cell vacuolization effectively; specific-
ally, lidocaine has been shown to be active in this respect
(2Æ5 mmol L)1).5 Thus, epidermal thickening induced by
EMLA cream could be mechanistically similar to the antiwrin-
kle effect of DMAE.
The termination of DMAE action presumably involves sys-
temic absorption following the vanishing of the reservoir
applied to the skin; this was modelled here by cell washout in
in vitro experiments and led to a relatively rapid regression of
vacuolization in fibroblasts. Of note, vacuolization regressed
less rapidly in cells treated with the higher concentration level
of DMAE (10 mmol L)1, Fig. 2), which is considerably lower
than that applied to the skin. An ethanolamine closely related
to DMAE, N-methyldiethanolamine, was sequestered in the skin
and only slowly released into the bloodstream following topical
administration to rats,18 supporting the possibility of a reser-
voir formation by ion trapping for extended periods of time in
the skin cells. To our knowledge, no information is available
on the systemic absorption of DMAE, but animal toxicology
data for triethanolamine suggest efficient transdermal absorp-
tion, low systemic toxicity or carcinogenicity, rapid elimination
in urine and ‘cloudy swelling’ (which may be synonymous
with koilocytosis) in liver and renal tubule cells at high doses.4
Diethanolamine, also commonly used in cosmetic formulations
(1–25%), caused fetal wastage, decreased mitosis and increased
apoptosis in fetal brain tissue when applied to the skin of preg-
nant mice.19 The vacuolar cytopathology induced by concentra-
ted organic amines and its consequences (mitotic arrest,
inhibition of the secretory pathway, minor cytotoxicity) may
not be dissociable from the improvement of the skin appear-
ance that is rapidly produced by topically administered DMAE.
Acknowledgments
We thank Ms Johanne Bouthillier for technical help, Dr Marc
Pouliot (CHUQ-CHUL) for facilitating the access to micro-
scopic equipment and Amelie Lavoie and Dr Danielle Larouche
(LOEX, Hopital du Saint-Sacrement) for help with human
keratinocyte culture. This work was supported by the Cana-
dian Institutes of Health Research (operating grant MOP-
74448, Canada Graduate Scholarships Doctoral Award to
G.M.).
References
1 Perricone N. The Wrinkle Cure: Unlock the Power of Cosmeceuticals for Supple,
Youthful Skin. Emmaus, PA: Rodale Books, 2000.2 Grossman R. The role of dimethylaminoethanol in cosmetic derma-
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tensile effect of 2-dimethylaminoethanol (deanol) gel. Skin ResTechnol 2002; 8:164–7.
4 Knaak JB, Leung HW, Stott WT et al. Toxicology of mono-, di-,and triethanolamine. Rev Environ Contam Toxicol 1997; 149:1–86.
5 Morissette G, Moreau E, C-Gaudreault R, Marceau F. Massive cellvacuolization induced by organic amines such as procainamide.
J Pharmacol Exp Ther 2004; 310:395–406.6 Morissette G, Moreau E, C-Gaudreault R, Marceau F. N-substituted
4-aminobenzamides (procainamide analogs): an assessment ofmultiple cellular effects concerning ion trapping. Mol Pharmacol
2005; 68:1576–89.7 Kim T, Holley GP, Lee JH et al. The effects of intraocular lidocaine
on the corneal endothelium. Ophthalmology 1988; 105:125–30.8 Atilla H, Tekeli O, Can B et al. Effects of intracameral lidocaine on
ocular tissues. Clin Experiment Ophthalmol 2003; 31:73–7.9 Michalik M, Pierzchalska M, Pabianczyk-Kulka A et al. Procaine-
induced enhancement of fluid-phase endocytosis and inhibition ofexocytosis in human skin fibroblasts. Eur J Pharmacol 2003; 475:1–
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CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07581.x
Expression of p16, CD95, CD95L and Helix pomatiaagglutinin in relapsing and nonrelapsing very thinmelanomaL.A. Fearfield,*�� J.M.G. Larkin,� A. Rowe,* R. A’Hern,� C. Fisher,� N. Francis,§ R. MacKie,– B. McCann,**M.E. Gore� and C.B. Bunker*�
*Department of Dermatology, Imperial College School of Medicine (START Laboratories), Chelsea and Westminster Hospital, London, U.K.
�Department of Dermatology, Royal Berkshire Hospital, London Road, Reading RG1 5AN, U.K.
�Royal Marsden Hospital, Fulham Road, London, U.K.
§Department of Histopathology, Charing Cross Hospital, London, U.K.
–Department of Dermatology, University of Glasgow, Glasgow, U.K.
**Department of Histopathology, Norfolk and Norwich Hospital, Colney Lane, Norwich, U.K.
CorrespondenceLonise A. Fearfield.
E-mail: louise.fearfield@royalberkshire.nhs.uk
Accepted for publication14 July 2006
Key wordsbiological markers, prognosis, skin neoplasms
Conflicts of interestNone declared.
Summary
Background The incidence of malignant melanoma is increasing worldwide andpatients are being diagnosed earlier with thinner primary lesions. Most patientswith very thin melanoma (Breslow thickness < 0Æ76 mm) are cured by surgerybut 2–18% relapse locally or with distant metastases.Objectives The objective of this study was to establish potential new prognosticmarkers in very thin melanoma.Methods We identified a group of subjects with relapsing very thin primary cuta-neous melanoma and a matched control group who had not relapsed. We inves-tigated the expression of p16, Helix pomatia agglutinin (HPA), CD95 and CD95ligand (CD95L) by immunohistochemistry on paraffin-embedded tissue sectionsfrom the subject group, their subsequent metastases and the control group.Results Reduced p16 expression was significantly associated with relapse in verythin melanoma (P ¼ 0Æ0129). Loss of p16 expression was also found in 76% ofmetastases. There was no significant association between HPA, CD95 or CD95Lexpression and subsequent relapse.Conclusions This work is the first to show a significant loss of p16 in relapsing verythin melanoma.
Melanoma has been increasing in incidence in caucasian popu-
lations around the world.1 This has been attributed by some
authors to an increased number of thin tumours.2,3 Although
the prognosis for patients presenting with very thin melanoma
(Breslow thickness < 0Æ76 mm) is good with an estimated 5-
year survival of 93Æ2%,4 no reliable prognostic markers exist
to identify those patients at increased risk of relapse. Identifi-
cation of such patients would allow close clinical follow-up
and consideration of entry into trials of adjuvant therapy.
Mutations of the p16 tumour suppressor gene [also called the
cyclin-dependent kinase inhibitor-2A (CDKN2A), p16INK4A or
MTS1 gene] have been identified in many different tumours
including melanoma, pancreatic adenocarcinoma, oesophageal
carcinoma and various haematological malignancies. The gene
has been mapped to human chromosome region 9p215 and
encodes the 40-kDa p16 protein. The p16 protein is a kinase
inhibitor that blocks the CDK4 and CDK6 kinases. These kinases
phosphorylate the retinoblastoma protein (pRb) allowing pro-
gression through the G1–S cell-cycle checkpoint. Loss of p16
consequently allows cells to escape cell-cycle arrest in the G1
phase. Immunohistochemical studies have generally shown a
correlation between reduced p16 protein expression and the
progression of melanoma6–9 although this association has not
been investigated specifically in very thin melanoma.
The CD95/CD95 ligand (CD95L) (or FAS/FAS ligand) system
is involved in various immune functions10–12 including cyto-
toxic T-cell-mediated apoptosis.13,14 A number of solid tumours
express CD95L15 and hepatocellular tumour cells expressing
CD95L are able to kill infiltrating T lymphocytes, thereby
evading immune attack.16 Furthermore, CD95L expressed by
melanoma cell lines is functionally active against FAS-sensitive
A20 B lymphoma cells.17 A plausible association between the
expression of CD95L and tumourigenicity in melanoma there-
fore exists and this is generally supported by immunohisto-
chemical studies on melanoma samples from patients.18,19
However, only small numbers of very thin melanoma are
� 2006 The Authors
440 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp440–447
reported in the literature and differences in methodology and
patient populations make comparison between published stud-
ies difficult.
Although the identity of the ligand for the Roman snail Helix
pomatia agglutinin (HPA) lectin in malignant cells is unknown,
binding of the lectin to tissue sections from breast, colon and
stomach carcinomas has been associated with a poor progno-
sis.20–23 Few studies have looked at the expression and bio-
logical significance of HPA binding in melanoma although it
has been reported that the extent of HPA lectin binding by
three human melanoma cell lines is related to their potential
for metastasis formation in an animal model,24 and HPA bind-
ing to primary melanoma sections has been reported as a mar-
ker for the risk of relapse in a clinical study.25 However, no
published studies have investigated the relationship between
HPA binding and risk of relapse in very thin melanoma.
In this study we have characterized a group of patients with
primary cutaneous relapsing very thin melanoma (Breslow
thickness < 0Æ76 mm) and a matched control group who have
not relapsed. We have investigated the expression of the po-
tential prognostic markers p16, CD95, CD95L and HPA by
immunohistochemistry on paraffin-embedded tissue sections
from the subject group, their subsequent metastases and the
control group and correlated marker expression with clinical
outcome.
Materials and methods
Subject recruitment
Ethical approval for the study was obtained both locally from
the Royal Marsden Hospital (RMH) Research Ethics Committee
and from the North Thames Multicentre Research Ethics Com-
mittee (MREC). Subjects (alive and deceased) with relapsing
very thin melanomas (Breslow thickness < 0Æ76 mm) were
identified from: (i) a clinical database at RMH spanning 1990–
1998; (ii) a histopathology database at RMH spanning January
1980 to July 1999; and (iii) via clinicians in the U.K. Melanoma
Study Group. The sample size was determined by the number of
subjects available. Patients with more than one primary cuta-
neous melanoma were excluded. Forty-five subjects with relap-
sing very thin primary cutaneous melanoma were initially
identified. Twenty-five of these subjects were excluded from the
data set after clinicopathological review due to insufficient bi-
opsy of a large melanoma (n ¼ 2), histologically unconfirmed
metastatic disease (n ¼ 1), vulval melanoma (n ¼ 1), thick
melanomas erroneously entered into the database as thin (n ¼2), original tissue blocks not available (n ¼ 7) and Breslow
thickness > 0Æ76 mm on review of original histological material
(n ¼ 12). Controls were identified as patients with very thin
melanomas that had not relapsed after at least 5 years of follow-
up from the RMH, Charing Cross and Chelsea and Westminster
Hospitals histopathology databases and the U.K. Melanoma
Study Group. Controls were selected to match individually the
subjects in terms of age, sex, melanoma primary site and hist-
ology. Forty-eight controls were initially identified. Fifteen
controls were subsequently excluded after review due to inad-
equate initial biopsy (n ¼ 1), death from other causes within
5 years of diagnosis of primary melanoma (n ¼ 3), follow-up
information not available (n ¼ 5), more than one primary
melanoma (n ¼ 2), no primary tissue available for re-assess-
ment (n ¼ 2) and Breslow thickness > 0Æ76 mm (n ¼ 2). Ori-
ginal paraffin-embedded blocks of primary tumours and
metastases were obtained from participating institutions.
Immunohistochemistry
Standard methods were used to prepare 3-lm sections. Heat-
mediated antigen retrieval by pressure cooker or water bath
method was performed prior to staining for CD95L and p16
using antigen unmasking solution (Vector Laboratories, Bur-
lingame, CA, U.S.A.). Proteolytic pretreatment with porcine
trypsin (1 mg mL)1; Sigma-Aldrich Chemical Co, Poole, U.K.)
for 20 min at 37 �C was performed prior to staining with
HPA. The following primary antibodies were incubated over-
night at 4 �C: mouse monoclonal IgG2a antihuman p16; rabbit
polyclonal IgG antihuman CD95L and mouse monoclonal IgG1
antihuman CD95 (all Santa Cruz Biotechnology, Santa Cruz,
CA, U.S.A.). HPA (Sigma-Aldrich) was used at 10 lg mL)1
followed by antibiotin monoclonal mouse antibody (Vector
Laboratories), both for 1 h at room temperature. Antibiotin
monoclonal mouse (biotin–HPA), anti-p16 and anti-CD95
antibodies were detected with the mouse IgG Vectastain� and
CD95L with the rabbit IgG Vectastain� ABC alkaline phospha-
tase kits and Vector Red alkaline phosphatase solution was
used as the cromophore (all Vector Laboratories).
Analysis of immunostaining
Immunostaining of sections was measured in three ways:
immune reactivity (p16, CD95 and CD95L), percentage of
positive cells or nuclei (HPA and p16, respectively) and stain-
ing index (p16). For the assessment of immune reactivity, at
least two nonadjacent sections were used for each antibody
from both primary tumours and metastases. Staining intensity
of primary tumours from subjects and controls was scored
‘blinded’ and metastases ‘unblinded’ by two investigators
(L.A.F. and A.R.). Tumours were scored according to the in-
tensity of stain compared with the normal adjacent epidermis,
sebaceous and eccrine glands (Table 1). Intensity was scored
throughout a section and summed to produce an overall score,
Table 1 Quantitation of immune reactivity
GradeIntensity of staining compared with the normal adjacentepidermis and/or sebaceous glands
0 Weak: absent to less than a quarter of the intensity
1 Mild: quarter to less than half the intensity2 Moderate: half to less than three-quarters of the intensity
3 Strong: three-quarters to less than equal to the intensity4 Very strong: equal or more intense
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp440–447
p16, HPA and CD95/CD95L in very thin melanoma, L.A. Fearfield et al. 441
i.e. 10% of a nonuniformly stained tumour might be graded
0, 70% graded 2 and 20% graded 4 for an overall score of
2Æ2 [(10/100 · 0) + (70/100 · 2) + (20/100 · 4)]. For
the assessment of percentage of positive cells or nuclei, 5–10
high power fields (150 · 150 lm) were selected randomly
and scored throughout each representative section for the
number of HPA-positive cells and the number of p16-positive
nuclei. A staining index, combining immune reactivity and
percentage of positive cells, adapted from Straume et al.,9 was
calculated as follows: staining index ¼ staining intensity
grade · percentage of positive cells grade (Table 2).
Statistical methods
Nonparametric tests were used to compare continuous variables
due to the non-normality of the data. The Mann–Whitney U-test
was used to compare the potential markers in subjects at base-
line and controls, and the Wilcoxon signed-rank sum test was
used to compare the paired values in subjects at baseline with
their metastases. Median differences and their 95% confidence
intervals (CIs) were calculated. Analyses were carried out using
statistical programmes written in Microsoft Excel.
Results
Forty-five subjects with relapsing very thin primary cutaneous
melanoma (Breslow thickness < 0Æ76 mm) and 48 controls
with nonrelapsing very thin primary cutaneous melanoma were
initially identified. Twenty-five subjects and 15 controls were
excluded from the data set after clinicopathological review (see
Materials and methods). The clinicopathological features of
patient and control groups included in the study are summar-
ized in Table 3. The only statistically significant difference
(Mann–Whitney U-test) between the subjects and controls with
regard to the clinicopathological parameters was the mitotic
rate: significantly more controls had mitoses of < 1 mm)2
(P < 0Æ01). Sixty-four percent of controls were Clark level II or
less compared with 40% of subjects but this was not significant.
Exclusions of subjects and controls after review therefore did
not result in notable differences between the two groups and
Table 3 demonstrates the comparability of the two groups.
p16
Immunostaining for p16 was performed on sections from 18
subjects, their 17 subsequent metastases and 28 controls (Fig. 1). Nuclear staining in sebaceous and eccrine glands was
used as a positive internal control as in other studies.9 Three
indices were used to quantitate immunostaining: immune re-
activity, percentage of p16-positive cells and staining index
(Table 4).
Subjects vs. controls
Median immune reactivity scores were 1Æ9 [interquartile range
(IQR) 1Æ3–2Æ1] for subjects and 2Æ4 (IQR 2Æ1–3Æ0) for controls
(median difference )0Æ7, 95% CI )1Æ2 to )0Æ2, P ¼ 0Æ01).
Table 2 Calculation of staining index
Percentage of
positive cells
Percentage of
positive cells grade
Immune
reactivity
Staining
intensity grade
0 0 0–1 0< 10% 1 > 1–2 1
10–50% 2 > 2–3 2> 50% 3 > 3–4 3
Table 3 Summary of clinicopathological characteristics of subjects andcontrols
Subjects Controls
Total number 20 33Sex, female, n (%) 13 (65) 22 (67)
Age, years, median (range) 47 (23–63) 44 (22–84)Site, n (%)
Head and neck 1 (5) 1 (3)Arm 4 (20) 8 (24Æ2)
Leg 7 (35) 12 (36Æ4)Trunk 8 (40) 12 (36Æ4)
Breslow mm, median (range) 0Æ55 (0Æ2–0Æ7) 0Æ5 (0Æ2–0Æ7)Clark level, n (%)
I 1 (5) 0II 7 (35) 21 (64)
III 12 (60) 8 (24)IV 0 4 (12)
V 0 0
VGP, n (%) 16 (80) 26 (79)Regression, n (%) 11 (55) 16 (48)
Mitoses per mm2, n (%)0 7 (35) 6 (18)
< 1a 9 (45) 27 (82)2 4 (20) 0
Tumour type, n (%)Superficial spreading 17 (85) 32 (97)
Lentigo 1 (5) 1 (3)Nodular 1 (5) 0
Unclassified 1 (5) 0TIL, n (%)
No 6 (30) 10 (30Æ3)Mild 7 (35) 12 (36Æ4)
Moderate 0 7 (21Æ2)Brisk 7 (35) 4 (12Æ1)
aP ¼ 0Æ004 for the subject vs. the control group. TIL, tumour-
infiltrating lymphocytes; VGP (vertical growth phase), the focalappearance within the radial growth phase of a new cell popula-
tion growing as an expanding spheroidal nodule with metastaticpotential; regression, the complete or partial disappearance of
the primary lesion spontaneously, heralded by TILs, melanin-containing macrophages, vascular ectasia and at end stage, fibro-
sis and scarring; brisk, TIL noted throughout the substance ofthe VGP or infiltrating across the entire base of the VGP; non-
brisk, TIL present in one or more foci of the VGP; absent,lymphocytes present but not infiltrating the melanoma or any
part of the VGP.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp440–447
442 p16, HPA and CD95/CD95L in very thin melanoma, L.A. Fearfield et al.
The median percentage of p16-positive cells was 28Æ8% (IQR
16–35Æ3%) for subjects and 35Æ5% (IQR 32–48%) for controls
(median difference )11Æ5%, 95% CI )20% to )1Æ5%, P ¼
0Æ02) and median staining index was 2 (IQR 2–4) for subjects
and 4 (IQR 2–6) for controls (median difference )2, 95% CI
)2 to 0, P ¼ 0Æ03).
Fig 1. Representative sections (· 250) stained for p16, CD95, CD95 ligand (CD95L) and Helix pomatia agglutinin (HPA) to demonstrate mild/
moderate [(a) p16, (c) CD95, (e) CD95L and (g) HPA] and moderate/intense staining [(b) p16, (d) CD95, (f) CD95L and (h) HPA].
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp440–447
p16, HPA and CD95/CD95L in very thin melanoma, L.A. Fearfield et al. 443
Subjects vs. metastases
The median immune reactivity score for metastases was 0Æ8(IQR 0–1Æ4, median difference –0Æ9, 95% CI )1Æ3 to )0Æ6,
P < 0Æ01 vs. subjects). The median percentage of p16-positive
cells was 0Æ5% for metastases (IQR 0–7Æ5%, median difference
)23Æ2%, 95% CI )30Æ3% to )13%, P < 0Æ001 for subjects vs.
metastases). Median staining index was 0 for metastases (IQR
0–2, median difference )2, 95% CI )2 to )2, P < 0Æ001 for
subjects vs. metastases). Furthermore, 22% of primary mela-
noma sections had a staining index of zero in comparison
with 76% of metastases.
These results demonstrate, in all scoring systems, a signifi-
cant reduction in p16 staining in very thin melanoma in sub-
jects who subsequently relapsed in comparison with controls
who had not relapsed. Further significant loss of p16 staining
was seen in metastases in comparison with the corresponding
primary tumours.
CD95 and CD95L
Tissue blocks were available for sectioning in 18 subjects,
their 17 subsequent metastases and 32 controls for CD95 and
in 18 subjects, 17 subsequent metastases and 30 controls for
CD95L. Due to diffuse immunostaining it was not possible to
count individual cells positive for CD95 and CD95L and so
only immune reactivity was measured; normal epidermis and
adnexal structures adjacent to tumours were used as internal
controls (Fig. 1, Table 5). For CD95 staining, median immune
reactivity score was 1Æ1 (IQR 1Æ0–1Æ6) for subjects, 1Æ5 (IQR
1Æ0–1Æ8) for controls and 1Æ5 (IQR 0Æ7–2Æ3) for metastases
(P not significant for all comparisons). For CD95L staining,
median immune reactivity score was 2Æ3 (IQR 1Æ8–2Æ6) for
subjects, 1Æ8 (IQR 1Æ4–2Æ3) for controls and 1Æ9 (IQR 1Æ6–2Æ7)
for metastases (P not significant for all comparisons). These
results show no significant association between staining for
CD95 or CD95L in very thin melanoma and the likelihood of
subsequent relapse and no significant difference in CD95 or
CD95L expression between primary very thin melanomas and
their subsequent metastases.
Helix pomatia agglutinin
HPA immunostaining was performed on sections from 15
subjects, their 15 metastases and 27 controls. The intensity of
immune reactivity was uniform throughout the sections and
therefore only the percentage of positive cells stained per high
power field was recorded (Fig. 1, Table 5). The median per-
centage of positive cells per high power field was 40 (IQR
33–50) for subjects, 27 (IQR 21–43) for controls and 21
(IQR 0–68) for metastases. The median difference in percent-
age of positive cells per high power field was 10% for subjects
vs. controls (95% CI )3Æ5 to 20, P ¼ 0Æ10) and )17Æ3% for
subjects vs. metastases (95% CI )24 to 5Æ5, P not significant).
These results demonstrate no significant association between
staining for HPA in very thin melanoma and the likelihood of
subsequent relapse and no significant difference in HPA
expression between primary very thin melanomas and their
subsequent metastases.
Discussion
In this study we have shown that the expression of p16 is sig-
nificantly reduced in relapsing very thin melanoma compared
with nonrelapsing controls and in relapsing primary mela-
noma compared with subsequent metastases in terms of the
immune reactivity score, percentage of p16-positive nuclei
and the combined staining index. This is the first study to spe-
cifically analyse various immunohistochemical markers in this
important although rare group of patients that at present have
no other significant prognostic indicators available to them
except Breslow thickness. However, the limitations of this
study include the small number of subjects and the fact that
the analysis was retrospective although the databases were
prospectively maintained. As the study is small, even quite
large differences between subjects and controls will not reach
statistical significance. Relapsing very thin melanoma is rela-
tively rare and as a result small numbers are inevitable; how-
ever, the number of subjects reported here is comparable to
other similar published studies.26–29
The results reported here are similar to other studies show-
ing that p16 expression is frequently reduced in relapsing
sporadic melanoma. For example, in a series of 102 primary
invasive cutaneous melanomas, the 5-year survival rate for
Table 4 Summary of p16 staining scores in subjects, controls andmetastases; medians and interquartile ranges are shown
p16 Subjects Controls Metastases
Immune reactivity 1Æ9 (1Æ3–2Æ1) 2Æ4 (2Æ1–3Æ0)a 0Æ8 (0–1Æ4)b
Percentage
positive cells
28Æ8 (16–35Æ3) 35Æ5 (32–48)c 0Æ5 (0–7Æ5)d
Staining index 2 (2–4) 4 (2–6)e 0 (0–2)f
aP ¼ 0Æ01 for subjects vs. controls. bP < 0Æ01 for subjects vs.
metastases. cP ¼ 0Æ02 for subjects vs. controls. dP < 0Æ001 forsubjects vs. metastases. eP ¼ 0Æ03 for subjects vs. controls.fP < 0Æ001 for subjects vs. metastases.
Table 5 Summary of CD95, CD95L and HPA staining scores in
subjects, controls and metastases; medians and interquartile ranges areshown
Subjects Controls Metastases
CD95: immunereactivity
1Æ1 (1Æ0–1Æ6) 1Æ5 (1Æ0–1Æ8) 1Æ5 (0Æ7–2Æ3)
CD95L: immunereactivity
2Æ3 (1Æ8–2Æ6) 1Æ8 (1Æ4–2Æ3) 1Æ9 (1Æ6–2Æ7)
HPA: % positivecells
40 (33–50) 27 (21–43) 21 (0–68)
P not significant for comparisons between subjects, controls andmetastases for all scores.
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp440–447
444 p16, HPA and CD95/CD95L in very thin melanoma, L.A. Fearfield et al.
patients with a significant reduction or loss of p16 staining
was zero compared with a 5-year survival rate of 67% in
those patients with moderate or strong p16 staining.7 In a fur-
ther series of 202 vertical growth phase primary melanomas
and 68 corresponding metastases, absent or minimal nuclear
staining significantly predicted poor patient survival with 37%
and 67% estimated 10-year survival rates for subjects with
absent or present p16 expression, respectively.9 However, our
report is the first to show that loss of p16 expression is a poor
prognostic factor in very thin melanoma.
The loss of p16 expression in melanoma metastases that we
report has been observed previously. In our study, 76% of
metastases had a staining index score of 0 for p16. A similar
proportion of metastases (77%) with a staining index of 0–1
has been reported in one previous study9 and a further report
demonstrated absent p16 expression in 44% of metastatic
melanoma lesions compared with 9% of primary invasive
melanomas.6 Loss of p16 in metastases in comparison with
primary tumours suggests that subclones of p16-deficient
tumour cells may be more likely to escape from the primary
tumour and to survive and proliferate at distant sites.
A role for p16 mutations in familial melanoma is well
recognized;30 however, its role in sporadic melanoma is less
clear. Mutations in the p16 gene have been identified in up to
33% of sporadic melanomas31–38 and therefore it is likely to
be an important factor in a proportion of sporadic melanomas.
A higher frequency of mutations is found in melanoma cell
lines. A number of other mechanisms of p16 inactivation such
as large heterozygous and homozygous deletions and silencing
by methylation of CpG islands in the promoter region have
been characterized. Their frequency and role in pathogenesis
and progression of melanoma are still unclear.
No significant differences between subjects, controls or
metastases were found in the immune reactivity scores for
CD95 or CD95L. However, the immune reactivity scores for
CD95 were generally reduced in all subjects and we have also
demonstrated that CD95L is expressed in most subjects and
controls with very thin melanoma. A previous study39 found
CD95L expression in only two of 12 very thin melanomas; a
further study reported the staining of frozen sections from pri-
mary melanomas (Breslow thickness 1Æ8–8Æ6 mm) and subse-
quent cutaneous metastases, demonstrating CD95L expression
in only 10% of primaries and 55% of the cutaneous metasta-
ses.19 Maeda et al.18 performed staining on paraffin-embedded
tissue from various benign melanocytic lesions, melanoma in
situ, stage I melanoma (Clark level 2 or 3), advanced mela-
noma (Clark level 4 or 5) and lymph node metastases. All
advanced melanomas and lymph node metastases of cutaneous
origin showed CD95L expression but melanoma in situ, six
very thin melanomas, benign or dysplastic naevi did not. This
latter discrepancy may be explained, in part, by the different
scoring methods used, as tumours were scored positive in the
Maeda et al. study only if the intensity of the CD95L signal
was greater than that of keratinocytes. In contrast, we have
compared signal intensity between tumour and epidermis,
sebaceous and eccrine glands. However, Sprecher et al.40 did
find CD95L expression in normal melanocytic naevi, Spitz
naevi and melanomas—including very thin lesions.
Three ideas have been advanced to explain how alterations
in the CD95/CD95L system may account for the escape from
immune surveillance by melanoma cells, which leads to diffi-
culties in interpretation of CD95 and CD95L immune staining
in melanoma. Experimental data interpreted to support all
three hypotheses have been published. The first such idea is
that CD95 may be downregulated and CD95L upregulated
on melanoma cells.41,42 The consequence of this would be
the induction of apoptosis in incoming CD95-expressing T
lymphocytes. The two other hypotheses that have been
suggested are: first, that CD95L may be downregulated, CD95
upregulated or normally expressed and that melanoma cells
may survive by escaping autocrine suicide/apoptosis;43,44 and
second, that expression of CD95 and CD95L may be normal
but the receptor may be nonfunctional or be blocked, for
example by soluble CD95, again resulting in escape from
apoptosis.45–47
We have shown that HPA binding is higher in relapsing
very thin melanomas than in controls, although the difference
is not statistically significant. This result is compatible with a
study24 that demonstrated that the extent of lectin binding by
three human melanoma (LOX, FEMX-1 and SESX) cell lines
was related to their ability to form metastases in nude mice.
Furthermore, Thies et al.25 reported a series of 100 patients
with cutaneous malignant melanoma followed up for at least
10 years. Tumour sections were stained with several lectins in-
cluding HPA. Kaplan–Meier analysis of time to first metastasis
showed a positive correlation between HPA binding and meta-
stasis formation and multivariate analysis demonstrated that
HPA binding to primary melanoma was an independent mar-
ker for the risk of metastasis. Small numbers and methodolo-
gical differences in the current study may in part account for
the lack of a significant association. For example, we used bi-
otinylated HPA as per the method of Schumacher et al.22
whereas Thies et al.25 used a native unconjugated HPA; differ-
ences in the fixing and processing of samples within our study
may also have caused variation in HPA binding between
samples (see Schumacher et al.48).
Our results support the hypothesis that expression of N-ace-
tyl-galactosamine (GalNAc)/-glucosamine (GlcNAc) residues
on the cell surface of malignant cells, as detected by HPA
binding, are involved in the process of metastatic spread.22,25
As the expression of HPA-binding glycoconjugates by primary
tumours is associated with metastatic potential, the metastases
themselves might also be expected to be invariably positive
for HPA binding. Investigations of HPA binding to metastases
have clearly demonstrated that this is not the case; for exam-
ple, about 20% of breast cancer metastases are negative for
HPA binding.49,50 Although the specific HPA-binding glyco-
conjugate associated with metastatic behaviour has yet to be
elucidated, some explanations have been advanced to explain
the increased expression of HPA-binding saccharides in malig-
nant cells. One possibility is the neo-expression of GalNAc/-
GlcNAc binding sites.25 Another possibility is that terminal
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp440–447
p16, HPA and CD95/CD95L in very thin melanoma, L.A. Fearfield et al. 445
carbohydrate residues are lost in malignant cells with the con-
sequent unmasking of subterminal HPA-binding regions.51
In conclusion, we have shown that there is no significant
association between HPA, CD95 or CD95L expression and
subsequent relapse although reduced p16 expression is shown
for the first time to be significantly associated with relapse in
very thin melanoma.
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CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07604.x
Inactivation of the CDKN2A and the p53 tumour suppressorgenes in external genital carcinomas and their precursorsN. Soufir, S. Queille,* M. Liboutet,* O. Thibaudeau,* F. Bachelier,� G. Delestaing,* B.C. Balloy,� J. Breuer,§A. Janin,� L. Dubertret,� C. Vilmer� and N. Basset-Seguin*�
Laboratoire de Biochimie Hormonale et Genetique, Hopital Bichat Claude Bernard, 46 rue Henri Huchard, 75018 Paris, France
*INSERM U532, �Department of Dermatology and �Department of Pathology, Hopital Saint-Louis, Paris, France
§Department of Medical Microbiology, St Barts and the Royal London Hospital Medical School, London, U.K.
CorrespondenceNadem Soufir.
E-mail: nsoufir@yahoo.com
Accepted for publication25 May 2006
Key wordsCDKN2A, genital carcinoma, human
papillomavirus, p53
Conflicts of interestNone declared.
C.V. and N.B-S. have codirected the work.
Summary
Background p53 has been extensively studied in external genital carcinoma (EGC),and is frequently inactivated, but little is known about the role of the CDKN2Atumour suppressor gene in the oncogenesis of EGC.Objectives To investigate the role of CDKN2A and p53 in the pathogenesis of EGCsand their precursor lesions vulval intraepithelial neoplasia (VIN3), penile intra-epithelial neoplasia and lichen sclerosus (LS).Methods By means of CDKN2A and p53 mutation screening (single-strand conform-ational polymorphism analysis and sequencing), methylation analysis of alternat-ive CDKN2A promoters (methylation-specific polymerase chain reaction) and p53immununochemistry, we analysed eight invasive EGCs (five from vulva and threefrom penis) and 25 precancerous lesions (two undifferentiated VIN3 and 23 vul-val/penile lesions of LS) from 33 patients.Results p53 mutations (mainly transversions) and CDKN2A mutations (includingone hot spot) were present in 75% and 50% of invasive tumours, respectively,but were absent in all precancerous lesions. Remarkably, all CDKN2A-mutatedtumours also harboured a p53 mutation. CDKN2A or p53 mutations wereobserved more frequently in LS-derived EGCs than in human papillomavirus-derived EGCs (P ¼ 0Æ053). A positive anti-p53 staining, but without p53 muta-tions, was also detected in 30% of LS lesions, suggesting a p53 stabilization inresponse to inflammation and carcinogenic insult. Methylation of p16INK4a andp14ARF promoters was not a frequent mechanism of CDKN2A inactivation.Conclusions Our study shows a high prevalence of co-inactivating mutations of p53and/or CDKN2A genes in EGC, that seem to occur preferentially in LS-derivedtumours and late in oncogenesis.
Squamous cell carcinomas (SCCs) of the external genital
region (vulva and penis) [mucous external genital carcinoma
(EGC)] and their precursor lesions can be separated into two
main subtypes that differ in terms of pathological and clinical
presentations.
Human papillomavirus (HPV)-associated carcinomas are of
basaloid or warty type, whereas tumours unrelated to HPV are
usually keratinizing and differentiated, usually associated with
lichen sclerosus (LS).1,2 Precancerous high-grade external
genital lesions are defined as vulval or penile intraepithelial
neoplasia (VIN3 or PIN3) and can also be classified as
HPV-dependent (undifferentiated VIN3 or PIN3) or HPV-
independent lesions (differentiated VIN3 or PIN3, mainly
associated with LS lesions).3,4
The role of the p53 gene has been extensively studied in
EGC.5–12 In EGC associated with oncogenic HPV infection,
inactivation of the p53 protein occurs through binding with
HPV E6 protein and subsequent degradation. In HPV-negative
carcinomas, mainly developed in the context of genital LS le-
sions,13 p53 is frequently inactivated by somatic mutations.7
Degradation of the p53 protein by viral oncoprotein leads to
the absence of p53 protein detection in HPV-positive EGC,
whereas somatic mutations of the p53 gene are often associ-
ated with increased stability of the mutant inactive p53 pro-
tein in HPV-negative EGC.
In contrast, little is known on the role of the CDKN2A gene
in EGC oncogenesis. CDKN2A encodes two tumour suppressor
proteins p16INK4a and p14ARF, that control cell growth
� 2006 The Authors
448 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp448–453
through the Rb-CDK4 and p53 pathways, respectively,14 and
is frequently inactivated in human cancer.15 Inactivation of
CDKN2A can occur by deletion, point mutation or methylation
of the two CpG-rich promoter regions.
CDKN2A is an important regulator of the proliferation of
both normal and tumorigenic squamous epithelial cells.16
p16INK4a accumulates in normal human keratinocytes under-
going replicative senescence17 and is inactivated during kera-
tinocyte immortalization,18 and p14ARF is also implicated in
cellular senescence19 and immortalization.20,21
To our knowledge, only few studies have been performed
on the role of CDKN2A in EGCs, and none has compared this
with the status of p53. In this work, we have analysed in a
series of EGCs and precancerous lesions the status of CDKN2A
and that of p53. The importance of the two main pathways
involved in the control of cell proliferation in the transform-
ation of external genital lesions is discussed.
Materials and methods
Patients and DNA extraction
Thirty-three skin specimens were collected from 33 patients in
an outpatient department dedicated to genital diseases. All pre-
senting patients with HPV, benign LS lesions or malignant
tumours were asked to participate in the study with their
informed consent (Dermatology Department, Hopital Saint-
Louis, Paris, France). Skin samples included eight invasive
SCCs of the external genital mucosa (five from vulva and three
from penis), two undifferentiated VIN3 and 23 vulval or
penile LS. Invasive SCC was defined as an invasive proliferation
made of sheets of atypical, sometimes dyskeratotic, squamous
cells, with a variable number of concentric layers and kerati-
nizing pearls. Undifferentiated in situ neoplasia (VIN3 or PIN3)
was defined as full-thickness atypia with disordered matur-
ation and atypical mitosis throughout the epithelium. LS was
characterized by a band of hyalinized collagen in the upper
dermis with a variable lymphocytic infiltrate. Genital carci-
noma was histologically and clinically defined as HPV-related
when associated with lesions of undifferentiated VIN3 or PIN3
and as LS-derived when associated with typical LS lesions.
Tissues were divided into two parts. One part was snap fro-
zen in liquid nitrogen and stored at )80 �C. The other part
was fixed in formalin, embedded in paraffin, and subjected to
histopathological assessment performed by the same patholo-
gist. DNA was prepared from frozen samples as previously de-
scribed.22
Immunohistochemistry
Skin samples fixed in 10% formalin were deparaffinized, and
stained after antigen retrieval in a microwave oven at 450 W
for 15 min in Tris buffer (pH 7Æ3) with monoclonal antibody
D-07 (code M7001; Dako, Glostrup, Denmark), which reacts
with both wild-type and mutant forms of the human p53 pro-
tein, at a dilution of 1 : 50, and revealed by avidin–biotin-
coupled immunoperoxidase staining and diaminobenzidine. A
p53-mutated tumour was always added as a positive control.
Staining was nuclear, and was negative when omitting the pri-
mary antibody. Mayer’s haematoxylin was used for counter-
staining. p53 positivity was evaluated by the percentage of
p53 nuclear staining on two independent microscopic fields
(magnification ·200) including at least 100 cells. Slides were
independently examined by two pathologists.
Single-strand conformational polymorphism analysis and
sequencing
The coding sequence and flanking intronic sequences of exon
1a, exon 1b and exon 2 of the CDKN2A gene were analysed
by polymerase chain reaction (PCR)–single-strand conforma-
tional polymorphism analysis as previously described. Primer
sequences for exons 1a and 2 were previously published.23,24
Exon 1b was analysed through two overlapping PCR products
generated by using the following primers: P14F1 5¢-TCAGGG-
AAGGGCGGGTGCG-3¢, P14R1 5¢-GCCGCGGGATGTGAACCA-3¢(PCR product 245 bp), P14F2 5¢-GCCGCGAGTGAGGGTTTT-
3¢, P14R2 5¢-CACCGCGGTTATCTCCTC-3¢ (PCR product
257 bp). Exons 4–9 of the p53 gene were amplified using the
same conditions as previously described.23 Products from 50-
lL PCR reactions from reamplified shifted bands and tumoral
genomic DNAs were purified using an Amicon Microcon 100
(Millipore, Beverly, MA, U.S.A.) and sequenced using a dye-
labelled terminator on an automated sequencer 310 (Applied
Biosystems, Foster City, CA, U.S.A.) according to the manufac-
turer’s instructions.
p16INK4a and p14
ARF promoter methylation analysis
Methylation analysis of p16INK4a and p14ARF promoters was
performed as methylation-specific PCR assay (MSP).25 In brief,
DNA modification by bisulphite converts exclusively unmeth-
ylated cytosines to uracil. Subsequent PCR amplification with
primers specific for unmethylated vs. methylated DNA reveals
the methylation status of investigated DNA sections.
Bisulphite-modified DNA was purified using a commercially
available PCR purification kit according to the manufacturer’s
recommendations (Qiagen, Valencia, CA, U.S.A.). Modified and
purified DNA was precipitated by ethanol for 12 h, resuspended
in 100 lL of water and used immediately or stored at )20 �C.
Primer pairs for PCR amplification are given in Table 1 and
were purchased from MWG-Biotech (Courtaboeuf, France).
Amplification was performed for 35 cycles (denaturing
95 �C/5 min, annealing 60 �C/90 s, extension 72 �C/60 s)
and was concluded by a final 8-min extension at 72 �C. A
positive control using universally methylated (or unmethylat-
ed) DNA was added for each reaction. All specimens were
analysed at least twice. A negative lane was added for each
run. Methylation was confirmed if at least two experiments
demonstrated an unequivocal MSP amplification product. In
addition, all methylation patterns were confirmed by sequen-
cing the PCR products of the corresponding bisulphite DNAs.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp448–453
CDKN2A and p53 in external genital carcinomas, N. Soufir et al. 449
Human papillomavirus typing
HPV typing was performed blindly by J.B., without informa-
tion regarding the type of lesion analysed, as previously de-
scribed for the detection of HPV in genital lesions. The primer
pair MY09–MY11 was originally designed by Manos et al.26
for the detection of HPV in genital lesions. To increase the
level of sensitivity, a second established primer, GP6, was
seminested within MY11.27,28
Statistical methods
We used the v2 test (Fisher’s exact unconditional test when
necessary) to evaluate the association between the occurrence
of CDKN2A and p53 mutations in external genital tumours. We
also compared the frequency of the association of mutations
in p53 and CDKN2A between external genital tumours and
sporadic ultraviolet (UV)-induced skin carcinomas, and com-
pared the HPV16 status between VIN3/PIN3-related tumours
or lesions and LS lesions. Two-sided tests were computed, and
P < 0Æ05 was considered statistically significant. The SAS soft-
ware (SAS Institute, Cary, NC, U.S.A.) was used for statistical
analysis.
Results
Thirty-three patients were included in the analysis. Eight
patients had invasive EGCs (five women and three men; mean
age at diagnosis 72 years, range 59–88). Of these carcinomas,
four were LS-derived EGC as associated with typical LS lesions,
three were HPV-related EGC as associated with undifferentiat-
ed VIN3 or PIN3 lesions, and one was an EGC of undeter-
mined origin as no associated lesion was seen in pathological
samples. In addition, 25 patients with precancerous lesions
were studied, including two women with undifferentiated
VIN3 and 23 patients with LS (21 women and two men). The
mean age at diagnosis was 58Æ6 years.
Seven p53 mutations were observed in exons 5, 7 and 8 of
the p53 gene, in six of eight tumours (75%), one tumour
harbouring two different mutations (Table 2). All mutations
were of missense type. Overall, p53 mutations were present
in all LS-derived carcinomas (four of four), but in only one of
three of the HPV-derived carcinomas (P ¼ 0Æ053). No p53
mutation was observed in the precancerous lesions (undiffer-
entiated VIN3 or LS lesions).
Two different CDKN2A mutations were found in four of
eight invasive tumours (50%): two LS-derived tumours, one
HPV-derived tumour and one of undetermined origin. These
mutations were C fi T transitions located in exon 2 of
CDKN2A and had different consequences on p16INK4a and
p14ARF reading frames. Both were p16INK4a nonsense muta-
tions (Arg58Ter in one case, Arg80Ter in three cases), leading
to a truncated p16INK4a protein. In addition, these mutations
also induced p14ARF amino acid substitutions at residues
located either on conserved codons or in the nucleolar local-
ization domain of the p14ARF protein (Table 2). Remarkably,
all CDKN2A-mutated tumours also harboured a mutation of the
p53 gene. No CDKN2A mutations were found in precancerous
lesions, but a previously described p16INK4a exon 2 poly-
morphism was observed in two cases (Table 2).
Methylation of the p16INK4a gene promoter was not detec-
ted in any of the eight tumours analysed but was present in
one of 11 LS lesions studied. Methylation of the p14ARF gene
promoter was observed in one tumour that was neither
mutated for CDKN2A nor for p53, and was also observed in
one of 11 LS lesions studied (Table 2).
A strong (> 50%) anti-p53 staining was observed in two of
five p53-mutated tumours but in none of three HPV-derived
tumours without p53 mutation (Fig. 1a). In addition, a posi-
tive anti-p53 staining ranging from 10% to > 50% was seen
in seven of 19 LS lesions (Fig. 1b).
HPV16 was detected in four of eight invasive tumours:
three of three undifferentiated VIN3/PIN3 lesion-derived
tumours and one tumour of undetermined origin. HPV16 was
also detected in the two undifferentiated VIN3. In contrast,
HPV was not detected in the two LS-derived carcinomas ana-
lysed (Table 2). HPV16 was statistically more frequent in un-
differentiated VIN3/PIN3-related tumours or in VIN3 lesions
Table 1 Polymerase chain reaction (PCR)primers for methylation-specific PCR of the
CDKN2A genePrimer
set
PCRproduct
size (bp)
Annealingtemperature
(�C)
P16-Ma TTATTAGAGGGTGGGGCGGATCGCGACCCCGAACCGCGACCGTAA
150 60
P16-Ua TTATTAGAGGGTGGGGTGGATTGTCAACCCCAAACCACAACCATAA
151 60
P14-Mb GTGTTAAAGGGCGGCGTAGCAAAACCCTCACTCGCGACGA
122 60
P14-Ub TTTTTGGTGTTAAAGGGTGGTGTAGTCACAAAAACCCTCACTCACAACAA
132 60
Primer sequences are written 5¢ fi 3¢. M represents methylation-specific primers and U,
unmethylation-specific primers. aPrimers used by Herman et al.25 bPrimers used by Estelleret al.35
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp448–453
450 CDKN2A and p53 in external genital carcinomas, N. Soufir et al.
Tab
le2
CDKN
2Aan
dp5
3m
utat
ions
,m
ethy
lation
stat
usof
p16IN
K4aan
dp1
4ARF
prom
oter
s,p5
3im
mun
oche
mistry
(IH
C)
and
hum
anpa
pillo
mav
irus
(HPV
)ty
ping
ofex
tern
alge
nita
lca
rcin
omas
(EGCs)
and
thei
rpr
ecur
sor
lesion
s
Patien
t
Age
(yea
rs)
Sex
Dia
gnos
is
p53
IHC
p53
sequ
ence
chan
ge
p53
amin
oac
id
chan
ge
CDKN
2Ase
quen
ce
chan
ge
p16IN
K4aam
ino
acid
chan
ge
p14A
RF
amin
oac
id
chan
ge
HPV
stat
us
Met
hylation
p16IN
K4a
Met
hylation
p14A
RF
Inva
sive
tum
ours
188
FEG
C+
undi
fVIN
3N
D–
––
––
16
––
282
MEG
C+
undi
fPI
N3
ND
Tgc
fiGgc
Ex5
Cys
135G
lyCga
fiTga
Ex2
Arg
80Ter
Pro9
4Leu
16
––
365
FEG
C+
undi
fVIN
3–
––
––
–16
+6
–+
459
MEG
Cun
d50
%aa
Gfi
aaC
Ex5
Lys1
32Asp
Cga
fiTga
Ex2
Arg
80Ter
Pro9
4Leu
16
––
560
FEG
C+
LS>
80%
Ggg
fiTgg
Ex8
Gly
356T
rp–
––
––
–
672
FEG
C+
LSN
DGgc
fiAgc
Ex7
Gly
245S
erCga
fiTga
Ex2
Arg
80Ter
Pro9
4Leu
ND
ND
ND
771
MEG
C+
LSN
DcG
cfi
cAc
Ex5
Cgg
fiTgg
Ex7
Arg
175C
ys
Arg
248T
rp
Cga
fiTga
Ex2
Arg
58Ter
Pro7
2Leu
ND
ND
ND
879
FEG
C+
LSN
DtG
cfi
tTc
Ex7
Cys
252P
he
––
––
––
Prec
ance
rous
lesion
s9
71F
undi
fVIN
3–
––
––
–16
+6
––
1046
Fun
difVIN
3–
––
––
–16
––
1163
FLS
––
––
––
–N
DN
D
1279
FLS
––
––
––
–+
–13
64F
LS–
––
––
–N
DN
DN
D
1466
FLS
––
––
––
ND
–+
1545
FLS
>10
%–
––
––
16
––
1669
FLS
––
–c.44
2G
fiA
Ex2
Ala1
48Th
r–
ND
––
1762
FLS
––
––
––
––
–
1884
FLS
ND
––
––
–6
ND
ND
1946
FLS
>10
%–
––
––
ND
ND
ND
2062
FLS
>50
%–
––
––
ND
––
2136
FLS
>50
%–
––
––
ND
ND
ND
2261
MLS
ND
––
––
–N
D–
–
2365
FLS
10%
––
––
–6
––
2466
FLS
>50
%–
––
––
ND
ND
ND
2540
FLS
––
––
––
ND
ND
ND
2668
FLS
30%
––
––
–16
ND
ND
2763
FLS
––
––
––
–N
DN
D28
67F
LS–
––
––
–N
D–
–
2950
FLS
––
––
––
ND
ND
ND
3062
FLS
ND
––
c.44
2G
fiA
Ex2
Ala1
48Th
r–
16
+6
––
3154
FLS
–N
DN
DN
DN
DN
DN
D–
–32
48M
LSN
D–
––
––
ND
ND
ND
3328
FLS
––
––
––
ND
ND
ND
The
mut
ated
base
isw
ritten
inup
per
case
.Si
gnifi
cant
resu
lts
are
show
nin
bold
face
,an
dpo
lym
orph
ism
sar
esh
own
initalic
s.LS
,lic
hen
scle
rosu
s;Ex
,ex
on;N
D,no
tde
term
ined
;un
difVIN
3or
PIN
3,un
differ
entiat
edvu
lval
orpe
nile
insit
une
oplasia;
und,
unde
term
ined
.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp448–453
CDKN2A and p53 in external genital carcinomas, N. Soufir et al. 451
(five of five) than in LS lesions (three of nine; P ¼ 0Æ0157,
Fisher’s exact test ¼ 0Æ031).
Discussion
In this work we analysed the status of the CDKN2A and p53
genes in benign and malignant external genital lesions. Our
results show for the first time and distinctively on a small ser-
ies of invasive tumoral samples two distinct molecular patterns
according to the origin of the EGC: mutations of both the p53
gene and the CDKN2A locus in 100% of LS derived-tumours
and a lower frequency (33%) of p53 or CDKN2A mutations in
undifferentiated VIN/PIN3-related tumours.
p53 has been extensively studied in EGCs. In our study,
p53 mutations were statistically more frequent in LS-derived
carcinomas than in HPV-derived carcinomas. Although our
work included a small series of carcinomas, our results are in
light with previous findings, particularly a meta-analysis that
revealed significantly more p53 mutations in HPV-negative
vulval SCC than in HPV-positive vulval SCC.29
In vulval carcinomas, p53 mutations were mostly transver-
sions (Table 2), in contrast to the p53 mutational spectrum
observed in UV-induced skin carcinomas, which comprises
mostly single or double transitions at dipyrimidic sites
(C fi T or CC fi TT).30 No LS lesions (n ¼ 23) showed any
evidence of p53 mutation, highly suggesting that p53 muta-
tions are a late event in external genital tumours, which has
already been proposed by several authors. This is in contrast
with UV-induced skin carcinomas, for which p53 mutations
are believed to be early events as they are usually present in
chronically sun-exposed skin. However, p53 immunostaining
was present in seven of 19 (37%) LS lesions, with a staining
that was nuclear and mostly basal and suprabasal (Fig. 1). Im-
munostaining intensity varied among these positive samples
but was always > 10% of cells analysed (see Table 2). This
observation was previously reported and was considered to be
an important feature of vulval LS compared with LS from
other sites;11 it was attributed to overexpression of the TP53
wild-type protein reflecting stress response to inflammation
and carcinogenic insult. In our series, the p53 positivity in LS
lesions was not associated with mutations of the p53 gene,
therefore favouring the hypothesis of wild-type protein stabil-
ization, the significance of which awaits further studies.
The CDKN2A gene has been much less studied in EGC. A
decreased expression of p16INK4a protein and p16INK4a promo-
ter methylation have been reported in vulval carcinomas and
their precursors.31,32 In our series, we found CDKN2A muta-
tions in four of eight invasive EGCs (50%), with mutations af-
fecting both p16INK4a and p14ARF. A hot spot (Arg80Ter)
present in three tumours was previously described in a mela-
noma cell line33 and in nonmelanoma skin cancers (NMSCs)
occurring in patients with xeroderma pigmentosum,24 but not
in sporadic NMSC. No mutation was detected in LS, suggest-
ing that, as for the p53 gene, inactivation of CDKN2A by muta-
tions is a late phenomenon in vulval carcinogenesis.
Methylation of CDKN2A promoters was rare in our series
compared with gene inactivation by point mutation. This con-
trasts with a recent work that reported a much higher propor-
tion of p16INK4a gene promoter methylation in vulval
carcinoma as well as in LS lesions (68% and 42Æ8%, respect-
ively).32 This difference might be due (i) to the high propor-
tion of CDKN2A-mutated tumours in our series, in which
methylation of promoters and mutations of CDKN2A were not
associated, and (ii) to differences in sensitivity or specificity of
gene promoter methylation detection. This awaits further an-
alysis. However, we also detected methylation of p16INK4a and
p14ARF promoters in LS lesions, which could mean that epi-
genetic inactivation of the CDKN2A locus most probably repre-
sents an early event, insufficient for malignant transformation,
that may occur in clinically benign lesions such as LS.32 All
CDKN2A-mutated tumours also harboured a mutation in the
p53 gene, suggesting that co-inactivation of both pathways is
important in EGCs, and may be indicating a cooperative effect.
In previous studies we observed a similar finding in cutaneous
SCC from patients with xeroderma pigmentosum,24 but not in
sporadic skin carcinomas.23 In animal models, a cooperative
effect between inactivation of CDKN2A and p53 gene inactiva-
tion in cancer development has been demonstrated.34 p16INK4a
mutations destabilize the G1/S checkpoint by inactivating pRb
and releasing E2F transcription factors that directly activate
p14ARF. Induction of p14ARF then stabilizes p53 and permits
p53-mediated apoptosis. p14ARF or p53 mutants would inhibit
p53-mediated apoptosis. Therefore mutations in both genes
could confer a selective cellular growth advantage, allowing
mutations in other genes to accumulate and promoting tu-
morigenesis.
In conclusion, our study shows a high prevalence of inac-
tivating mutations of p53 and/or CDKN2A genes preferentially
observed in EGC derived from LS (100%), that seem to
occur late in oncogenesis as they were not present in LS
lesions. The carcinogenic insult responsible for the mutations
involving these two crucial genes still remains to be eluci-
dated.
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CDKN2A and p53 in external genital carcinomas, N. Soufir et al. 453
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07601.x
Serum chemokine profile in patients with bullouspemphigoidH. Nakashima, M. Fujimoto,* N. Asashima, R. Watanabe, Y. Kuwano, N. Yazawa, N. Maruyama,� H. Okochi,�A. Kumanogoh§ and K. Tamaki
Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
*Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan
�Division of Biostatistics, School of Pharmaceutical Science, Kitasato University, Tokyo, Japan
�Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan
§Department of Immunopathology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
CorrespondenceM. Fujimoto.
E-mail: fujimoto-m@umin.ac.jp
Accepted for publication7 August 2006
Key wordsautoimmunity, bullous pemphigoid, chemokine,
multiplex protein analysis system, pemphigus
vulgaris, Th1/Th2
Conflicts of interestNone declared.
Summary
Background Bullous pemphigoid (BP) is an autoimmune inflammatory disease caus-ing blister formation at the dermoepidermal junction. Cutaneous infiltration ofactivated CD4+ T cells and eosinophils is an early event in blister formation dur-ing the disease process, suggesting that the trafficking of circulating leucocytesthrough the sites of inflammation is crucial in the pathogenesis of the disease.While the accumulated evidence suggests that some cytokines are involved in thepathogenesis, there have been few reports about serum chemokine profiles inpatients with BP.Objectives To determine serum profiles of various chemokines and their clinical as-sociation in patients with BP.Methods Concentrations of 10 chemokines – interferon (IFN)-c-inducible protein-10 (IP-10), monokine induced by IFN-c (MIG), macrophage inflammatory pro-tein (MIP)-1a, MIP-1b, RANTES, eotaxin, monocyte chemoattractant protein(MCP)-1, MCP-2, MCP-3 and growth-regulated oncogene-a – were measured inserum samples from 38 patients with BP, 16 with pemphigus vulgaris (PV) and17 normal controls using a sandwich immunoassay-based multiplex protein arraysystem.Results While there was no significant increase in any serum chemokine levels inpatients with PV, serum levels of IP-10 and MCP-1 were significantly increasedin patients with BP compared with healthy controls. Furthermore, serum levelsof IP-10, MIG, MCP-1 and eotaxin in patients with BP increased significantlywith disease severity as determined by the area affected.Conclusions These observations suggest that an elaborately orchestrated network ofchemokines, especially MCP-1 and IP-10, contributes to the pathomechanism ofBP.
Bullous pemphigoid (BP) is an autoimmune subepidermal
blistering disease, usually occurring in the elderly, which is
characterized by large, tense blisters. The production of auto-
antibodies directed to a 180-kDa hemidesmosomal protein
(BP180) of the basement membrane zone is the initiating
event of the pathomechanism.1 Once the autoantibodies bind
to the basement membrane, a cascade of inflammatory events
occurs, resulting in subsequent blister formation at the dermo-
epidermal junction. During this process, cytokines and chem-
okines are considered to play crucial roles in inflammatory
cell recruitment, deposition and perpetuation.2 Therefore, clar-
ifying the serum and local levels of cytokines and chemokines
may help in understanding the immune dysregulation in the
pathomechanism.
It has been demonstrated that various cytokines including
interleukin (IL)-1b, IL-2, IL-4, IL-5, IL-6, IL-8, interferon
(IFN)-c and tumour necrosis factor-a are increased in sera
of patients with BP.3–8 By contrast, while serum levels of
several chemokines including eotaxin,9–11 RANTES (regulated
on activation, normal T-cell expressed and secreted),6 thy-
mus and activation-regulated chemokine (TARC)12 and mu-
cosae-associated epithelial chemokine13 have been reported,
� 2006 The Authors
454 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp454–459
the involvement of most chemokines in the disease remains
unknown. In this study, we utilized a recently developed
protein array system and assessed the serum levels of 10
chemokines: IFN-c-inducible protein-10 (IP-10), monokine
induced by IFN-c (MIG), macrophage inflammatory protein
(MIP)-1a, MIP-1b, monocyte chemoattractant protein
(MCP)-1, MCP-2, MCP-3, growth-regulated oncogene-a(GRO-a), eotaxin and RANTES.
Materials and methods
Subjects
Serum samples obtained from 38 patients with BP (17 women
and 21 men; mean age 71Æ0 years), 16 patients with pemphi-
gus vulgaris (PV; nine women and seven men; mean age
55Æ7 years) and 17 healthy individuals (eight women and nine
men; mean age 61Æ7 years) were examined. BP and PV were
diagnosed on the basis of clinical features, and findings of skin
biopsy examination by light microscopy, direct immunofluo-
rescence and indirect immunofluorescence. Patients with BP
and PV had not been treated with oral corticosteroids, mino-
cycline/tetracycline or immunosuppressive drugs. As an index
of disease severity, the percentage of the body surface area
covered with skin lesions (oedema, erythema, blisters, ero-
sions) was estimated when the serum samples were obtained.
Patients with < 20%, 20–40% and > 40% of their body sur-
face affected were classified into group 1 (mild), 2 (inter-
mediate) and 3 (severe), respectively. Institutional review
board approval and informed consent from all patients and
controls were obtained. The samples collected were stored at
)80 �C until use.
Multiplex chemokine assay
The 10 chemokines measured simultaneously were IP-10, MIG,
MIP-1a, MIP-1b, RANTES, eotaxin, MCP-1, MCP-2, MCP-3 and
GRO-a. A sandwich immunoassay-based protein array system
(Biosource, Camarillo, CA, U.S.A.), which contains dyed micr-
ospheres conjugated with a monoclonal antibody specific for a
target protein, was used. Serum samples were thawed and run
in duplicate. Antibody-coupled beads were incubated with the
plasma sample after which they were incubated with biotinylat-
ed detection antibody before finally being incubated with
streptavidin-conjugated phycoerythrin. A broad sensitivity
range of standards (Biosource) ranging from 1Æ95 to
32 000 pg mL)1 was used for the quantification of a dynamic
wide range of chemokine concentrations and to provide the
greatest sensitivity. Standards included all the chemokines
assayed. This captured immunoassay was then read by the Bio-
plex array reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.)
which uses Luminex fluorescent bead-based technology (Lum-
inex Corporation, Austin, TX, U.S.A.) with a flow-based dual
laser detector with real-time digital signal processing to facili-
tate the analysis of up to 100 different families of colour-coded
polystyrene beads and to allow multiple measurements of the
sample, ensuring the effective quantification of chemokines.
The concentrations of chemokines analysed in these assays were
quantified using a calibration or standard curve. A regression
analysis was performed to derive an equation that was then
used to predict the concentration of the unknown samples.
Statistical analysis
Statistical differences between measured values in patients with
BP, patients with PV and controls were analysed using the
Steel–Dwass’ multiple comparison test. Trends in the measured
values according to the affected area (disease severity) were
analysed by the two-sided Jonckheere–Terpstra test. Correla-
tions between each serum chemokine level were analysed
using Spearman’s correlation coefficient by rank test. P < 0Æ05
was considered statistically significant.
Results
Serum levels of 10 chemokines in patients with bullous
pemphigoid and pemphigus vulgaris
Concentrations of 10 chemokines, IP-10, MIG, MIP-1a,
MIP-1b, RANTES, eotaxin, MCP-1, MCP-2, MCP-3 and GRO-a,
were simultaneously measured in the sera from patients with
BP, patients with PV and healthy controls. Serum levels of IP-10
and MCP-1 were significantly increased in patients with BP
compared with healthy controls (P ¼ 0Æ011 for IP-10 and P ¼0Æ014 for MCP-1, Fig. 1). Serum levels of MIG, MIP-1a, MIP-1band eotaxin were also slightly higher in patients with BP than in
controls, although the differences were not significant. By con-
trast, all chemokines except for eotaxin showed comparable lev-
els between patients with PV and healthy controls (Fig. 1).
Serum eotaxin levels in patients with PV were higher than those
in controls although the difference was not significant (Fig. 1).
When chemokine levels higher than the mean + 2 SD of
the control serum samples were considered to be elevated, IP-
10 level was elevated in 39%, MIG in 29%, MIP-1a in 18%,
MIP-1b in 29%, eotaxin in 32% and MCP-1 in 36% of
patients with BP (Table 1). In patients with PV, 38% had ele-
vated eotaxin level, 25% had elevated MCP-1 level and 19%
had elevated IP-10 level. While an increased RANTES level was
observed in one control sample, all the other chemokine levels
in control samples were less than the mean + 2 SD levels. Col-
lectively, patients with BP showed more prominent abnormal-
ities in chemokine levels than patients with PV.
Positive correlation between serum levels of monokine
induced by interferon-c and macrophage inflammatory
protein-1 and disease severity
To assess the association of chemokine levels with disease
severity, patients with BP were classified into three groups by
their affected percentage body surface area. Significant disease
severity-related increases in serum levels of IP-10, MIG,
MCP-1 and eotaxin (P ¼ 0Æ008 for IP-10, P ¼ 0Æ0007 for
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp454–459
Chemokine profile in BP, H. Nakashima et al. 455
MIG, P < 0Æ0001 for MCP-1 and P ¼ 0Æ005 for eotaxin by the
Jonckheere–Terpstra test) were present (Fig. 2). By contrast,
serum levels of MIP-1a, MIP-1b, MCP-2, MCP-3, GRO-a and
RANTES showed no such tendency (data not shown). Thus,
serum levels of MIG, IP-10, MCP-1 and eotaxin appear to
reflect the disease severity.
Fig 1. Comparison of serum levels of interferon (IFN)-c-inducible protein-10 (IP-10), monokine induced by IFN-c (MIG), macrophage
inflammatory protein (MIP)-1a, MIP-1b, RANTES, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3 and growth-regulated
oncogene-a (GRO-a) in patients with bullous pemphigoid (BP), pemphigus vulgaris (PV) and normal controls (Control). The measured values
from individual patients were plotted by dots. The boxes and whiskers indicate the median ± (or +) 25% and maximum/minimum values in
each group. A broken horizontal line indicates the cut-off value (mean + 2 SD of the control samples). *P < 0Æ05 by Steel–Dwass’ multiple
comparison test. Significant differences were also confirmed using the Mann–Whitney U-test with Bonferroni correction.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp454–459
456 Chemokine profile in BP, H. Nakashima et al.
Correlation between serum chemokine levels
We further analysed the correlation between serum levels of
measured chemokines. Serum IP-10 levels in patients with BP
correlated positively with serum levels of MIG (r ¼ 0Æ385;
P ¼ 0Æ018), MCP-1 (r ¼ 0Æ328; P ¼ 0Æ041) and eotaxin (r ¼0Æ345; P ¼ 0Æ031). Serum eotaxin levels also correlated posi-
tively with serum concentration of MCP-1 (r ¼ 0Æ490; P ¼0Æ001). There was a strongly positive correlation between
serum levels of MIP-1a and MIP-1b (r ¼ 0Æ979, P < 0Æ0001).
Thus, several chemokines were concomitantly elevated, which
suggests that these cooperatively contribute to the disease
development.
Discussion
The aim of this study was to determine the profile of various
chemokines (IP-10, MIG, MIP-1a, MIP-1b, RANTES, eotaxin,
MCP-1, MCP2, MCP-3 and GRO-a) in sera from patients with
BP. While no chemokine was significantly elevated in sera
from patients with PV, patients with BP exhibited significantly
increased serum levels of IP-10 and MCP-1 compared with
healthy controls. Serum levels of IP-10, MIG, MCP-1 and eo-
taxin in patients with BP also showed a significant increase
with disease severity as determined by affected area. Addition-
ally, there were positive correlations between IP-10 and MIG,
IP-10 and eotaxin, IP-10 and MCP-1, eotaxin and MCP-1, and
MIP-1a and MIP-1b. Collectively, a variety of chemokines is
likely to contribute to the development of BP cooperatively.
Pathogenesis of the inflammatory skin disease is often rela-
ted to the T helper (Th) 1/Th2 balance. BP is considered to
be dominantly mediated by Th2 responses. Consistently, Kaki-
numa et al.12 have reported that serum levels of TARC, a major
Th2 chemokine, were elevated in patients with BP. While
there has been no literature describing Th1 chemokines of BP
sera, IP-10 and MIG measured in this study are considered as
chemoattractants of Th1 lymphocytes.14 Serum IP-10 levels
were significantly elevated in patients with BP compared with
controls. Also, there was a trend for MIG levels in sera from
patients with BP to increase in association with the affected
area. By contrast, serum levels of MIP-1a and MIP-1b, which
are also known as Th1 chemokines,15,16 showed neither signi-
ficant differences between patients with BP and controls nor
trends to increase with increasing disease severity. Positive
correlations between IP-10 and MIG levels suggest that IP-10
and MIG may work at adjacent parts of intricate inflammatory
cascades in the development of BP. Also, considering that in-
creased serum MIG levels were limited to patients with severe
disease, MIG may especially contribute to exacerbation of BP.
Alternatively, insufficient sensitivity of MIG detection in this
Table 1 Frequency of elevated samples of each chemokine
IP-10 MIG MIP-1a MIP-1b Eotaxin MCP-1 MCP-2 MCP-3 RANTES GRO-a
BP (n ¼ 38) 15 (39) 11 (29) 7 (18) 11 (29) 12 (32) 13 (36) 1 (3) 2 (6) 3 (8) 9 (24)
PV (n ¼ 16) 3 (19) 3 (19) 1 (6) 1 (6) 6 (38) 4 (25) 1 (6) 0 1 (6) 3 (19)Control (n ¼ 17) 0 0 0 0 0 0 0 0 1 (6) 0
BP, bullous pemphigoid; PV, pemphigus vulgaris; IP-10, interferon (IFN)-c-inducible protein-10; MIG, monokine induced by IFN-c; MIP,
macrophage inflammatory protein; MCP, monocyte chemoattractant protein; GRO-a, growth-regulated oncogene-a. Values are numbers(percentages) of the patients who showed elevated serum levels of each chemokine (> mean + 2 SD that of controls).
Fig 2. Serum levels of interferon (IFN)-c-inducible protein-10 (IP-10), monokine
induced by IFN-c (MIG), monocyte
chemoattractant protein (MCP)-1 and eotaxin
in patients with bullous pemphigoid classified
by affected area and in controls. Patients with
< 20%, 20–40% and > 40% of their body
surface affected were classified as group 1
(mild), 2 (intermediate) and 3 (severe),
respectively. The boxes and whiskers indicate
the median ± (or +) 25% and maximum/
minimum values in each group.
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp454–459
Chemokine profile in BP, H. Nakashima et al. 457
system may interfere with significant differences between
serum levels of patients with BP and controls. None the less,
serum IP-10 and MIG levels may also serve as indicators for
disease severity in BP.
Interestingly, T cells positive for CXC chemokine receptor
3, which is preferentially expressed on Th1-type cells, have
been demonstrated to exist beneath the bullae.17 Furthermore,
Budinger et al.18 have described that the autoreactive T cells,
which participate in the blister formation of BP and recognize
the extracellular domains of BP180, possess both Th2 and Th1
cytokines. Taken together, not only Th2 cells but also Th1
cells may be important for pathogenesis of BP.
BP lesions exhibit prominent eosinophil infiltration. Eotaxin
is considered to be a potent chemoattractant and activator of
eosinophils,19 and a main attractant of Th2 lymphocytes.20,21
Although serum eotaxin levels were not significantly increased
in patients with BP, serum eotaxin levels were associated with
disease severity and were also positively correlated with IP-10
and MCP-1. Correlation between eotaxin and these chemok-
ines implies that there may be a similar process for infiltration
of eosinophils and another subset of leucocytes including Th1
lymphocytes. Some previous reports have demonstrated eleva-
ted levels of eotaxin in sera and blister fluid as well as in-
creased expression in skin lesions of patients with BP.9–11 The
enhanced expression of eotaxin indicates that this chemokine
may also play an important role in the pathomechanism of
this autoimmune bullous skin disease by recruiting and activa-
ting eosinophils and lymphocytes.
MCP-1 is a chemoattractant and activator of monocytes,
lymphocytes and basophils, but lacks activity on neutrophils
and eosinophils.22,23 Serum MCP-1 levels were elevated in
patients with BP, and exhibited a tendency to increase accord-
ing to the disease severity. Furthermore, MCP-1 levels were
correlated positively with IP-10 and eotaxin, which act on
other types of inflammatory cells than those regulated by
MCP-1. Therefore, correlation between MCP-1 and these
chemokines suggests that there may be a similar process for
infiltration of different subsets of leucocytes utilizing a variety
of chemokines. While there is no literature regarding serum
MCP-1 levels in BP, some studies have demonstrated that
expression of CCR2, the chemokine receptor to which MCP-1
binds, is increased in chronically inflamed skin lesions of both
atopic dermatitis and psoriasis.24 Therefore, MCP-1 may play
an essential role in prolonged inflammation of BP lesions.
Serum RANTES levels did not show significant differences
between patients with BP and controls. RANTES is known as a
chemoattractant for monocytes, T lymphocytes and eosinoph-
ils.25 Our data are consistent with the findings of D’Auria et
al.6 Furthermore, they demonstrated that the RANTES concen-
trations in blister fluid were < 1/100 of those in the sera.
Therefore, RANTES may be rapidly degraded in the blister
fluid. Alternatively, RANTES derived from platelets may
increase the serum concentration.
In summary, the current study indicates that patients with
BP have a complex disorder of various chemokine levels in
their sera. This supports that various immune pathways are
involved in the pathogenesis of BP. It was also remarkable that
the chemokine profile of the sera from patients with BP
showed heterogeneity, which may suggest that the different
profiles correspond to distinct disease subsets or severity, or
may reflect different stages of the disease. Nonetheless, it
appears that especially MCP-1 and IP-10, then MIG and
eotaxin, play relatively more important roles than the other
chemokines in BP. Further profiling of immune mediators
including chemokines will elucidate the precise mechanism of
the disease development.
References
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2 Pastore S, Mascia F, Mariotti F et al. Chemokine networks in inflam-matory skin diseases. Eur J Dermatol 2004; 14:203–8.
3 Tamaki K, So K, Furuya T et al. Cytokine profile of patients withbullous pemphigoid. Br J Dermatol 1994; 130:128–9.
4 Sun CC, Wu J, Wong TT et al. High levels of interleukin-8, solubleCD4 and soluble CD8 in bullous pemphigoid blister fluid. The re-
lationship between local cytokine production and lesional T-cell ac-tivities. Br J Dermatol 2000; 143:1235–40.
5 Inaoki M, Takehara K. Increased serum levels of interleukin (IL)-5,IL-6 and IL-8 in bullous pemphigoid. J Dermatol Sci 1998; 16:152–7.
6 D’Auria L, Pietravalle M, Mastroianni A et al. IL-5 levels in theserum and blister fluid of patients with bullous pemphigoid: cor-
relations with eosinophil cationic protein, RANTES, IgE and diseaseseverity. Arch Dermatol Res 1998; 290:25–7.
7 D’Auria L, Mussi A, Bonifati C et al. Increased serum IL-6, TNF-
alpha and IL-10 levels in patients with bullous pemphigoid: rela-tionships with disease activity. J Eur Acad Dermatol Venereol 1999;
12:11–15.8 Ameglio F, D’Auria L, Bonifati C et al. Cytokine pattern in blister
fluid and serum of patients with bullous pemphigoid: relationshipswith disease intensity. Br J Dermatol 1998; 138:611–14.
9 Frezzolini A, Teofoli P, Cianchini G et al. Increased expression ofeotaxin and its specific receptor CCR3 in bullous pemphigoid. Eur J
Dermatol 2002; 12:27–31.10 Shrikhande M, Hunziker T, Braathen LR et al. Increased coexpres-
sion of eotaxin and interleukin 5 in bullous pemphigoid. Acta DermVenereol (Stockh) 2000; 80:277–80.
11 Wakugawa M, Nakamura K, Hino H et al. Elevated levels of eotaxinand interleukin-5 in blister fluid of bullous pemphigoid: correl-
ation with tissue eosinophilia. Br J Dermatol 2000; 143:112–16.12 Kakinuma T, Wakugawa M, Nakamura K et al. High level of
thymus and activation-regulated chemokine in blister fluid andsera of patients with bullous pemphigoid. Br J Dermatol 2003;
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in patients with atopic dermatitis, psoriasis vulgaris and bullouspemphigoid. J Invest Dermatol 2005; 124:1088–90.
14 Bonecchi R, Bianchi G, Bordignon PP et al. Differential expressionof chemokine receptors and chemotactic responsiveness of type 1
T helper cells (Th1s) and Th2s. J Exp Med 1998; 187:129–34.15 Koch AE, Kunkel SL, Harlow LA et al. Macrophage inflammatory
protein-1 alpha. A novel chemotactic cytokine for macrophages inrheumatoid arthritis. J Clin Invest 1994; 93:921–8.
16 Tanaka Y, Adams DH, Hubscher S et al. T-cell adhesion induced byproteoglycan-immobilized cytokine MIP-1 beta. Nature 1993;
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19 Ponath PD, Qin S, Post TW et al. Molecular cloning and characteri-zation of a human eotaxin receptor expressed selectively on eosin-
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Chemokine profile in BP, H. Nakashima et al. 459
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07631.x
Resolution of inflammatory acne vulgaris may involveregulation of CD4+ T-cell responses to PropionibacteriumacnesH.E. Wilcox, M.D. Farrar, W.J. Cunliffe,* K.T. Holland and E. Ingham
Skin Research Centre, Institute of Molecular and Cellular Biology, Garstang Building, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K.
*Department of Dermatology, Leeds General Infirmary, Leeds LS1 3EX, U.K.
CorrespondenceMark D. Farrar.
E-mail: m.d.farrar@leeds.ac.uk
Accepted for publication11 June 2006
Key wordsacne vulgaris, heat shock proteins, limiting dilution
analysis, lymphocyte transformation,
Propionibacterium acnes
Conflicts of interestNone declared.
Summary
Background Propionibacterium acnes has been strongly implicated in inflammatory acne.However, its role in the disease is unclear. It has been hypothesized that animmune response to P. acnes and/or P. acnes heat shock proteins (HSPs) may playa role in the pathogenesis of inflammatory acne.Objectives To compare the cell-mediated immune response to P. acnes and HSPs inacne patients, nonacne controls and individuals with resolved acne.Methods The proliferative response of peripheral blood mononuclear cells (PBMC)from acne patients, resolved acne donors and healthy controls to P. acnes, P. acnesHSP60 and HSP70, and mycobacterial HSPs was assessed by lymphocyte trans-formation assay (LTA). The proliferative response of purified CD4+ T cells wasfurther analysed by limiting dilution analysis (LDA). Contingency tables (G-test)were used to analyse the proportion of individuals in each group showing apositive proliferative response for LTA or data fitting single-hit kinetics forLDA.Results Analysis of stimulation of PBMC with P. acnes, P. acnes HSP60 and HSP70 inthe LTA showed the proportion of positive responders to be independent of sub-ject group. However, the proportion of acne patients with a positive response tomycobacterial HSPs was significantly higher than those for the other subjectgroups. Analysis of LDA data showed the proportion of resolved donors withresponses to P. acnes fitting the single-hit kinetics model to be significantly lowerthan those of the other groups. There were no significant differences in responsesto other antigens.Conclusions The significantly lower proportion of resolved donors demonstrating asingle-hit kinetics response to P. acnes by LDA may represent negative regulationof the CD4+ T-cell response to P. acnes in these subjects.
Acne vulgaris is a multifactorial, spontaneously resolving pleo-
morphic skin disease, characterized by a variety of nonin-
flamed and inflamed skin lesions. Propionibacterium acnes has been
strongly implicated in the pathogenesis of the disease due to
the clinical improvement seen following a reduction in bacter-
ial population density with antibiotic therapy and the finding
that a lack of clinical response was associated with the pres-
ence of antibiotic-resistant propionibacteria.1,2 However, the
P. acnes factors that contribute to the disease are still not
known. The bacterium has been shown to have mitogenic as
well as antigenic activity3 and the inflammatory infiltrate
within early lesions is predominantly CD4+ T cells.4,5
Antibodies in sera of acne patients have been shown to recog-
nize a greater number of P. acnes protein antigens than those in
sera from healthy controls.6 Antigens specifically involved in
acne have not been identified but we have previously hypothes-
ized that heat shock proteins (HSPs) may have a role to play.7–9
These proteins are highly conserved throughout prokaryotes
and eukaryotes, with roles in protein folding and the cellular
stress response.10,11 HSPs are thought to contribute to inflam-
matory diseases through the induction of cytokine production
by a variety of cells and these proteins are frequently dominant
antigens in bacterial infections.12–14 Conditions within some
pilosebaceous follicles may be such that P. acnes are subject to
physiological stress and upregulate production of HSPs leading
to a cell-mediated inflammatory response to these proteins.
Studies of the cell-mediated immune (CMI) response to
P. acnes and P. acnes products have been carried out but the CMI
� 2006 The Authors
460 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp460–465
response to P. acnes HSPs has not been investigated. Previous
investigations of cellular immunity to P. acnes assayed by leuco-
cyte migration inhibition showed that only individuals with
severe acne could be considered as demonstrating CMI to the
bacterium.15 Investigations employing the lymphocyte trans-
formation assay (LTA) provided conflicting results with
one study demonstrating a significantly greater proliferative
response in acne patients compared with nonacne controls16
and a second study demonstrating a significantly greater
response only after resolution of the disease following a course
of therapy.17 Acne is most often a naturally self-limiting dis-
ease and important information could be gained by the inclu-
sion of a group of individuals with resolved acne in further
studies into immune responses to P. acnes and its products.
Therefore this study was undertaken to assess the specific CMI
response to P. acnes and P. acnes HSPs of cells from the peripheral
blood of acne patients, nonacne controls and individuals with
resolved acne. Responses to HSPs from mycobacteria, which
are closely related to propionibacteria but are not implicated in
acne, were also assessed.
Patients, materials and methods
Acne patients, control subjects and resolved donors
Three groups of subjects were studied—acne patients, nonacne
controls and resolved donors. Acne patients were outpatients of
the Department of Dermatology, Leeds General Infirmary, and
suffered from mild to severe acne as assessed by the Leeds tech-
nique.18 The nonacne controls were healthy individuals who
had no previous history of acne. The resolved donor group
consisted of individuals who had suffered from mild to moder-
ate acne at some period in their life but were now acne-free.
To determine the resolved acne status of these individuals,
volunteers were asked to complete a brief questionnaire
accompanied by a set of photographs representing acne
grades. None of the volunteers in any group were taking anti-
microbial therapy during or 6 weeks prior to the study. For
the LTA, the subject groups consisted of 12 patients (age
range 14–24 years), 10 controls (age range 20–24 years) and
10 resolved donors (age range 18–36 years). For the limiting
dilution analysis (LDA), the subject groups consisted of 11
patients (age range 14–23 years), 12 controls (age range 21–
29 years) and 13 resolved donors (age range 18–36 years).
Volunteers were asked if they had received the BCG vaccin-
ation. Not all of the samples from the donors were tested
against all antigens due to the availability of donors, the will-
ingness to donate the required volume of blood and the num-
ber of cells recovered from each blood sample (n ‡ 10).
Ethical approval for this project was obtained from the local
Ethics Committee.
Culture media
Total peripheral blood mononuclear cells (PBMC) were main-
tained in Medium A, which consisted of RPMI-1640 supple-
mented with 20 mmol L)1 HEPES, 60 lg mL)1 penicillin,
100 U mL)1 streptomycin, 2 mmol L)1L-glutamine and 10%
(v/v) fetal calf serum. Purified CD4+ T cells were maintained
in Medium B consisting of RPMI-1640 supplemented with
60 lg mL)1 penicillin, 100 U mL)1 streptomycin, 2 mmol L)1
L-glutamine and 7Æ5% (v/v) autologous serum. Purified
CD14+ cells were maintained in Medium C, which consisted
of Medium B supplemented with 25 U mL)1 recombinant
interleukin (IL)-2 (Roche Diagnostics Ltd., Lewes, U.K.).
PBMC and purified cells were grown at 37 �C in 5% (v/v)
CO2 in air. P. acnes was routinely grown in TYEG broth (10%
w/v tryptone, 5% w/v yeast extract, 1% w/v glucose) and on
reinforced clostridial agar (Oxoid, Basingstoke, U.K.) at 34 �Cin an atmosphere of 80% (v/v) N2, 10% (v/v) CO2, 10%
(v/v) H2.
Propionibacterium acnes and heat shock proteins
Propionibacterium acnes strain P37 was grown to the mid-expo-
nential phase. Bacterial cells were harvested, washed three
times in phosphate-buffered saline (PBS) and stored in 1%
(v/v) formalin in PBS at 4 �C. Prior to use, bacterial cells were
washed three times in PBS, resuspended at 1 · 109 cells mL)1
and sonicated to disaggregate clumps. P. acnes HSP60 and
HSP70 were overproduced in Escherichia coli and purified as pre-
viously described.8 Mycobacterium bovis BCG HSP65 was obtained
from the World Health Organization antigen bank, Germany.
Mycobacterium smegmatis HSP70 was obtained from Sigma (Poole,
U.K.).
Lymphocyte transformation assay
Peripheral venous blood (10 mL) was collected from each
subject into tubes containing 100 U preservative-free heparin
then diluted with an equal volume of Hank’s balanced salt
solution (HBSS) supplemented with 60 lg mL)1 penicillin
and 100 U mL)1 streptomycin. The diluted sample was
layered onto Lymphoprep� (Nycomed, Oslo, Norway) and
centrifuged at 800 g for 20 min. PBMC were harvested from
the Lymphoprep�–HBSS interface, washed three times with
antibiotic-supplemented HBSS and diluted to a final concentra-
tion of 1 · 106 cells mL)1 in culture medium A. Six replicate
volumes (100 lL) of PBMC were co-cultured with equal vol-
umes of formalized P. acnes (1 · 107 cells mL)1), each HSP
(0Æ5 lg mL)1), phytohaemagglutinin (PHA, 10 lg mL)1) or
Medium A only in a 96-well plate. Cells were cultured for
3 days with PHA, 6 and 9 days with P. acnes, and 7 and 9 days
with each HSP.
Culture periods and antigen amounts had been optimized in
preliminary experiments. High-activity [3H]thymidine (0Æ25 lCi)
was then added to each well and cells incubated for a
further 4 h. DNA was extracted from cells and harvested onto
glass fibre filter mats using a semi-automatic cell harvester.
The amount of [3H]thymidine incorporated into cellular DNA
was determined by scintillation counting. Results were
expressed as the stimulation index calculated as the mean
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp460–465
CD4+ T-cell regulation in acne resolution, H.E. Wilcox et al. 461
counts per minute (cpm) of the test divided by the mean cpm
of the negative control. A stimulation index of ‡ 3Æ0 was con-
sidered to be a positive response.
Limiting dilution analysis
Isolation of CD14+ monocytes and CD4+ T cells
CD14+ monocytes to be used as antigen-presenting cells
(APCs) and CD4+ T cells required for LDA were isolated from
PBMC by magnetic cell sorting (MACS�). PBMC were separ-
ated from whole blood as described for LTA, then monocytes
positively selected using CD14+ Microbeads (Miltenyi Biotec
Ltd, Bisley, U.K.) according to the manufacturer’s instructions.
The remaining cell fraction was then used for purification of
CD4+ T cells using the CD4+ T-cell isolation kit (Miltenyi
Biotec Ltd) according to the manufacturer’s instructions. Puri-
fied cells were quantified and viability assessed by trypan blue
dye exclusion.
Assay setup
Isolated CD4+ T cells were diluted to 1Æ25 · 106, 1Æ0 · 106,
7Æ5 · 105, 5Æ0 · 105, 2Æ5 · 105, 1Æ25 · 105 and 0 cells mL)1
in Medium B. Replicate (18, 24 or 30) 10 lL volumes of each
dilution were dispensed into wells of Terasaki microculture
plates. Isolated APCs were diluted to 3 · 105 cells mL)1 in
Medium C alone, Medium C with antigen (P. acnes or each
HSP), or Medium C with PHA, and 10-lL aliquots added to
the wells containing the CD4+ T cells. The Terasaki plates
were inverted and cultured for 3 days. High-activity
[3H]thymidine (0Æ25 lCi) was then added to each well and
incubation continued for 4 h. Microcultures were harvested
using a semi-automatic cell harvester and the amount of
[3H]thymidine incorporated into cellular DNA determined by
scintillation counting. The number of cultures positively
responding to each stimulus was calculated for each concen-
tration of CD4+ T cells. A culture was deemed to be positive
if the cpm of the stimulated CD4+ T cells was equal to, or
greater than, the mean cpm of the unstimulated CD4+ T cells
plus three times the SD. Antigen-specific CD4+ T-cell precur-
sor frequencies were then calculated.
Statistical analyses
The number of subjects whose cells responded to each anti-
gen in the LTA was analysed using the G-test (R · C and
2 · 2 tables).19 For the LDA, precursor frequency was esti-
mated by linear regression using the least squares method,
in which the negative slope of the line is equal to the
reciprocal value of the cell dose containing an average of
one precursor cell.20,21 A v2 test for goodness of fit was
performed to determine whether the data conformed to the
single-hit model.20 A P-value of > 0Æ05 indicated an accept-
able fit to the model. For presentation of the data, the
negative logarithm of the fraction of nonresponding cultures
was plotted against the number of CD4+ T cells per
culture.
Results
Peripheral blood mononuclear cell proliferative response
to Propionibacterium acnes and heat shock proteins
Following stimulation of PBMC with mycobacterial HSPs,
58Æ3% of acne patients, 10Æ0% of resolved donors and 18Æ2%
of controls responded to M. bovis BCG HSP65; 66Æ7% of acne
patients, 20Æ0% of resolved donors and 9Æ1% of controls
responded to M. smegmatis HSP70 (Fig. 1). All patients, resolved
donors and controls had a positive response to PHA (data not
shown). The proportion of positive responders to each antigen
within each group was analysed using the G-test for independ-
ence. The number of subjects whose cells responded to each
antigen was not independent of whether the subject was a
patient, resolved donor or a control. There was a significantly
(P < 0Æ05) higher proportion of positive responders to HSP65
and HSP70 in the acne patient group compared with the
respective resolved acne donor group or control group for that
antigen. The BCG vaccination status of each volunteer was
ascertained. There was no significant difference in the propor-
tion of BCG-vaccinated individuals between the acne, resolved
and control groups (data not shown).
A high proportion of individuals in each group had a signi-
ficant PBMC response to P. acnes (66Æ7% of acne patients,
80Æ0% of resolved donors, 45Æ5% of controls). However, only
a small percentage responded to either P. acnes HSP60 (10Æ0%
of acne patients, no resolved donors or controls) or P. acnes
HSP70 (10Æ0% of acne patients, 20Æ0% of resolved donors,
10Æ0% of controls; Fig. 1). G-test analysis showed the propor-
tion of positive responders to P. acnes, P. acnes HSP60 or P. acnes
Fig 1. Proportion of acne patients, resolved donors and control
individuals with positive peripheral blood mononuclear cell (PBMC)
proliferative response to each antigen. PBMC from each donor were
cultured with antigen for up to 9 days and proliferation measured
by [3H]thymidine incorporation. A stimulation index ‡ 3Æ0 was
considered positive. Data were analysed by G-test of independence.
*Indicates a significant difference (P £ 0Æ05) in the proportion of
positive responders. Mb, Mycobacterium bovis BCG; Ms, Mycobacterium
smegmatis; Pa, Propionibacterium acnes; HSP, heat shock protein.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp460–465
462 CD4+ T-cell regulation in acne resolution, H.E. Wilcox et al.
HSP70 within each group to be independent of whether the
subject was an acne patient, resolved donor or control.
Limiting dilution analysis
Mean precursor frequencies were calculated for those data that
conformed to the model of single-hit kinetics. For responses
to P. acnes, mean precursor cell frequencies for acne patients,
controls and resolved donors were one in 112890, 133836
and 135208, respectively. For responses to P. acnes HSP60, fre-
quencies were one in 124774, 510788 and 87884, respective-
ly. Frequencies for responses to P. acnes HSP70 were one in
147036, 262036 and 144243, respectively. For responses to
M. bovis HSP65, frequencies were one in 266368, 89683 and
116037, respectively. Finally, for responses to M. smegmatis
HSP70, frequencies were one in 93497, 244318 and 213560,
respectively. The mean precursor frequencies could not be
analysed for any difference between groups as the number of
donors in each group that resulted in data that fitted the
single-hit kinetics model was too small.
As a failure of the data to fit the single-hit kinetics model
could indicate regulation of the CD4+ T-cell responses, the
proportion of subjects with data that fitted the model was
recorded for each antigen (Fig. 2) and the results analysed
with the G-test of independence. The proportion of subjects
with data that fitted the single-hit kinetics model following
LDA of P. acnes-specific CD4+ T cells was not independent of
subject group, with a significantly lower number of resolved
donors (8%) having LDA data that fitted the single-hit kinetics
model compared with acne patients (36%) and controls
(50%). In contrast, the proportion of acne patients, controls
and resolved donors with data that fitted the single-hit kinetics
model following LDA of CD4+ T-cells specific for P. acnes
HSP60, P. acnes HSP70, M. bovis BCG HSP65 or M. smegmatis
HSP70 was independent of subject group. As with the LTA,
there was no significant difference in the proportion of BCG-
vaccinated individuals between the acne, resolved and control
groups (data not shown).
Owing to the increased complexity of the curves generated
by the data that did not conform to the single-hit kinetics
model, interpretation of the results was difficult. However, a
number of general patterns emerged. Several samples had data
points that appeared to form a nonlinear negative slope
(Fig. 3a) with the data points forming a rough curve as they
decreased in value. In other cases, sample data appeared to
result in a dominant positive slope (Fig. 3b). The negative
section visible on these charts occurred only between two data
points, due to the inclusion of the zero cell concentration at
the origin. Further dilutions between these points may have
revealed more of a negative slope. Other sample data appeared
to have a distinct negative slope at the lower cell concentra-
tions and a positive slope at the higher cell concentrations, as
illustrated in Figure 3c. Finally, some of the samples had data
points that formed a jagged (zigzag) pattern between adjacent
points or over a number of points as illustrated in Figure 3d.
Discussion
The early developing inflammatory lesions in acne vulgaris are
characterized by a predominantly CD4+ T-lymphocytic cellular
infiltrate and there is evidence to suggest that this represents a
type IV hypersensitivity reaction.4,5,22 Epitopes derived from
P. acnes components are thought to be the main driver of this
inflammatory response although the identity of these antigens
is unknown. We have previously hypothesized that HSPs may
have a role due to their association with other inflammatory
conditions.7–9 Therefore, this work aimed to identify differ-
ences in the proliferation of PBMC and isolated CD4+ T cells
from acne patients, controls and resolved individuals in
response to P. acnes, P. acnes HSPs and the related mycobacterial
HSPs. For both LTA and LDA, the BCG vaccination status was
compared between groups as this may have affected the results
of these assays. No significant difference was found in the vac-
cination status of each group.
Data from the LTAs showed that the proportion of acne
patients with a positive response to M. bovis BCG HSP65 and
M. smegmatis HSP70 was significantly higher than that for con-
trols or resolved donors. There was no significant difference
in the proportion of individuals with a positive response to
P. acnes or P. acnes HSPs between the three donor groups. The
increased proliferative response to mycobacterial HSPs may be
as a result of the presentation of immunoreactive species-
specific epitopes not present in the P. acnes HSPs or a lack of
regulation to shared epitopes in acne patients. P. acnes and
mycobacterial HSPs demonstrate 45–50% identity to their
human homologues at the amino acid level. The homology
between the bacterial HSPs is greater with an identity of 78%
between the P. acnes and mycobacterial HSP60 and 67% for
HSP70.8 Species-specific regions of these proteins may there-
fore be responsible for the differences in responses.
Fig 2. Proportion of acne patients, resolved donors and control
individuals with limiting dilution analysis data fitting the single-hit
kinetics model for determination of precursor frequency to each
antigen. Purified CD4+ T cells were cultured for 3 days with antigen,
proliferation measured by [3H]thymidine incorporation and precursor
frequencies calculated. Data were analysed by G-test of independence.
*Indicates a significant difference (P £ 0Æ05) in the proportion of
positive responders. Mb, Mycobacterium bovis BCG; Ms, Mycobacterium
smegmatis; Pa, Propionibacterium acnes; HSP, heat shock protein.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp460–465
CD4+ T-cell regulation in acne resolution, H.E. Wilcox et al. 463
These results do not necessarily indicate that P. acnes HSPs
do not have a role to play in acne. Individuals should have the
potential to respond to P. acnes-specific epitopes of the HSPs
but this response appeared to be regulated in all three groups
of individuals in this study. Regulation of the response to
P. acnes HSPs in resolved individuals and controls may explain
their lack of acne. Regulatory cells may inhibit inflammatory
immune responses to P. acnes HSP60 and may represent per-
ipheral tolerance to this antigen as a mechanism of preventing
cross-reactivity with human HSPs.23 The low proportion of
patients with acne responding to P. acnes HSPs may have been
due to a degree of regulation having already developed in
these individuals who were at the upper end of the age range
(when acne is most likely to resolve). A further explanation
for these results is that this study used cells from peripheral
blood and the regulation of responses locally in the skin may
occur only at a later stage in the disease process. Regulation
could be detected in peripheral blood prior to resolution of
the disease. Therefore, further studies are required using
younger volunteers, who have only recently developed acne,
to determine if P. acnes HSPs have a role in the disease.
Analysis of T-cell responses by LDA allows the frequency
of T cells responding to a particular antigen to be deter-
mined and can also reveal the activity of regulatory cells.
The proliferative response of purified CD4+ T cells from
individuals in each subject group to P. acnes, P. acnes HSPs
and mycobacterial HSPs was tested by LDA. Mean precursor
frequencies could be calculated only for those data that fit-
ted the single-hit kinetics model. From the results it can be
seen that the proportion of samples that did fit this model
was small. Therefore, differences between groups could not
be analysed. The failure of data to fit single-hit kinetics can
indicate regulatory effects within the cell population being
tested. Therefore, the proportion of subjects in each group
that demonstrated single-hit kinetics was analysed by the
G-test as for LTA. The finding that a significantly lower
number of resolved donors had LDA responses to P. acnes
that fitted single-hit kinetics compared with other groups
could indicate the presence of a regulatory subset of CD4+
T cells within the purified CD4+ T-cell population. This is
an important finding and gives support to our hypothesis
that downregulation of the CD4+ T-cell-mediated immune
(a) (b)
(d)(c)
Fig 3. Examples of titration curves obtained following limiting dilution analysis of CD4+ T cells with P. acnes and heat shock proteins. Dilutions
of purified CD4+ T cells were stimulated with antigen and proliferation was measured by [3H]thymidine incorporation. Graphs are the negative
logarithm of the fraction of nonresponding cultures plotted against the number of CD4+ T cells per culture. (a) Nonlinear negative slope,
(b) dominant positive slope, (c) V-shaped with negative slope at the lower cell concentrations and positive slope at the higher concentrations,
(d) zigzag. The linear regression line (dashed line) was calculated by the least squares method and the goodness of fit of the data to the single-hit
kinetics model was assessed using the v2 test.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp460–465
464 CD4+ T-cell regulation in acne resolution, H.E. Wilcox et al.
response to P. acnes is associated with the spontaneous reso-
lution of acne.
Differences in the shape of LDA titration curves is not
unique to this study. Zigzag, V-shaped, saw-tooth curves and
other descriptions have been reported.20,24,25 Concave nega-
tive slopes (Fig. 3a) have been described as multi-hit and
reflect the need for an interaction between two or more cells
of the same type in order to produce a measurable positive
response. Positive slopes can result from interaction between a
high frequency of stimulatory cells and a lower frequency of
inhibitory (regulatory) cells as demonstrated in Figure 3b. The
presence of regulatory cells in the cell populations tested could
therefore account for these nonlinear plots. No particular pat-
tern was dominant and the data indicate that the response to
P. acnes is complex and varies between individuals.
Further analysis of CD4+ T-cell responses to P. acnes is
required to define in greater detail the proliferative response
and the involvement of different T-cell subsets. If the presence
of regulatory T cells was confirmed to have a role in acne
resolution then this would have important clinical implica-
tions. The identification of those P. acnes antigens important in
mediating the inflammatory response, and to which this
response is regulated in resolution, would represent a signifi-
cant step in the development of an immunotherapy for acne
whereby the induction of tolerance to the antigen(s) could be
a therapeutic option.
Acknowledgments
This study was funded by the Leeds Foundation for Dermato-
logical Research.
References
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CD4+ T-cell regulation in acne resolution, H.E. Wilcox et al. 465
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07642.x
Tight junction components occludin, ZO-1, and claudin-1,-4 and -5 in active and healing psoriasisS. Peltonen,* J. Riehokainen,*� K. Pummi*� and J. Peltonen*�
*Department of Dermatology, University of Turku, PL 52, 20521 Turku, Finland
Departments of �Medical Biochemistry and �Anatomy, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland
CorrespondenceSirkku Peltonen.
E-mail: sirkku.peltonen@tyks.fi
Accepted for publication25 August 2006
Key wordsclaudin, involucrin, occludin, psoriasis, tight
junction, ZO-1
Conflicts of interestNone declared.
Summary
Background Cells of the granular layer are interconnected by tight junctions (TJs) innormal epidermis. The structural proteins of epidermal TJs include occludin, ZO-1,and claudin-1 and -4.Objectives Our aim was to correlate the expression of TJ components with keratino-cyte differentiation using psoriasis as a model of premature keratinization.Methods The distribution of TJ proteins was evaluated in the skin of nine patientswith psoriasis. Punch biopsies were taken from perilesional skin, from active pso-riasis plaques, and from healed, previously lesional locations. The punch biopsieswere analysed using indirect immunolabelling for ZO-1, occludin and claudin-1,-4 and -5. In addition, epidermal samples were analysed by reverse transcrip-tion–polymerase chain reaction for claudin-1, -4 and -5 mRNAs.Results Claudin-5 was localized to the granular cell layers of normal control skinas well as perilesional and lesional psoriatic epidermis. This was unexpected, asprevious studies have not detected claudin-5 in the epidermis. Occludin andZO-1 were expressed in the granular cell layer in psoriatic perilesional epidermis.In the psoriasis plaques, ZO-1 and occludin were detected in a wider zoneextending from the granular layer to the middle spinous cell layers. In healedpsoriasis plaques, the expression of occludin and ZO-1 resumed a normal-lookingprofile, being restricted to the upper epidermis only. Claudin-1 and -4 did notshow marked changes in psoriasis compared with normal skin.Conclusions The results demonstrate claudin-5 in normal epidermis and psoriaticskin, and abnormal distribution of occludin and ZO-1 in psoriasis plaques. Clin-ical healing of aberrant keratinization is associated with restoration of the normaldistribution of occludin, ZO-1 and also involucrin.
Molecular characterization of numerous tight junction (TJ)
proteins1–3 and the availability of antibodies to various TJ
components have focused increasing attention on TJs in var-
ious tissues, and have resulted in recognition of TJs also in
stratified epithelia such as the epidermis.4–7 Subsequently, TJs
have been studied in human diseases, including those affecting
the epidermis.5,8–12 Immunoelectron microscopy and immu-
nohistochemistry have shown that TJs are located in the gran-
ular cell layer and that they contain occludin, ZO-1, ZO-2 and
claudin-1 and -4.4–7 The above findings, and results obtained
from transgenic mice, have prompted re-evaluation of the role
of TJs in the regulation of the paracellular diffusion barrier of
the epidermis.13–18 The role of TJs in the formation of the
epidermal barrier has been emphasized by experiments using
transgenic mouse models for claudin-1 and claudin-6.14,15
The results showed that claudin-1 knockout mice and mice
overexpressing claudin-6 had epidermal barrier dysfunc-
tion.14,15 On the other hand, occludin knockout mice did not
show signs of affected barrier function or of morphological
alterations in the ultrastructure of intestinal TJs, indicating a
crucial function for claudins in the function of TJ seals.17 The
role of claudins in the assembly of TJs has further been under-
lined by studies showing that ZO-1–3 can be recruited to
claudin-based TJs in the absence of occludin.19
We and others have previously described altered localization
of TJ proteins in conditions where the horny layer is missing
or abnormal, such as in developing skin, healing wounds, or
psoriasis.5,6,10–13 Observations on psoriatic skin or the hyper-
proliferative zone at the border of healing blisters also indicate
that aberrant keratinocyte differentiation is linked with chan-
ges in the expression profile of TJ components. More specific-
ally, ZO-1 and occludin have been detected in the acanthotic
� 2006 The Authors
466 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp466–472
spinous cell layers in psoriasis lesions.5,11 Previous results have
also suggested that the expression of occludin may be used as
a differentiation marker for keratinocytes.11 To date, the
expression of claudin-1, -4 and -5 has not been analysed in
psoriasis. The present work adds to our knowledge of psoriasis
and TJs by extending the study to healing of psoriasis and
expression of claudin-1, -4 and -5. This study also correlates
the expression of TJ proteins with the expression of involu-
crin, another differentiation marker.
Materials and methods
Biopsy samples
Nine patients with psoriasis, five women and four men, age
range 35–67 years, were included in the study. The study
took place at the Department of Dermatology, Turku Univer-
sity Central Hospital, with the approval of the joint Ethic
Committee of the University of Turku and the Turku Univer-
sity Hospital, and with written consent of the patients. All
patients had an activation of previously stable plaque-type pso-
riasis. Punch biopsies, 3 mm in diameter, were taken from:
(i) a typical scaly psoriasis plaque, (ii) perilesional skin at
~7 mm distance from the edge of the biopsied lesion, and
(iii) the location of the original lesion after therapy, 6–
8 weeks later. The treatment included potent corticosteroid
cream for 1–2 weeks, followed by narrowband (wavelength
311 nm) ultraviolet B phototherapy for 15–18 sessions during
5–6 weeks. If the plaque showed no scaling even though it
displayed slight erythema or postinflammatory pigmentation it
was considered as a healed plaque. Three control samples were
obtained from otherwise healthy patients undergoing plastic
surgery. The skin samples were frozen in liquid nitrogen and
stored at )70 �C.
Primary antibodies
The following primary antibodies were used: affinity-purified
rabbit polyclonal antibodies to human claudin-1 (51–9000),
claudin-5 (34–1600), ZO-1 (61–7300) and occludin (71–
1500), and mouse monoclonal antibodies to human claudin-4
(32–9400), claudin-5 (35–2500) and ZO-1 (33–9100), all
from Zymed Laboratories (South San Francisco, CA, U.S.A.).
Mouse monoclonal antibody to involucrin (MS-126-P0) was
obtained from NeoMarkers (Fremont, CA, U.S.A.). Nonimmu-
nized mouse (2025) and rabbit (2027) IgG were purchased
from Santa Crutz Biotechnology (Santa Cruz, CA, U.S.A.).
Indirect immunofluorescence labelling
Punch biopsy samples were cut into 7-lm cryosections on
silanated glass slides and fixed in 100% methyl alcohol at
)20 �C for 10 min. To prevent nonspecific binding, the sam-
ples were preincubated in 1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS) for 15 min. Antibodies were
diluted in 1% BSA in PBS, and incubated on the samples for
20 h at 4 �C. Following six 5-min washes in PBS, the samples
were incubated with secondary antibodies for 1 h at 20 �C.
The samples were washed in PBS and mounted with Glycergel
(Dako, Glostrup, Denmark). Secondary antibodies for indirect
immunofluorescence (IIF) were tetramethylrhodamine isothio-
cyanate (TRITC)-conjugated swine antirabbit (R0156) and
rabbit antimouse IgG (R0270) from Dako, or Alexa Fluor
488-conjugated goat antimouse IgG (A-11029) from Molecu-
lar Probes (Eugene, OR, U.S.A.). In double labellings, involu-
crin antibody was mixed with polyclonal antibodies to ZO-1
or occludin. After the incubation and washes, TRITC-conju-
gated swine antirabbit IgG was mixed with Alexa Fluor 488-
conjugated goat antimouse IgG (A-11029). In control immu-
noreactions, primary antibodies were replaced with 1% BSA in
PBS or nonimmunized mouse or rabbit IgG.
Microscopy
IIF labellings were examined and photographed using a Leica
DMRB fluorescence microscope, equipped with a Leica DC
300F camera and Leica IM50 (version 1.20) software (Leica
Microsystems AG, Heerbrugg, Switzerland). Resolutions of
images were 1950 · 1545 pixels and images were saved in
tiff format. Confocal laser scanning microscopy was carried
out using a Zeiss LSM 510 META confocal microscope
equipped with argon ion and helium–neon lasers (Zeiss, Jena,
Germany) and LSM 3.0 software. The objective magnification
used was · 20 (numeric aperture 0Æ5), · 40 (oil immersion,
numeric aperture 1Æ3) or · 63 (oil immersion, numeric aper-
ture 1Æ4). For excitation of TRITC the 543 line was used. The
resolutions of original images were 1024 · 1024 pixels and
images were saved in tiff format and assembled into panels
using Adobe Photoshop or Corel Draw software.
RNA isolation and reverse transcription–polymerase
chain reaction
To isolate RNA from whole skin, a 4-mm punch biopsy was
taken from the upper arm of an otherwise healthy woman.
RNA was isolated by acid guanidinium isothiocyanate–chloro-
form–phenol extraction.20 Epidermal samples for RNA isola-
tion were obtained from three healthy controls (age range
35–49 years). In analogy, five psoriasis plaques from five
patients with psoriasis were chosen for sampling. Four of the
patients (men aged 31–45 years) had plaque-type psoriasis
that had been untreated for several months. The fifth patient
(a woman aged 41 years) had guttate-type psoriasis. The
extensor aspect of the lower arm close to the elbow, which is
a typical location for psoriasis plaques, was first wiped with
70% ethanol. Then the normal skin of the control individuals
and a psoriasis plaque of patients with psoriasis were gently
scraped using a disposable curette, carefully avoiding bleeding,
on an area of about 10 cm2. The corneal layer and the epider-
mal keratinocytes that were detached by scraping were imme-
diately submerged from the curette into the RNAlater RNA
Stabilization Reagent (76104; Qiagen, Valencia, CA, U.S.A.).
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp466–472
Tight junctions in psoriatic skin, S. Peltonen et al. 467
RNA was then isolated using RNeasy Mini Kit (74104;
Qiagen) according to the protocol provided by the manufac-
turer. The RNA was transcribed into single-stranded DNA in a
20-lL reaction containing 200 U of SuperScript II Reverse
Transcriptase (18064–022; Invitrogen, Carlsbad, CA, U.S.A.),
1 lmol L)1 of each of the four deoxynucleotides, Random
Hexamers (C1181; Promega, San Luis Obispo, CA, U.S.A.),
20 U of RNasin� (N2111; Promega) and 0Æ01 mol L)1 di-
thiothreitol in the first-strand buffer. The reaction was incuba-
ted at 42 �C according to the first-strand synthesis protocol
provided by the manufacturer. The cDNA yielded by reverse
transcription (RT) reactions was used as a template for poly-
merase chain reaction (PCR). PCRs for claudin-1, -4 and -5
and glyceraldehyde-3-phosphate dehydrogenase were per-
formed using a MultiGene-12 RT–PCR profiling kit (PH-083B;
SuperArray Bioscience Co., Frederick, MD, U.S.A.) detecting
claudin-1–11, according to the protocol provided by the
manufacturer.
Results
Immunohistochemical mapping of five TJ proteins, claudin-1,
-4, -5, ZO-1 and occludin, was performed in skin samples of
nine patients with psoriasis and three control samples. Punch
biopsies of apparently normal perilesional skin, psoriasis
plaque, and the same psoriasis plaque in the clinically healed
phase, were analysed. In perilesional epidermis the localization
of occludin, ZO-1 and claudin-1 and -4 corresponded to that
described earlier for normal skin (Figs 1, 2).4–6 Specifically,
occludin and ZO-1 were located to the granular cell layer
(Fig. 2a,g) while claudin-1 was distributed to all living cell
layers, the basal cell layer being only faintly labelled (Fig. 1a).
Claudin-4 was mainly localized to the upper epidermis
(Fig. 1d). Unexpectedly, all samples from patients with psori-
asis, both lesional and perilesional, displayed fine intercellular
labelling for claudin-5 in the cell–cell contact zones of the
granular cell layer (Fig. 1g–i). To confirm the existence of
claudin-5 in epidermis, the same psoriasis samples were im-
munolabelled for polyclonal rabbit antibody to claudin-5. This
resulted in similar findings as obtained with the monoclonal
antibody (Fig. 3a). The parakeratotic foci and their surround-
ings were especially intensely labelled (Fig. 3a,b). The pres-
ence of claudin-5 in psoriatic epidermis prompted us to
perform further labelling for skin samples of healthy patients
with monoclonal and polyclonal antibodies to claudin-5. Con-
focal laser scanning microscopy revealed thin lines of positive
labelling for claudin-5 in the granular cell layer (Fig. 3c,d). As
described earlier,6,19 claudin-5 was also detected in the dermal
blood vessel walls in psoriasis and control samples. Control
immunoreactions of normal skin and psoriasis samples with
isotype-matched mouse and rabbit antibodies did not show
labelling in intercellular contact zones in normal or psoriatic
epidermis (Fig. 3e–h).
Further to confirm the expression of claudin-5 in psoriatic
epidermis, RT–PCR was performed using RNA isolated
from normal skin, normal epidermis and psoriatic epidermis.
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Perilesional skin
Claudin1
Claudin4
Claudin5
Psoriasis plaque Healed psoriasis plaque
Fig 1. Indirect immunofluorescence labelling
of perilesional skin (left column), psoriasis
plaque (middle column) and healed psoriasis
plaque (right column) for claudin-1, -4 and
-5. (a–c) Claudin-1 labels the granular and
spinous cell layers, the basal cell layer being
mostly negative in all samples. (d–f) Claudin-
4 is mainly expressed in the granular cell
layer but is detectable also in spinous cell
layers. (g–i) Claudin-5 is expressed only in
the granular cell layer in perilesional, lesional
(arrows) and healed psoriatic epidermis, in
addition to being present in dermal blood
vessel walls. Scale bars ¼ 50 lm.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp466–472
468 Tight junctions in psoriatic skin, S. Peltonen et al.
A commercial kit designed for the detection of claudins was
utilized for PCR and showed expression of claudin-1 and -4
in all samples. Claudin-5 was detected in the whole skin sam-
ple and three of the five psoriasis samples, but not in any of
the control epidermis samples (Fig. 4).
In the psoriasis plaque, the distribution of claudin-1 was
extended to all living cell layers, excluding the basal layer,
thus resembling the distribution profile described for healthy
skin (Fig. 1b). Anticlaudin-4 antibody labelled more numer-
ous spinous cell layers in psoriasis plaque than in normal skin
(Fig. 1e). In analogy to our previous results, ZO-1 and occlu-
din in psoriasis plaques displayed a wider distribution than in
control or perilesional epidermis. Specifically, ZO-1 and occlu-
din were detected in psoriasis plaques in the granular and in
the middle spinous cell layers (Fig. 2b,h).
The expression of involucrin, an early differentiation mar-
ker, was compared with the localization of ZO-1 and occlu-
din. Double immunolabellings showed that in perilesional
epidermis, involucrin, ZO-1 and occludin colocalized in the
granular cell layer (Fig. 2d,j). The expression of involucrin by
granular cells is in agreement with previous findings showing
that involucrin can be expressed by granular cells in psoriasis.
The results showed that, in psoriasis plaques, involucrin dis-
played a more extended shift of expression to the spinous
layer than ZO-1 or occludin. In more detail, the expression of
ZO-1 and occludin was localized to the middle spinous cell
layers while involucrin was detected also in the lower spinous
cell layers (Fig. 2e,k).
Healing of psoriasis was associated with the restoration of
the expression of involucrin, ZO-1 and occludin exclusively in
the granular cell layers (Fig. 2f,l). The expression patterns of
claudin-1, -4 and -5 remained essentially the same in the
healed and in the active psoriasis plaques (Fig. 1c,f,j,i).
Discussion
The results of the present study demonstrate markedly altered
expression profiles of occludin and ZO-1 in the epidermis of
psoriasis plaques compared with normal skin. Interestingly,
claudin-5 was detected in the epidermis of normal skin,
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j) (k) (l)
Normal perilesional skin Psoriasis plaque Healed psoriasis plaque
Occludin
Occl+INV
ZO1+INV
ZO1
Fig 2. Indirect immunofluorescence labelling
of perilesional skin (left column), psoriasis
plaque (middle column) and healed psoriasis
plaque (right column) for ZO-1, occludin and
involucrin. (a,d,g,j) In perilesional skin, ZO-1
and occludin are expressed in the granular cell
layer, corresponding to the localization of
involucrin. (b,e,h,k) In psoriasis plaque, ZO-1
and occludin are spread to the upper spinous
cell layers while expression of involucrin is
detectable even in the lowest spinous cell
layers. (c,f,i,l) In healed psoriasis plaque, the
expression of ZO-1 and occludin is mainly
detected in the granular cell layer, along with
involucrin. However, tight junction proteins
can still be seen scattered between some
uppermost spinous cell layers. Scale bars ¼50 lm.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp466–472
Tight junctions in psoriatic skin, S. Peltonen et al. 469
psoriasis plaques and perilesional skin. This is an unexpected
finding as previous studies have reported that normal epidermis
is negative for claudin-5, while claudin-5 is a component of TJs
of dermal endothelial cells.6,7 In the present study, claudin-5
was expressed more intensely in the upper epidermis of the pso-
riasis plaque than in the normal epidermis. Specifically, most
intense labelling was detected in and near the vicinity of the
parakeratotic foci in psoriatic epidermis. In perilesional and
healed epidermis claudin-5 was strictly restricted to the granular
cell layer, the distribution resembling that of occludin. Claudin-
5 immunolabelling was detectable only using a microscope with
high-quality immunofluorescence optics and confocal laser
scanning modality. As the amount of claudin-5 in the epidermis
is very low compared with claudin-1 and -4, it is understand-
able that epidermis has been considered to be negative for clau-
din-5. The localization of claudin-5 to the cell–cell contact sites
of the granular cells suggests that claudin-5 can be a component
of the epidermal TJs in epidermis.
(a)
(c) (d)
(e) (f)
(g) (h)
Fig 3. Indirect immunofluorescence labelling of lesional psoriatic skin
and normal control skin for claudin-5 and nonimmunized mouse
and rabbit IgG. (a,b) Claudin-5 in psoriasis plaque as demonstrated
using polyclonal rabbit antibody to claudin-5. (a) Indirect
immunofluorescence and (b) phase contrast image showing claudin-5
in the granular cell layer in the vicinity of a focally parakeratotic area.
(c) Normal skin labelled with a polyclonal antibody to claudin-5.
(d) Normal skin labelled with a monoclonal antibody to claudin-5.
Both antibodies reveal claudin-5 in the granular cell layer.
(e–h) Control labellings using IgG from nonimmunized mouse and
rabbit. (e,f) Psoriasis plaque and (g,h) normal skin labelled for rabbit
(e,g) or mouse (f,h) IgG do not show specific labelling patterns. Scale
bars ¼ 50 lm (a,b,e–h), 25 lm (c,d).
(a)
Wholeskin
GAPDH
Cl-5Cl-4Cl-1
Normalepidermis
Psoriaticepidermis
Psoriaticepidermis
(b)
(c)
(d)
Fig 4. Reverse transcription–polymerase chain reaction (PCR) for
claudin (Cl)-1, -4 and -5 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) in samples of (a) normal whole skin,
(b) normal epidermis and (c,d) two psoriatic epidermis samples.
PCR for claudin-1 and -4 and GAPDH shows PCR products with an
expected size of 160–210 bp, claudin-5 yielding a larger fragment.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp466–472
470 Tight junctions in psoriatic skin, S. Peltonen et al.
For RT–PCR, RNA was isolated from the material scraped
from the epidermis with a disposable curette. To our know-
ledge, this technique to obtain cells for RNA isolation from
the upper epidermis has not been published previously. RT–
PCR expectedly revealed claudin-1 and -4 in all epidermal
samples. However, claudin-5 was detected only in samples
from active, untreated psoriatic skin and control samples of
normal skin including dermal blood vessels. This suggests that
the expression level of claudin-5 in normal epidermis was
below the detection level of this technique.
The function of claudins has been related to the size-select-
ive sieving of molecules by TJs. For example, endothelial cells
contributing to the blood–brain barrier express at least two
distinct claudins: claudin-5 and -12. In claudin-5-null mice,
the brain endothelium is size-selectively leaky for small pro-
teins (<~800 Da), but not for large proteins.21,22 It is feasible
to speculate that the upregulation of claudin-5 in psoriatic epi-
dermis may serve to compensate for the dysfunction of the
lipid barrier by adding to the tightness of the barrier against
small molecules.
The barrier function in psoriasis is impaired potentially due
to different reasons: (i) alterations in the lipid structures of
the cornified cell layer23–25 and (ii) abnormal differentiation
of keratinocytes leading to prematurity of the cornified cell
envelope.26–28 Previous results have shown that barrier dys-
function leads to premature expression of involucrin.29 In
addition, several other differentiation markers, such as small
proline-rich proteins, cystatin A and transglutaminase 1, are
more conspicuously expressed in psoriasis while late differen-
tiation markers such as profilaggrin and loricrin are dimin-
ished or absent.29,30
In a recent report, experimental overexpression of claudin-6
in transgenic mouse epidermis induced the expression of clau-
din-5 and -8 in the same tissue.16 These mice also displayed
aberrations in epidermal development including hyperkeratosis
and atypical patterns of keratin expression.15,16 The results of
the present and previous studies thus suggest that there is
cross-talk between the different components of the epidermal
barrier: a defect in the lipid barrier can alter expression pro-
files of genes encoding TJ components and other proteins of
the epidermis.
Our earlier results have shown the spreading of ZO-1 to the
spinous cell layers in psoriasis plaques.5 In addition to being
located in TJs, ZO-1 may also be distributed with adherens
junctions in spinous cell layers as ZO-1 can associate with
a-catenin at the adherens junction and occludin at the TJ.31,32
The results of the present study also showed that healing of
psoriasis is associated with the reconstitution of expression of
ZO-1 and occludin to the granular cell layer.
The expression of claudin-1 is not altered in psoriasis, in
the perilesional psoriatic skin, or in the healed plaque, com-
pared with normal skin. Claudin-1 and -4 are abundantly
expressed in the intercellular contacts of upper epidermis.
These claudins may be associated with TJ-like structures that
have been described in the suprabasal cell layers of several
stratified epithelia.32 Claudin-4 was detected in more numer-
ous spinous cell layers in psoriasis than in normal epider-
mis, which may be explained by the increased number of
spinous cell layers in psoriasis. The expression profile of TJ
proteins in the perilesional skin resembled that in normal
skin.
Taken together, the results of the present study show over-
expression of three different types of TJ proteins which, in
combination, are sufficient to form functional TJs. The biolo-
gical significance of the potentially increased number of TJs
may relate to the defective diffusion barrier of the horny layer
in psoriasis plaques. Thus, the expression of occludin and
ZO-1 in acanthotic spinous cell layers and of claudin-5 in the
granular cell layer suggests a compensatory mechanism for TJs
under conditions when the horny layer is not fully function-
ing as a barrier.
The results of the present study highlight the fact that the
expression of the different TJ proteins is separately regulated
but, on the other hand, coordinated expression of selected TJ
components may serve to compensate for the defective barrier
function observed in psoriasis. The results of the present study
also demonstrate that changes in the expression profiles of TJ
components studied here are transient and are associated with
active psoriasis, with the exception of claudin-5 which is
expressed in perilesional and healed psoriasis. These findings
on altered gene expression profiles of TJ proteins may reflect
differentiation/dedifferentiation of epidermal cells in psoriasis,
or compensatory responses to impaired barrier function in
psoriasis plaques. The present study provides evidence for
both lines of thinking.
Acknowledgments
This work was supported by the Department of Dermatology,
Turku University Central Hospital, and Finnish Society of Der-
matology. We thank Maria Alanne and Jouko Sandholm for
assistance with confocal microscopy.
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp466–472
472 Tight junctions in psoriatic skin, S. Peltonen et al.
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07652.x
Cutaneous mucinosis associated with dermatomyositis andnephrogenic fibrosing dermopathy: fibroblast hyaluronansynthesis and the effect of patient serumM. Edward, L. Fitzgerald,* C. Thind,* J. Leman� and A.D. Burden*
Section of Squamous Cell Biology and Dermatology, Division of Cancer Sciences and Molecular Pathology, Robertson Building, University of Glasgow, Glasgow
G12 8QQ, U.K.
*Department of Dermatology, Western Infirmary, Dumbarton Road, Glasgow, U.K.
�Falkirk and District Royal Infirmary, Majors Loan, Falkirk, U.K.
CorrespondenceMichael Edward.
E-mail: m.edward@clinmed.gla.ac.uk
Accepted for publication12 September 2006
Key wordsdermatomyositis, hyaluronan, mucinosis,
nephrogenic fibrosing dermopathy
Conflicts of interestNone declared.
Summary
Background Dermal mucin is an amorphous gelatinous substance composed primar-ily of hyaluronan (HA) and sulphated glycosaminoglycans (GAGs). In primarycutaneous mucinosis, accumulation of mucin is a characteristic feature of lichenmyxoedematosus, scleromyxoedema and reticular erythematous mucinosis. Sec-ondary mucinoses are disorders where mucin deposition is an additional finding,and deposition is associated with lupus erythematosus, dermatomyositis, sclero-derma and granuloma annulare. The underlying cause of the abnormal mucindeposition is unknown. An increasing number of cases of a fibromucinousscleromyxoedema-like disorder associated with renal dysfunction, recently termednephrogenic fibrosing dermopathy (NFD), is being reported.Objectives To examine the synthesis of sulphated GAGs and HA by fibroblastsderived from uninvolved and involved skin of a patient with dermatomyositisand two patients with NFD, and the effect of patient serum.Methods GAGs were quantified by a radiometric assay and HA was determined byan enzyme-linked HA-binding protein assay.Results We found that fibroblasts derived from active lesions of NFD synthesizeelevated levels of GAGs, and in particular HA, compared with normal controls,while serum from the patient with dermatomyositis and the two patients withNFD stimulates GAG synthesis, including HA synthesis, by both control andpatient fibroblasts.Conclusions Fibroblasts from patients with active NFD are either activated to syn-thesize elevated levels of HA or contain another cell type, possibly derived fromcirculating fibrocytes. In both disorders, there is additionally a serum-derived fac-tor that stimulates production of sulphated GAGs and HA by fibroblasts.
The cutaneous mucinoses are a highly heterogeneous group
of disorders in which elevated levels of mucin are found in
the skin, predominantly in the dermis. Dermal mucin is an
amorphous gelatinous substance that is thought to be
composed primarily of hyaluronan (HA) and sulphated
glycosaminoglycans (GAGs), and is normally visualized
histochemically by staining tissue sections with alcian blue at
pH 2Æ5.
Primary cutaneous mucinoses are characterized by mucin
deposition as the major histological feature, such as in lichen
myxoedematosus (papular mucinosis), scleromyxoedema,
reticular erythematous mucinosis, and many more. Secondary
mucinoses are disorders where mucin deposition is an
additional finding, and deposition is found associated with
disorders including lupus erythematosus, dermatomyositis,
scleroderma and granuloma annulare.1 Many of these primary
and secondary mucinoses have considerable overlap in their
characteristics, but the underlying cause of the abnormal
mucin deposition is unknown.
Increased levels of serum immunoglobulins in mucinoses
such as lichen myxoedematosus, scleromyxoedema and Graves
disease-associated pretibial myxoedema suggest that these anti-
bodies may be involved in the increased mucin deposition.
However, it has been reported that fibroblast mucin
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp473–479 473
production is not affected by removal of the IgG paraprotein
from lichen myxoedematosus patient serum2 or removal of
the autoantibodies in Graves disease-associated pretibial myx-
oedema.3 This suggests that other factors present in serum are
responsible. Pandya et al.4 have demonstrated that fibroblasts
from a patient with papulonodular mucinosis associated with
systemic lupus erythematosus produced more GAGs than con-
trol cells, and in addition, GAG synthesis was increased in
control cells exposed to patient serum.
Recently there have been reports of scleromyxoedema-like
cutaneous changes appearing following renal dialysis,5,6 now
termed nephrogenic fibrosing dermopathy (NFD), and the
two patients from whom we have obtained biopsies and
serum both receive dialysis, although it likely that it is renal
failure rather than dialysis that is the trigger as some lesions
develop in patients not on dialysis.7 NFD fibromucinosis may
also occur in patients with liver disease,8 suggesting that there
may be an identical aetiological factor in renal and liver dis-
ease. In patients on peritoneal dialysis, levels of transforming
growth factor (TGF)-b1 in dialysate increase while levels of
basic fibroblast growth factor (bFGF) increase in dialysate
during peritonitis.9 In addition, several studies have documen-
ted an interaction between TGF-b1 and bFGF resulting in
stimulation of skin fibroblasts and irreversible fibrosis.10
Reports of mucinosis associated with dermatomyositis are
rare, although mucin deposits in cutaneous lesions are prob-
ably frequent when adequate staining techniques are
employed.11
Here we have examined the synthesis of sulphated GAGs
and HA by fibroblasts derived from a patient with dermato-
myositis and from two patients with NFD, and the effect of
patient serum on GAG synthesis by patient and control fibro-
blasts.
Materials and methods
Patient with dermatomyositis
A 31-year-old woman presented with a slightly itchy rash on
the flexural aspects of the forearms and anterior chest of
6 months’ duration. This consisted of small coalescing, skin-
coloured dermal papules. A skin biopsy revealed dermal
accumulation of mucin without any epidermal changes or
inflammatory infiltrate. A diagnosis of papular mucinosis was
made.
On review several weeks later she had developed a viola-
ceous rash involving the forehead, eyelids, cheeks and chin.
Plaques consistent with Gottron’s papules were seen overlying
metacarpophalangeal (MCP) and interphalangeal joints of both
hands in addition to a violaceous rash streaking down the
extensor tendons. Nail fold telangiectases were also evident.
The papular eruption remained unchanged. Biopsy of a Got-
tron’s papule overlying the fifth MCP joint showed smudging
of the dermoepidermal junction and a chronic inflammatory
cell infiltrate within the dermis. Staining with alcian blue was
positive for mucin. Clinical examination revealed no apparent
muscle weakness. Creatine kinase was within the normal
range. Magnetic resonance imaging of the pelvic girdle
musculature did not reveal any evidence of an inflammatory
myopathy. Antinuclear antibody was not significantly raised at
a titre of 1 : 40. A diagnosis of amyopathic dermatomyositis
was made. Extensive screening including pelvic examination,
Pap smear, computed tomography of the abdomen and pelvis,
and serum tumour markers failed to uncover any underlying
internal malignancy. Both the dermatomyositis and papular
mucinosis have been controlled by a prolonged course of oral
steroids.
Nephrogenic fibrosing dermopathy patient 1
A 49-year-old woman presented with nephrotic syndrome
with rapid progression to advanced renal failure over a
2-month period. She had a history of ulcerative colitis for
which she was receiving treatment with mesalazine. Renal
biopsy showed focal segmental glomerulonephritis. Within
weeks of starting haemodialysis, she developed induration of
the skin of the anterior abdominal wall, anterior upper thighs,
antecubital fossae and dorsal forearms. Skin biopsy showed an
increased number of fibroblasts in the dermis, fibrosis, and
the deposition of extracellular material that stained positively
with alcian blue. Her skin condition has remained largely
unchanged over 4 years of observation.
Nephrogenic fibrosing dermopathy patient 2
A 61-year-old woman developed acute on chronic renal fail-
ure associated with renal calculi requiring haemodialysis.
Shortly after beginning haemodialysis, she developed pain and
stiffness in the hands associated with marked swelling and
induration of the skin. Skin biopsy revealed dermal accumu-
lation of mucin. Over the subsequent 4 years, the skin
improved slightly without any specific intervention.
Controls
Fibroblasts were established from forearm punch biopsies
taken from a healthy 29-year-old woman and a healthy
42-year-old man. Serum was prepared from blood taken from
these two healthy control volunteers and from two women
(aged 52 years and 59 years) on dialysis who did not exhibit
any fibrotic disorders.
Fibroblasts
Fibroblasts were established from patient and control biopsies
using the explant technique. Tissue was finely chopped, placed
in a 25-cm2 flask with 1Æ5 mL medium [minimum essential
medium (MEM)/10% fetal calf serum (FCS); Invitrogen, Paisley,
U.K.], and incubated at 37 �C for 3 days before addition of
a further 3 mL of medium. Once established, fibroblasts
were routinely subcultured and expanded before freezing.
Fibroblasts were subsequently used between passages 5 and 9.
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474 Nephrogenic fibrosing dermopathy and hyaluronan, M. Edward et al.
Patient and control serum
Blood from controls and patients was withdrawn into poly-
propylene tubes, and allowed to clot overnight at 4 �C. The
serum was separated from the clot, and centrifuged at
2000 g for 20 min and filtered through 0Æ45 lm filters prior
to freezing.
Glycosaminoglycan analysis
Total GAG synthesis (sulphated GAGs and HA) was deter-
mined by incubating the cells with 3H-glucosamine and 35SO4
(Amersham Biosciences, Amersham, U.K.). Twenty-four-well
plates containing confluent patient or control skin fibroblasts
were washed with 1Æ0 mL serum-free (SF)-MEM/well, and
incubated with SF-MEM for 2 h prior to radiolabelling to
ensure there was no residual FCS present. Then 0Æ6 mL of
medium containing either SF-MEM (for comparison of GAG
synthesis by different fibroblasts) or appropriate patient or
control serum, 3H-glucosamine (5 lCi mL)1) and 35SO4
(20 lCi mL)1) was added to triplicate wells and the cultures
incubated for 24 h at 37 �C, after which the contents of each
well, together with 2 · 100 lL phosphate-buffered saline
(PBS) washes, were transferred to 1Æ5-mL Eppendorf tubes.
The cells were detached with trypsin and counted in a Coulter
counter. A sample of the harvested medium was diluted 1 in
15 with SF-MEM, and retained for subsequent HA determin-
ation. To the remaining radioactive medium was added
100 lL of carrier GAG solution (3Æ5 mg chondroitin sulphate,
3Æ0 mg mL)1 HA in H2O; Sigma Chemical Company, Poole,
U.K.), followed by boiling for 5 min. Pronase (100 lL of
3 mg mL)1 50 mmol L)1 Tris–HCl pH 7Æ6; Sigma) was
added, and the samples incubated for 24 h at 50 �C followed
by heat inactivation and centrifugation at 13 000 g for 5 min.
To the supernatant was added 1/5 volume of 10% cetylpyridi-
nium chloride (CPC) in 0Æ03 mol L)1 NaCl, and the mixture
was incubated for 20 min at 40 �C, followed by centrifuga-
tion at 13 000 g for 5 min. The resultant precipitate was
washed twice with 0Æ1% CPC in 0Æ03 mol L)1 NaCl, and the
pellet finally dissolved in 2 mol L)1 NaCl. Samples were taken
and suspended in Optiphase HiSafe 3 scintillant, and radioacti-
vity determined using a Packard Tricarb scintillation counter.
Hyaluronan assay
HA present in various media was determined using the Corge-
nix HA test kit that is based on an enzyme-linked HA-binding
protein (HABP) assay. Briefly, diluted media samples and
appropriate controls were incubated in HABP-coated micro-
wells, allowing HA present in the samples to react with the
immobilized binding protein. After the removal of unbound
material by washing, HABP conjugated with horseradish per-
oxidase solution was added to form complexes with the
bound HA. Following another washing step, the chromogenic
substrate tetramethylbenzidine with hydrogen peroxide was
added to develop a colour reaction. The intensity of colour
was measured in an enzyme-linked immunosorbent assay plate
reader at 450 nm. Levels of HA were determined from a refer-
ence curve obtained using reference HA solutions provided
with the kit.
Statistical analysis
Comparisons were performed using one-way ANOVA and Dun-
nett’s post hoc test.
Results
Glycosaminoglycan synthesis by control fibroblasts and
fibroblasts derived from uninvolved and involved patient
skin
It is apparent from Figure 1a that fibroblasts derived from
both involved and uninvolved skin of the patient with derma-
tomyositis did not demonstrate any significant difference in
GAG synthesis, as measured by 3H-glucosamine and 35SO4
incorporation, from that of two control fibroblast cultures.
The fibroblasts derived from NFD patient skin did, however,
exhibit elevated levels of both 3H-glucosamine and 35SO4
incorporation into GAGs, suggesting that synthesis of sulphat-
ed GAGs and possibly HA was somehow stimulated in these
patient fibroblasts. While the fibroblasts from NFD uninvolved
skin exhibited elevated GAG synthesis, the greatest increase
was observed with the fibroblasts from involved skin, where
NFD patient 2 fibroblasts exhibited a 3Æ1-fold increase in3H-glucosamine incorporation and a 3Æ3-fold increase in35SO4 incorporation compared with control 1 fibroblasts.
Levels of HA were also determined using a specific HABP
plate assay. Again, the fibroblasts from the patient with
dermatomyositis did not differ in their expression of HA from
the control fibroblasts, while the fibroblasts from NFD unin-
volved and involved skin synthesized elevated levels of HA,
particularly in the cells derived from involved skin where a
10Æ7-fold increase was observed in patient 2 fibroblasts com-
pared with control 1 fibroblasts (Fig. 1b).
Effect of patient serum on glycosaminoglycan synthesis
by control fibroblasts
It has previously been suggested that the serum of patients
with pretibial myxoedema and papulonodular mucinosis asso-
ciated with systemic lupus erythematosus may contain certain
factors responsible, at least in part, for the observed fibrosis/
mucinosis.3,4 To determine if the serum from our patients
with dermatomyositis and NFD contained any GAG-stimula-
ting activity, the effect of serum from these patients and from
two normal control volunteers was examined on GAG synthe-
sis by normal fibroblasts. Two additional serum samples from
unaffected patients on dialysis were also included, as the two
patients with NFD were on dialysis. A modest but significant
(P < 0Æ01) stimulation of GAG synthesis, as measured by3H-glucosamine incorporation by the control fibroblasts
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp473–479
Nephrogenic fibrosing dermopathy and hyaluronan, M. Edward et al. 475
exposed to the dermatomyositis and NFD patient serum, was
observed when compared with normal and control dialysis
patient serum (Fig. 2a). This suggests that sera from both the
patient with dermatomyositis and the two patients with NFD
contain factors capable of stimulating GAG synthesis by fibro-
blasts. Samples taken from the radiolabelled medium were
analysed for HA. To ensure that possible variations in HA con-
tent of the serum did not influence the levels of synthesis by
the fibroblasts, HA levels in the serum were determined, and
subtracted from that present in the medium following incu-
bation with the control fibroblasts. Figure 2b demonstrates
that the serum from the patients with dermatomyositis and
NFD contained potent HA-stimulating activities, with the
NFD patient 1 serum stimulating a 2Æ75-fold increase in HA
synthesis compared with the control 1 fibroblasts (P < 0Æ01).
Notably, one of the patients with NFD (230 ng HA mL)1
serum) and one of the dialysis control patients (242 ng
HA mL)1 serum) had elevated levels of serum HA compared
with the controls (mean 17Æ6 ng HA mL)1 serum). Elevated
levels of serum HA occur in a number of diseases including
liver disease,12 arthritis13 and certain cancers.14
Effect of patient serum on glycosaminoglycan synthesis
by fibroblasts from uninvolved patient skin
Fibroblasts established from uninvolved skin of the patients
with NFD exhibited elevated levels of GAG synthesis compared
with control fibroblasts in the presence of control serum,
while fibroblasts from uninvolved skin of the patient with der-
matomyositis did not exhibit any elevated GAG synthesis
(a)
(b)
Fig 1. Glycosaminoglycan (a) and hyaluronan (b) synthesis by
fibroblasts from patients with dermatomyositis and nephrogenic
fibrosing dermopathy. Glycosaminoglycan synthesis was determined
by measuring the incorporation of 3H-glucosamine (solid bars) and35SO4 (cross-hatched bars) into cetylpyridinium chloride-precipitable
material from the medium of control (Con1 and Con2),
dermatomyositis uninvolved and involved (Derm un and Derm in),
and nephrogenic fibrosing dermopathy uninvolved and involved (NFD
un and NFD in) fibroblasts. Hyaluronan from the corresponding
cultures was determined using a highly specific plate assay utilizing a
biotinylated hyaluronan-binding protein. Values are mean ± SD for
three separate cultures. ANOVA, F-test P < 0Æ001; Dunnett’s post hoc test
*P < 0Æ05, **P < 0Æ01 in comparison with Con1.
(a)
(b)
Fig 2. Effect of serum from patients with dermatomyositis and
nephrogenic fibrosing dermopathy on normal fibroblast
glycosaminoglycan (a) and hyaluronan (b) synthesis.
Glycosaminoglycan synthesis was determined by measuring the
incorporation of 3H-glucosamine into cetylpyridinium chloride-
precipitable material, and hyaluronan synthesis was determined by
measuring the levels present in the culture medium utilizing a
hyaluronan-binding plate assay. Dermatomyositis (Derm) and
nephrogenic fibrosing dermopathy (NFD1 and NFD2) patient sera
were compared with normal control sera (Con1 and Con2) and with
control dialysis patient sera (Dial1 and Dial2). Values are mean ± SD
for three separate cultures. ANOVA, F-test P < 0Æ001; Dunnett’s post hoc
test **P < 0Æ01 in comparison with Con1.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp473–479
476 Nephrogenic fibrosing dermopathy and hyaluronan, M. Edward et al.
(Fig. 3a). Fibroblasts derived from uninvolved skin of the
patients with dermatomyositis and NFD all exhibited signifi-
cantly higher levels of GAG synthesis compared with the
control cultures when incubated in the presence of the corres-
ponding patient serum. It is therefore apparent that fibroblasts
from the patient with dermatomyositis are not activated to
synthesize increased levels of GAG in the presence of control
serum, but do respond to the dermatomyositis patient serum
with a 2Æ3-fold (P < 0Æ01) increase in 3H-glucosamine incor-
poration and a 1Æ4-fold increase in incorporation of 35SO4. In
contrast, the fibroblasts from uninvolved NFD skin exhibited
elevated levels of both 3H-glucosamine and 35SO4 in the
presence of control serum, and even higher levels of GAG
synthesis in the presence of the corresponding NFD patient
serum, where NFD patient 1 fibroblasts exhibited a 4Æ3-fold
increase in 3H-glucosamine incorporation and a 2Æ8-fold
increase in 35SO4 incorporation (P < 0Æ01). Similarly, HA
synthesis was significantly increased in uninvolved fibroblasts
from patients with NFD in the presence of control and corre-
sponding NFD patient serum (Fig. 3b), with the greatest
increase being observed in the presence of patient serum (9Æ9-
fold, P < 0Æ01). The fibroblasts from uninvolved dermatomyo-
sitis skin showed a slight but not significant increase in HA
compared with control fibroblasts in the presence of control
serum, while HA synthesis was stimulated 4Æ4-fold in the pre-
sence of dermatomyositis patient serum compared with the
normal control (P < 0Æ05).
Effect of patient serum on fibroblasts from involved
patient skin
The pattern of GAG stimulation in fibroblasts from involved
skin was similar to that observed with fibroblasts from unin-
volved skin (Fig. 4a). Fibroblasts from involved dermatomyo-
sitis skin exhibited a similar level of GAG synthesis to that of
control fibroblasts in the presence of control serum, but
enhanced their synthesis significantly in the presence of serum
from the patient with dermatomyositis. Again, the fibroblasts
from involved NFD skin exhibited elevated GAG synthesis in
the presence of both control and corresponding NFD patient
serum, with the greatest increase being observed with the
NFD patient serum (5Æ1-fold increase in 3H-glucosamine
incorporation, and 3Æ7-fold increase in 35SO4 incorporation in
NFD patient 2 compared with control fibroblasts; P < 0Æ01).
The HA synthesis was similar to the 3H-glucosamine incorpor-
ation (Fig. 4b), with the greatest increase in HA synthesis
being observed in fibroblasts from NFD patient 2 in the pres-
ence of NFD patient serum (17Æ5-fold compared with control
fibroblasts in control serum; P < 0Æ01).
Discussion
Mucinous lesions are probably more widespread than currently
reported, primarily due to fixation techniques in which one of
the main molecules involved, HA, is depleted. Mucin is nor-
mally visualized histochemically by staining tissue sections with
alcian blue at pH 2Æ5. However, this technique requires to be
modified as fixation of tissue in 10% formalin in PBS does not
fix HA, which can subsequently be lost by simple PBS washing.
In addition, alcian blue does not readily stain HA at pH 2Æ5, so
most of the observed staining with alcian blue is presumably
attributable to sulphated GAGs.15 The most effective and speci-
fic method of visualizing HA in tissue is by utilizing a biotinyl-
ated HABP, isolated from articular cartilage. Indeed, Neudecker
et al.16 have recently shown that scleromyxoedema-like lesional
biopsies of patients in renal failure are associated with marked
deposition of HA, particularly in the papillary dermis, as visual-
ized using a biotinylated HABP.
Dermatomyositis is an inflammatory disorder of both mus-
cle and skin; however, in practice, dermatomyositis represents
(a)
(b)
Fig 3. Effect of patient serum on glycosaminoglycan (a) and
hyaluronan (b) synthesis by fibroblasts from uninvolved skin of
patients with dermatomyositis and nephrogenic fibrosing dermopathy.
Glycosaminoglycan synthesis was determined by measuring the
incorporation of 3H-glucosamine (solid bars) and 35SO4 (cross-
hatched bars) into cetylpyridinium chloride-precipitable material, and
hyaluronan synthesis was determined by measuring the levels present
in the culture medium utilizing a hyaluronan-binding plate assay.
Dermatomyositis (Derm) and nephrogenic fibrosing dermopathy (NFD
1 and NFD 2) fibroblast glycosaminoglycan and hyaluronan synthesis
was examined in the presence of patient and control serum. Values
are mean ± SD of three separate cultures. ANOVA, F-test P < 0Æ001;
Dunnett’s post hoc test *P < 0Æ05, **P < 0Æ01 compared with control
fibroblasts in the presence of control serum.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp473–479
Nephrogenic fibrosing dermopathy and hyaluronan, M. Edward et al. 477
a disease continuum ranging from inflammatory myopathy in
isolation to cutaneous disease in the absence of muscle
involvement. There are several reports documenting the occur-
rence of macroscopic mucinous plaques in dermatomyositis
and, indeed, our patient exhibited papular mucinosis. The
underlying cause of the mucinosis is unknown, but immuno-
logically mediated fibroblast stimulation has been postulated
in secondary mucinosis.17 The presence of a tumour has fre-
quently been associated with dermatomyositis,18 and in such
cases it may be that the tumour is releasing factors into serum
that promote the development of the mucinous lesions. Exten-
sive screening of our patient including pelvic examination,
Pap smear, computed tomography of the abdomen and pelvis,
and tumour markers including a-fetoprotein, chorionic gon-
adotrophin, carcinoembryonic antigen and C125 failed to
uncover an underlying internal malignancy over a 3-year per-
iod of clinical observation. It does appear likely that the muc-
inous lesions are caused, at least in part, by the presence of
certain serum-derived factors as we have demonstrated that
dermatomyositis patent serum stimulates both sulphated GAG
and HA synthesis by fibroblasts from both control and
involved skin. In contrast, the patient fibroblasts show similar
levels of GAG synthesis as control fibroblasts in the presence
of control serum, suggesting that these fibroblasts are not acti-
vated, nor contain a population of cells that overexpress GAGs.
There has been a report of epidermal changes in dermatomyo-
sitis patients with mucinosis, in whom there is moderate atro-
phy of the epidermis,11 while a lack of CD44, a receptor for
HA, in mouse epidermis, leads to an abnormal accumulation
of HA in the dermis.19 This suggests that disruption of kera-
tinocyte–HA interactions may affect both epidermal prolifer-
ation and HA synthesis within the dermis.
In contrast, the fibroblasts derived from involved and unin-
volved NFD patient skin show elevated levels of sulphated
GAG and HA synthesis in addition to patient serum actively
stimulating GAG synthesis. Again, it is unclear what the
serum-derived active factors are, or why the fibroblasts consti-
tutively synthesize excess levels of GAGs. The cells may be
permanently activated by certain factors, or some infiltrating
cell type may be actively synthesizing excess collagen and
GAGs and contributing to the development of the lesions. A
possible candidate cell type is the circulating CD34+ fibro-
cyte.20 Several cell types express CD34, including embryonic
fibroblasts, endothelial cells and bone marrow stromal cells,
but the haematological origin of fibrocytes within a tissue is
confirmed by the expression of the panleucocyte antigen
CD45.21 In addition to CD34, and CD45, the coexpression of
type I procollagen has also been utilized to identify fibro-
cytes.22 These circulating fibrocytes are likely to play an im-
portant role in wound healing as CD34+ cells appear at the
inflammatory stage, and apparently decrease with maturation
of the wound.23 However, it appears that the decreased
expression of CD34 is a result of differentiation of the fibro-
cytes into myofibroblasts, and subsequent elevated collagen
synthesis and contractile properties, and this is supported by
the observation that cultured fibrocytes are induced to express
a-smooth muscle actin and downregulate expression of CD34
in the presence of TGF-b1.24
Why the fibrocytes infiltrate the skin to create lesions char-
acteristic of the dermopathy is unknown. Are there certain
chemokines expressed in the skin attracting the fibrocytes, or
are the fibrocytes stimulated by some factor within the circula-
tion, and why do they apparently switch off in normal acute
wound healing as the wound matures, yet produce excess col-
lagen and GAGs in NFD lesions? Neudecker et al.16 have sug-
gested that circulating low molecular weight HA may play a
role in NFD. While liver is the main organ involved in deg-
radation of high molecular weight HA, the kidney may be
involved in degradation of lower molecular weight HA which,
following renal failure, may accumulate in the circulation.25
(a)
(b)
Fig 4. Effect of patient serum on glycosaminoglycan (a) and
hyaluronan (b) synthesis by fibroblasts from involved skin of patients
with dermatomyositis and nephrogenic fibrosing dermopathy.
Glycosaminoglycan synthesis was determined by measuring the
incorporation of 3H-glucosamine (solid bars) and 35SO4 (cross-
hatched bars) into cetylpyridinium chloride-precipitable material, and
hyaluronan synthesis was determined by measuring the levels present
in the culture medium utilizing a hyaluronan-binding plate assay.
Dermatomyositis (Derm) and nephrogenic fibrosing dermopathy (NFD
1 and NFD 2) fibroblast glycosaminoglycan and hyaluronan synthesis
was examined in the presence of patient and control serum. Values
are mean ± SD of three separate cultures. ANOVA, F-test P < 0Æ001;
Dunnett’s post hoc test *P < 0Æ05, **P < 0Æ01 compared with control
fibroblasts in the presence of control serum.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp473–479
478 Nephrogenic fibrosing dermopathy and hyaluronan, M. Edward et al.
HA is a molecule with a multitude of biological functions
depending upon its molecular size, with high molecular weight
HA being anti-inflammatory, and low molecular weight frag-
ments inflammatory.26 These low molecular weight fragments
may therefore induce an inflammatory wound healing reaction
resulting in fibrosis,27 possibly mediated by circulating fibro-
cytes. This hypothesis does not, however, explain why only a
few patients with renal failure develop NFD, or why the first
case was identified only in 1997. Recent reports suggest a cor-
relation between the development of NFD and exposure to the
magnetic resonance angiography contrast agent, gadolinium.28
The two patients with NFD described here were exposed to
gadolinium approximately 2 months prior to the development
of fibrotic lesions, thus further supporting the possibility that
gadolinium is acting as the trigger for the development of
NFD. Current studies are under way to examine in more detail
the relationship between gadolinium and NFD.
Acknowledgments
We gratefully acknowledge financial assistance from the British
Skin Foundation.
References
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2 Harper RA, Rispler J. Lichen myxedematosus serum stimulateshuman skin fibroblast proliferation. Science 1978; 199:545–7.
3 Cheung HS, Nicoloff JT, Kamiel MB et al. Stimulation of biosyn-thetic activity by serum of patients with pretibial myxedema.
J Invest Dermatol 1978; 71:545–7.4 Pandya AG, Sontheimer RD, Cockerell CJ et al. Papulonodular muci-
nosis associated with systemic lupus erythematosus: possible mech-anisms of increased glycosaminoglycan accumulation. J Am Acad
Dermatol 1995; 32:199–205.5 Cowper SE, Robin HS, Steinberg SM et al. Scleromyxedema-like
cutaneous disease in renal dialysis patients. Lancet 2000; 356:1000–1.6 McNeill AM, Barr RJ. Scleromyxedema-like fibromucinosis in a
patient undergoing hemodialysis. Int J Dermatol 2002; 41:364–7.7 Cassis TB, Jackson JM, Sonnier MD, Callen JP. Nephrogenic fibro-
sing dermopathy in a patient with acute renal failure never requi-
ring dialysis. Int J Dermatol 2006; 45:56–9.8 Banno H, Takama H, Nitta Y et al. Lichen myxedematosus associ-
ated with chronic hepatitis C. Int J Dermatol 2000; 39:212–15.9 Mlambo NC, Hylander B, Brauner A. Increased levels of transform-
ing growth factor beta 1 and basic fibroblast growth factor inpatients on CAPD. A study during non-infected steady state and
peritonitis. Inflammation 1999; 2:131–9.10 Takehara K. Growth regulation of skin fibroblasts. J Dermatol Sci
2000; 24:S70–7.
11 Del Pozo J, Almagro M, Martinez W et al. Dermatomyositis andmucinosis. Int J Dermatol 2001; 40:120–4.
12 Guechot J, Poupon RE, Poupon R. Serum hyaluronan as a markerof liver fibrosis. J Hepatol 1995; 22:103–6.
13 Emlen W, Niebur J, Flanders G, Rutledge J. Measurement of serumhyaluronic acid in patients with rheumatoid arthritis: correlation
with disease activity. J Rheumatol 1996; 23:974–8.14 Wilkinson CR, Bower LM, Warren C. The relationship between
hyaluronidase and hyaluronic acid concentration in sera fromnormal controls and from patients with disseminated neoplasm.
Clin Chim Acta 1996; 256:165–73.
15 Lin W, Shuster S, Maibach HI, Stern R. Patterns of hyaluronanstaining are modified by fixation techniques. J Histochem Cytochem
1997; 45:1157–69.16 Neudecker BA, Stern R, Mark LA, Steinberg S. Scleromyxedema-like
lesions of patients in renal failure contain hyaluronan: a possiblepathophysiological mechanism. J Cutan Pathol 2005; 32:612–15.
17 Igarashi M, Aizawa H, Tokudome Y, Tagami H. Dermatomyositiswith prominent mucinosis skin change. Histochemical and biochemi-
cal aspects of glycosaminoglycans. Dermatologica 1985; 170:6–11.18 Leow YH, Goh CL. Malignancy in adult dermatomyositis. Int J
Dermatol 1997; 36:904–7.19 Kaya G, Rodrigez I, Jorcano JL et al. Selective suppression of CD44
in keratinocytes of mice bearing an antisense CD44 transgene dri-ven by a tissue-specific promoter disrupts hyaluronate metabolism
in the skin and impairs keratinocyte proliferation. Genes Dev 1997;11:996–1007.
20 Quan TE, Cowper S, Wu S-P et al. Circulating fibrocytes: collagen-secreting cells of the peripheral blood. Int J Biochem Cell Biol 2004;
36:598–606.21 Bucala R, Spiegel LA, Chesney J et al. Circulating fibrocytes define a
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24 Abe R, Donnelly SC, Peng T et al. Peripheral blood fibrocytes: dif-ferentiation pathway and migration to wound sites. J Immunol
2001; 166:7556–62.25 Lipkin GW, Forbes MA, Cooper EH, Turney JH. Hyaluronic acid
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Nephrogenic fibrosing dermopathy and hyaluronan, M. Edward et al. 479
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07677.x
Sweet’s syndrome: a spectrum of unusual clinicalpresentations and associationsC.Y. Neoh, A.W.H. Tan and S.K. Ng
National Skin Centre, 1 Mandalay Road, Singapore 308205, Singapore
CorrespondenceDr C.Y. Neoh.
E-mail: cyneoh@nsc.gov.sg
Accepted for publication28 July 2006
Key wordsinfections, imatinib, neutrophilic dermatosis,
neutrophilic panniculitis, Sweet’s syndrome,
unusual presentations
Conflict of interestNone declared.
Summary
Background Sweet’s syndrome (SS) is the prototypic neutrophilic dermatosis. Firstdescribed in 1964, the characterization of new clinical associations, unique histo-pathological findings and clinical variants have stimulated much interest and dis-cussion recently. However, the prevalence of these unusual variants and clinicalassociations within a single cohort of patients, has not been described.Objectives To describe and evaluate the prevalence of unusual clinical and histo-pathological features, as well as the clinical associations of SS seen in patientsfrom the National Skin Centre, Singapore.Methods This is a retrospective study of all consecutive cases of SS seen at our cen-tre over a 5Æ5-year period (June 1999–December 2004). Data on associated sys-temic diseases was obtained from the medical records and matched withinformation from the National Cancer Registry, Singapore. Patients not activelyfollowed up for more than 3 months were contacted for their updated healthstatus.Results Thirty-seven patients were identified. Ten (27%) had non-idiopathic SS.These were associated with haematological disorders, connective tissue disorders,infections or a drug. Twenty-nine patients (78%) had at least one atypical clinicalor histopathological feature. Atypical clinical features included bullous lesions, SSwith hand involvement or neutrophilic dermatoses of the hands and the con-comitant existence of subcutaneous SS with pyoderma gangrenosum. SS was thepresenting feature in three patients with infections caused by atypical organisms,including Mycobacterium chelonae, Penicillium species and Salmonella type D. Uniquehistopathological variants included subcutaneous SS and lesions containing anadmixture of mature and immature neutrophils. Subcutaneous neutrophilicinflammation seemed to be more common in patients with an underlying haem-atological disorder. This group of patients also had a lower mean haemoglobinlevel.Conclusions Unusual clinical and histopathological variants of SS described in theliterature are similarly encountered in our cohort of patients, with some featuresbeing more common than others. We highlight and discuss some unique clinicaland histopathological observations seen in our patients with SS.
Sweet’s syndrome (SS) is the prototypic neutrophilic dermato-
sis. Its typical features consist of pyrexia, elevated neutrophil
counts, painful red plaques and a predominantly mature neu-
trophilic dermal infiltrate. Since its original description in
1964,1 many subsequent reports have expanded the clinical
and pathological features of this condition. Of special import-
ance is the identification of new malignancies and/or medica-
tions associated with SS and the description of variants such as
neutrophilic dermatosis of the hands. New histopathological
variants like histiocytoid SS, SS-associated leukaemia cutis and
neutrophilic panniculitis have also attracted particular attention
in recent years. Taking these evolving and new concepts into
account, we review patients diagnosed with SS, at the National
Skin Centre, Singapore over 5Æ5 years and highlight the atyp-
ical presentations and clinical associations of SS.
Patients and methods
Consecutive patients attending the National Skin Centre, a ter-
tiary dermatological referral centre in Singapore, between June
� 2007 The Authors
480 Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp480–485
1999 and December 2004, diagnosed with SS, were included
in this study. The diagnosis of SS was established through a
history of the abrupt onset of erythematous tender plaques
with corresponding dense neutrophilic infiltrates on histo-
pathology and a rapid therapeutic response to systemic corti-
costeroids.2,3 Medical and histopathological records were
reviewed. Patients with atypical clinical and histopathological
features, as well as those with underlying systemic diseases
were identified. Those who had not been followed up for
more than 3 months were contacted by phone for their updated
health status. The patients were also matched with data from
the National Cancer Registry, Singapore. The National Cancer
Registry is a comprehensive registry established in January
1968, with cancer notifications derived from medical
practitioners, pathology records, hospital records and death
certificates.
Results
Thirty-seven patients were diagnosed with SS during the study
period. They were aged 14–78 years (mean: 46 years). The
female : male ratio was 1Æ5 : 1. The mean duration of follow-
up was 14 months. Ten (27%) patients had nonidiopathic SS.
SS was associated with haematological malignancies in five
patients [two acute myeloid leukaemia (AML), one chronic
myeloid leukaemia (CML) and two myelodysplastic syndrome
(MDS)]. One patient with MDS had concomitant Penicillium
species bone marrow infection. SS was drug-induced (imati-
nib) in one of the patients with AML. Nontuberculous myco-
bacterial infections were found in two patients (Table 1).
Three patients had associated connective tissue disorders (CTD)
[undifferentiated CTD, systemic lupus erythematosus (SLE) and
SLE with autoimmune thrombocytopenic purpura (AITP)].
Eighty per cent of patients with idiopathic SS and 75% of
patients with nonidiopathic SS exhibited atypical features.
Unusual clinical presentations and associations
Neutrophilic dermatosis of the dorsal hands. Seven patients (19%) pre-
sented with lesions on the hands (Fig. 1). Involvement of the
dorsum of the hands was an isolated event in two patients
only. The rest had concomitant involvement of the lower
limbs, face or trunk. Of these, three patients had typical SS
lesions while two had bullous lesions. Histopathological find-
ings in all cases showed dense, neutrophilic infiltrates. Vasculi-
tis was seen in one specimen. No patients had associated
systemic diseases at presentation or on follow-up. All patients
were successfully treated with oral prednisolone, colchicine or
topical corticosteroids.
Sweet’s syndrome with bullous or pustular lesions. Eighteen patients
(49%) were documented with either bullous or pustular lesions
(Fig. 2). The dominant morphology could not be ascertained in
most patients because of the retrospective nature of this study.
Of these, five had associated underlying systemic conditions
(SLE with AITP, undifferentiated CTD, AML, group D Salmonella Tab
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� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp480–485
Unusual clinical feature in Sweet syndrome, C.Y. Neoh et al. 481
and Mycobacterium chelonae cervical lymphadenitis).4 Of five
patients with haematological disorders, only one patient with
AML presented with bullous lesions. Biopsies were performed
in all patients and consistently showed marked dermal oedema
with dense dermal neutrophilic infiltrates, compatible with SS.
Concomitant multiple neutrophilic dermatoses. A 47-year-old Indones-
ian man presented concurrently with lesions of SS on the
limbs and pyoderma gangrenosum (Fig. 3) on his chest. He
was subsequently diagnosed with MDS with refractory anae-
mia, as well as bone marrow infection with Penicillium species
(Table 1).
Imatinib-induced Sweet’s syndrome. A 36-year-old woman with a
1-year history of CML presented with fever and recurrent ten-
der nodules on the limbs. Imatinib mesylate was started
1 month before the appearance of SS. Spontaneous resolution
occurred after imatinib was stopped. Upon re-institution of
imatinib at progressively increasing doses, there was a
recurrence of SS. The lesions responded remarkably to oral
prednisolone but recurred upon stopping this treatment.
Sweet’s syndrome associated with infections. Three patients had SS
associated with infections (Table 1). These infections include
group D Salmonella lymphadenitis, Penicillium species bone mar-
row infection and Mycobacterium chelonae lymphadenitis. The first
two patients tested negative for human immunodeficiency
virus (HIV) infection while the last patient declined the test.
Unusual histopathological presentations
Subcutaneous neutrophilic inflammation. Subcutaneous neutrophilic
inflammation was seen in seven patients (Fig. 4). In six
patients, there was an overlying dermal inflammatory infiltrate
comprising mainly neutrophils. The only patient with isolated
diffuse lobular neutrophilic infiltrates within the subcutis
presented with typical erythematous plaques and was later
diagnosed with MDS. Two other patients had associated haem-
atological disorders (one MDS, one CML). One had an under-
lying undifferentiated connective tissue disease.
Sweet’s syndrome with infiltrates containing neutrophils and immature white
cells. Two of three patients with a mixture of mature neu-
trophils and immature white cells on histopathology (Fig. 5)
were diagnosed with acute myeloid leukaemia and MDS,
respectively. All three patients presented acutely with skin
lesions resembling SS with fever, in contrast to the more
Fig 2. Bullous lesions of Sweet’s syndrome.
Fig 1. Neutrophilic dermatosis of the dorsal hands.
Fig 3. Ulcerative lesion of pyoderma gangrenosum occurring in a
patient with concomitant Sweet’s syndrome.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp480–485
482 Unusual clinical feature in Sweet syndrome, C.Y. Neoh et al.
indolent lesions of leukaemia cutis. The last patient had a
repeat biopsy on a fresh SS lesion, 6 months after the initial
presentation, which did not show any atypical white cells.
Comprehensive investigations for haematological malignancy
are negative to date.
Vasculitis. Fibrinoid necrosis of the blood vessels within a
dense, neutrophilic infiltrate was seen in two specimens from
fresh SS lesions (Fig. 6). Both patients responded to treatment
with oral prednisolone and topical corticosteroids.
Features defining patients with Sweet’s syndrome
associated with haematological disorders
Subcutaneous neutrophilic inflammation was seen in three
of five patients with an underlying haematological disorder.
The mean haemoglobin level in the patients with associ-
ated haematological disorders was 8Æ6 g dL)1 compared with
13Æ0 g dL)1 in patients without.
Discussion
Sweet’s syndrome was originally described by Dr Robert
Douglas Sweet in 1964.1 In 1986, several diagnostic criteria
for SS were proposed by Su and Liu.5 It was subsequently
revised by von den Driesch,2 who classified them into two
categories: major and minor. The clinical and histopathologi-
cal concepts of SS have evolved over the years and are still
constantly being updated. We highlight in this study some
unusual features of SS seen in our patients.
The mean age of presentation of 46 years in our patients
falls within the previously reported age range of 30–60 years.6
The female : male ratio appears to be lower at 1Æ5 : 1. This
ratio remains fairly similar to that from a previous epidemi-
ological study of SS in Singapore.3 It is known that the
female : male ratio for malignancy-associated SS is 1 : 1 com-
pared with 4 : 1 in idiopathic SS.2 However, since the propor-
tion of malignancy-associated SS in both series from Singapore
was not much more than idiopathic SS, the lower female :
male ratio is unlikely to be attributable to this. SS may poss-
ibly be more common in Asian males from Singapore, in con-
trast with those seen in Western countries.
Neutrophilic dermatosis of the dorsal hands is a recently
described disorder that is now recognized to be a unique,
localized variant of SS with predominant or isolated hand
involvement.7,8 This relationship is supported by the similar
female preponderance of both conditions and their identical
incidence of associated haematopoietic and inflammatory dis-
eases.8 Initially termed as pustular vasculitis of the hands,9,10
it was later revised after recognizing that vasculitis was an
inconsistent finding and of secondary importance in its diag-
nosis. Frequently misdiagnosed as a cutaneous infection, these
signs were often found to be significantly associated with an
occult malignancy or systemic inflammatory disorder.8,9,11
Although none of our patients had associated disorders, larger
cohort studies are needed to establish a definite association
between this entity and systemic diseases.
Fig 5. Infiltrate of mature neutrophils admixed with immature white
cells (haematoxylin and eosin, original magnification · 600).
Fig 6. Fibrinoid necrosis of blood vessels with extravasation of red
blood cells. Dense neutrophilic infiltrate in upper field (haematoxylin
and eosin, original magnification · 400).
Fig 4. Lobular infiltrates of neutrophils between adipocytes:
Neutrophilic panniculitis (haematoxylin and eosin, original
magnification · 200).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp480–485
Unusual clinical feature in Sweet syndrome, C.Y. Neoh et al. 483
Despite the rare incidence of the bullous variant of SS,12
almost half of our patients appear to have presented with this.
The inclusion of the more commonly encountered pseudoves-
icular variant of SS due to pronounced oedema, may have
caused this group to be over-represented. Only a handful
of these patients had photographic documentation of their
lesions, making the verification of truly bullous lesions diffi-
cult. Patients with haematological disorders in our series did
not present more commonly with the bullous variant of SS,
contrary to previous reports.13,14 Another uncommon presen-
tation is the successive appearance of multiple neutrophilic
dermatoses in the same patient.15 One of our patients presen-
ted concurrently with pyoderma gangrenosum and SS.
Together, these cases provide evidence that these skin dis-
orders form a spectrum of entities, rather than isolated
diseases.16
Drug-induced SS has been reported with all-trans retinoic
acid,17 carbamazepine,18 trimethoprim-sulfamethoxazole,19
granulocyte colony-stimulating factor20 and recently with
imatinib.21–23 We report another patient who developed SS
after imatinib. Imatinib mesylate (Gleevec�, Novartis, Basel,
Switzerland) is approved by the United States Food and Drug
Administration (FDA) for treatment of CML, hypereosinophilic
syndrome, systemic mastocytosis, gastrointestinal stromal
tumours and dermatofibrosarcoma protuberans.24 Despite the
wide use of imatinib, reports of imatinib-induced SS have been
described only in CML patients. As SS can be associated with
CML itself, the time course of appearance of SS in relation to
any drugs must be examined carefully in any CML patient,
before attributing the lesions to a particular medication.
Sweet’s syndrome has been well described in association
with nontuberculous mycobacterial infections.25,26 Similarly,
reports of SS occurring together with Salmonella infections are
available.27,28 Penicilliosis, however, has not been described in
association with SS, despite it being a common opportunistic
pathogen in Southeast Asia.29 It is interesting that in the three
patients with infections associated with SS (Table 1), there
were either recurrent, multiple infections or infections second-
ary to atypical organisms, raising the possibility of an underly-
ing immunocompromised state. As demonstrated by our two
patients with SS in association with lymphadenitis, an underly-
ing atypical infection should be considered, particularly if no
clinical response to conventional treatment is observed.
Although SS is traditionally classified as a neutrophil-
predominant dermatosis without vasculitis, the presence of
vasculitis should not preclude SS. Seen in two of our patients,
vasculitis is considered an epiphenomenon occurring second-
ary to extensive neutrophilic dermal inflammation in SS.30 The
phenomenon in which immature granulocytes are found
mixed with mature neutrophils in SS has been termed SS-
associated leukaemia cutis.31 These immature white cells are
hypothesized to constitute the incipient presence of a specific
leukaemic infiltrate (primary leukaemia cutis) or may repre-
sent the recruitment of immature myeloid precursor cells in
response to chemotactic factors stimulated by inflammatory SS
lesions (secondary leukaemia cutis).31
Distinguishing true leukaemia cutis from neutrophilic derma-
toses in a patient with a haematological disorder can be diffi-
cult, since dermal infiltrates of immature white cells can also be
found in the latter, more benign condition.32–34 It is believed
that in cases of neutrophilic dermatoses occurring in the setting
of a myeloid malignancy, the mature neutrophils are in fact
derived from the same clonal differentiation of myeloblasts.35 A
common origin of these cells explains why the presence of leu-
kaemia cutis and neutrophilic dermatoses in haematological
malignancies, both herald an overall poorer prognosis for the
malignancy.35 A series of SS patients with biopsy specimens
containing immature myeloid cells resembling histiocytes has
also recently been described.36 The presence of these histopath-
ological variants make thorough clinicopathological correlation
and meticulous follow-up vital for patients with SS.
The entity of neutrophilic panniculitis is contentious. Whe-
ther it is a distinct neutrophilic dermatoses or subcutaneous
SS, is debatable. It has been proposed as a specific entity char-
acterized by: (i) lobular panniculitis on histopathology, (ii) a
significant association with myelodysplasia or (iii) the good
response to treatment with steroids.37 However, some con-
sider it to be subcutaneous SS, in which pathological changes
are localized either within the adipose tissue or involving both
the dermis and the subcutaneous fat.38 Neutrophilic panniculi-
tis, being a broad descriptive term, encompasses differential
diagnoses including alpha-1 antitrypsin deficiency syndrome,
infections, pancreatitis, rheumatoid arthritis and factitial pan-
niculitis.38 From our findings, we feel that an underlying
haematological malignancy should be considered when there
is subcutaneous involvement in SS and/or anaemia.
SS is an intriguing dermatosis with protean clinical associa-
tions and histological findings which are constantly being
revised. In this study, we have illustrated and reinforced these
evolving concepts and variants of SS with patients from Singa-
pore. Clinical awareness and recognition of these unique
features are essential in the initiation of appropriate investiga-
tions and follow-up of these patients.
References
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2 von den Driesch P. Sweet’s syndrome (acute febrile neutrophilicdermatosis). J Am Acad Dermatol 1994; 31:535–56.
3 Goh CL, Alora M, Kohar Y. Sweet’s syndrome in a skin clinic inSingapore: an epidemiology study. Ann Acad Med Singapore 1996;
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ated with Mycobacterium chelonae and herpes simplex virus infections:
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1986; 37:167–74.6 Moschella SL. Neutrophilic dermatoses. In: Dermatology (Bolognia JL,
Jorizzo JL, Rapini RP, eds), 1st edn. London: Elsevier Limited,2003; 411–23.
7 Galaria NA, Junkins-Hopkins JM, Kligman D, James WD. Neutro-philic dermatosis of the dorsal hands: pustular vasculitis revisited.
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study of eleven cases. Int J Dermatol 1994; 33:425–32.14 Cohen PR, Kurzrock R. Sweet’s syndrome and cancer. Clin Dermatol
1993; 11:149–57.15 Vazquez Garcia J, Almagro Sanchez M, Fonseca Capdevila E.
Multiple neutrophilic dermatoses in myelodysplastic syndrome.Clin Exp Dermatol 2001; 26:398–401.
16 Davies MG, Hastings A. Sweet’s syndrome progressing to pyoder-ma gangrenosum: a spectrum of neutrophilic skin disease in
association with cryptogenic cirrhosis. Clin Exp Dermatol 1991;16:279–82.
17 Cox NH, O’Brien HAW. Sweet’s syndrome associated with trans-retinoic acid treatment in acute promyelocytic leukemia. Clin Exp
Dermatol 1994; 19:51–2.18 Sitjas D, Cuatrecasas M, De Moragas JM. Acute febrile neutrophilic
dermatosis (Sweet’s syndrome). Int J Dermatol 1993; 32:261–8.19 Walker DC, Cohen PR. Trimethoprim-sulfamethoxazole-associated
acute febrile neutrophilic dermatosis: case report and review ofdrug-induced Sweet’s syndrome. J Am Acad Dermatol 1996; 34:918–
23.20 Shimizu T, Yoshida I, Eguchi H et al. Sweet syndrome in a child
with aplastic anaemia receiving recombinant granulocyte colony-stimulating factor. J Pediatr Hematol Oncol 1996; 18:282–4.
21 Ayirookuzhi SJ, Ma L, Ramshesh P, Mills G. Imatinib-inducedSweet syndrome in a patient with chronic myeloid leukaemia. Arch
Dermatol 2005; 141:368–70.22 Liu D, Seiter K, Mathews T et al. Sweet’s syndrome with CML cell
infiltration of the skin in a patient with chronic phase CML whiletaking imatinib mesylate. Leuk Res 2004; 28 (Suppl. 1):S61–3.
23 Scheinfeld N. Imatinib mesylate and dermatology part 2: a review
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24 Krause DS, Van Etten RA. Tyrosine kinases as targets for cancertherapy. N Engl J Med 2005; 353:172–87.
25 Choonhakam C, Chetchotisakd P, Jirarattanapochai K, Mootsika-pun P. Sweet’s syndrome associated with non-tuberculous myco-
bacterial infection: a report of five cases. Br J Dermatol 1998;139:107–10.
26 Chen HH, Hsiao CH, Chin HC. Successive development of cutane-ous polyarteritis nodosa, leucocytoclastic vasculitis and Sweet’s
syndrome in a patient with cervical lymphadenitis caused by Myco-bacterium fortuitum. Br J Dermatol 2004; 151:1096–100.
27 Florez A, Sanchez-Aguilar D, Roson E et al. Sweet’s syndrome asso-
ciated with Salmonella enteritidis infection. Clin Exp Dermatol 1999;24:239–40.
28 Zillikens D, Goldstein RK, Elsner P et al. Sweet’s syndrome associ-ated with Salmonella typhimurium infection. Acta Derm Venerol 1991;
71:77–9.29 Wong SSY, Wong KH, Hui WT et al. Differences in clinical and
laboratory diagnostic characteristics of penicilliosis marneffei inhuman immunodeficiency virus (HIV)- and non-HIV-infected
patients. J Clin Microbiol 2001; 39:4535–40.30 Cohen PR. Skin lesions of Sweet syndrome and its dorsal hand
variant contain vasculitis: an oxymoron or an epiphenomenon?Arch Dermatol 2002; 138:400–2.
31 Cohen PR, Kurzrock R. Sweet’s syndrome revisited: a review ofdisease concepts. Int J Dermatol 2003; 42:761–78.
32 Urano Y, Miyaoka Y, Kosaka M et al. Sweet syndrome associatedwith chronic myelogenous leukaemia: demonstration of leukaemic
cells within a skin lesion. J Am Acad Dermatol 1999; 40:275–9.33 Morgan KW, Callen JP. Sweet’s syndrome in acute myelogenous
leukaemia presenting as periorbital cellulitis with an infiltrate ofleukaemic cells. J Am Acad Dermatol 2001; 45:590–5.
34 Pozo J, Martinez W, Pazos JM et al. Concurrent Sweet’s syndrome andleukaemia cutis in patients with myeloid disorders. Int J Dermatol
2005; 44:677–80.35 Vignon-Pennamen MD, Aracting S. Sweet’s syndrome and leukae-
mia cutis: a common skin homing mechanism? Dermatology 2003;206:81–4.
36 Requena L, Kutzner H, Palmedo G et al. Histiocytoid Sweet syn-drome: a dermal infiltration of immature neutrophilic granulo-
cytes. Arch Dermatol 2005; 141:834–42.37 Sutra-Loubet C, Carlotti A, Guillemette J et al. Neutrophilic pannic-
ulitis. J Am Acad Dermatol 2004; 50:280–5.38 Cohen PR. Subcutaneous Sweet’s syndrome: a variant of acute feb-
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Unusual clinical feature in Sweet syndrome, C.Y. Neoh et al. 485
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2007.07682.x
Cutaneous side-effects in patients with rheumatic diseasesduring application of tumour necrosis factor-a antagonistsH-H. Lee, I-H. Song,* M. Friedrich, A. Gauliard,* J. Detert,* J. Rowert, H. Audring, S. Kary,* G.-R. Burmester,*W. Sterry and M. Worm
Department of Dermatology and Allergy and *Department of Rheumatology and Clinical Immunology, Charite-Universitatsmedizin Berlin, D-10117 Berlin,
Germany
CorrespondenceMargitta Worm.
E-mail: margitta.worm@charite.de
Accepted for publication13 September 2006
Key wordsatopic eczema, biologics, psoriasis, rheumatic
disease, tumour necrosis factor-a antagonists
Conflicts of interestDuring the time of recording cutanous adverse
events under the patients’ treatment with TNF
antagonists, S.K. was employee of the Charite
University Medicine, Clinic for Rheumatology and
Clinical Immunology. Since September 2004, S.K.
has been working as contractor for Abbott GmbH
& Co. KG in Germany. This status has not
influenced the presentation of data or the discussion
in this article.
H.H.L. and I.H.S. contributed equally to this
study.
Summary
Background Patients with rheumatic diseases receiving antitumour necrosis factor(TNF)-a-based treatment may develop cutaneous reactions.Objectives To analyse the new onset or aggravation of skin lesions in patients witha rheumatic disease during treatment with TNF-a antagonists.Methods We conducted a prospective analysis of 35 of 150 patients with a longhistory of rheumatic disease, including rheumatoid arthritis, ankylosing spondy-litis (Bechterew’s disease) and psoriatic arthritis, to test for the development ofcutaneous manifestations during anti-TNF-a (infliximab, adalimumab or etaner-cept) treatment.Results Chronic inflammatory skin diseases such as psoriasis and eczema-like mani-festations represented the majority of cases (16 of 35). Cutaneous infectionscaused by viral, bacterial and fungal agents were also observed in many patients(13 of 35). Skin diseases such as dermatitis herpetiformis, leucocytoclastic vascu-litis and alopecia occurred in single cases only.Conclusions We observed a broad, diverse clinical spectrum with a majority ofchronic inflammatory and infectious skin diseases. However, we did not identifyindividual risk factors and a discontinuation of the anti-TNF-a treatment was notnecessary if adequate dermatological treatment was performed. The onset ofcutaneous side-effects in anti-TNF-a-based treatments should be determined bynationwide registries.
Rheumatic diseases are chronic inflammatory autoimmune dis-
orders and may lead to physical disability. The most common
form is rheumatoid arthritis (RA), which affects approximately
1% of adults worldwide.1 Other forms of arthritis include
ankylosing spondylitis (AS, Bechterew’s disease) and psoriatic
arthritis (PsA). Tumour necrosis factor (TNF)-a plays a central
role in the pathogenesis of rheumatic diseases and has become
a target molecular structure for antibody or TNF receptor
(TNF-R)-based treatment in the past few years. Several rand-
omized controlled clinical trials have been performed in which
biologics against TNF-a and/or its receptor showed a high
clinical efficacy and a good tolerability.2–7
The following biologics targeting the action of TNF-a have
been registered since 2002 by the U.S. Food and Drug Admin-
istration: infliximab (Remicade�; licensed by Centocor Inc.,
Horsham, PA, U.S.A.) is a chimeric IgG1j monoclonal anti-
body with approximately 10% mouse sequence in the variable
antigen-binding region. It binds with high affinity to the
soluble and transmembrane forms of TNF-a and inhibits
the binding of the cytokine to its receptor. By contrast,
adalimumab (Humira�; licensed by Abbott Laboratories,
Abbott Park, IL, U.S.A.) is a recombinant, fully human IgG1
monoclonal antibody that binds specifically to human TNF-aand neutralizes the activity of this cytokine. Etanercept
(Enbrel�; licensed by Immunex Corp., Thousand Oaks, CA,
U.S.A.) is a subcutaneously administered biological response
modifier and is a dimeric fusion protein consisting of the
extracellular ligand-binding portion of the human TNF-R. This
molecule is linked to the Fc portion of human IgG1 and pre-
vents the interaction of both TNF-a and TNF-b with their
respective receptors. Currently, these and other biologics
against TNF-a are in various stages of clinical studies and/or
are in the stage of approval in dermatology, especially for pso-
riasis and PsA.8 This indicates that anti-TNF-a antibodies are
effective for the immunological treatment of psoriasis. How-
ever, only approximately 30% of treated patients respond suf-
ficiently. It is not yet clear which are the responding subtypes.
On the other hand, infections, the occurrence of autoanti-
bodies and hypersensitivity reactions have been reported
during TNF-a-targeted treatment.9,10 Some recent single case
� 2007 The Authors
486 Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp486–491
reports have demonstrated cutaneous side-effects in patients
with a rheumatic disease during anti-TNF-a treatment.11–15
Such reports included the onset of skin lesions such as hyper-
keratosis, eczema, blistering and dry skin. Also, local reactions
at the injection side (urticaria and pruritus) were detected
during clinical studies.16 However, larger studies evaluating
cutaneous side-effects in these patients are not available.
In this prospective analysis, we examined 35 patients with
an active rheumatic disease receiving anti-TNF-a antibodies.
The clinical work-up shows that a wide spectrum of chronic
inflammatory but also infectious skin disorders may develop
in patients receiving anti-TNF-a treatment.
Materials and methods
In total, 150 adult patients who were sequential attendees at
the clinic and who had an active rheumatic disease were trea-
ted with different TNF-a antagonists in the Department of
Rheumatology and Clinical Immunology (Charite-Universitats-
medizin Berlin, Berlin, Germany). During the treatment, 35 of
150 patients developed skin eruptions (Table 1) and were
referred to the Department of Dermatology and Allergy
between November 2002 and November 2004.
A detailed history and dermatological examination was per-
formed in all 35 patients. Eleven of these patients underwent
a skin biopsy for histological examination, and seven had an
additional diagnostic work-up regarding type I and/or type IV
sensitizations.
Results
Examination of the 35 patients (18 women and 17 men, aged
19–76 years) receiving TNF-a antagonists revealed a broad
spectrum of skin diseases. These included the new onset or
Table 1 Details of the study population (n ¼ 35), including demographics, rheumatic disease, antitumour necrosis factor (TNF)-a treatment, and
time between treatment initiation and development of the skin eruptions
Patient Sex
Age
(years)
Rheumatic
disease
Anti-TNF-atreatment Skin eruption
Time after initiation of
anti-TNF-a treatment
New onset or
exacerbation
1 F 71 RA Etanercept Palmoplantar pustular psoriasis 10 months New onset2 F 44 RA Etanercept Psoriasis vulgaris 6 months New onset
3 M 39 RA Etanercept Eczematous skin lesion 12 months New onset4 F 41 AS Infliximab Atopic dermatitis-like eruption 1 month New onset
5 M 39 NC Adalimumab Psoriasis vulgaris 6 months Exacerbation6 F 49 RA Adalimumab Perivascular neutrophilic dermatitis 1 week New onset
7 M 45 PsA Infliximab Acute staphylococcal infection 6 months New onset8 F 74 RA Adalimumab Herpes simplex virus II infection 4 months New onset
9 M 64 RA Adalimumab Pityriasis versicolor 18 months New onset10 F 68 RA Infliximab Eczematous skin lesion 4 months New onset
11 M 19 AS Infliximab Psoriasis-like eruption 2 months New onset12 M 28 AS Infliximab Pityriasis versicolor 9 months New onset
13 F 23 RA Adalimumab Alopecia areata 7 months Exacerbation14 F 45 RA Adalimumab Microbial eczema 6 months New onset
15 F 62 RA Adalimumab Dermatitis herpetiformis 6 months Exacerbation16 M 51 AS Infliximab Eczematous skin lesion 10 months New onset
17 F 39 RA Adalimumab Androgenetic alopecia 8 months New onset18 F 44 RA Etanercept Tinea 6 months New onset
19 F 39 AS Infliximab Microbial eczema 6 months New onset20 M 63 RA Etanercept Psorasis vulgaris 2 weeks Exacerbation
21 F 34 AS Infliximab Microbial eczema 4 months New onset22 F 76 RA Adalimumab Pityriasis rosea 26 months New onset
23 M 63 RA Infliximab Dermatitis sicca 1 month New onset24 F 52 RA Etanercept Eczematous skin lesion 10 months New onset
25 F 54 RA Adalimumab Palmoplantar pustular psoriasis 8 months New onset26 F 27 AS Infliximab Psoriasis-like lesion 17 months New onset
27 M 45 AS Infliximab Palmoplantar pustular psoriasis 4 months New onset28 M 43 AS Infliximab Pityriasis versicolor 1 month New onset
29 M 46 AS Infliximab Eczematous skin lesion 2 weeks New onset30 M 56 RA Adalimumab Eczematous skin lesion 1 week New onset
31 M 34 AS Infliximab Eczematous skin lesion 10 months New onset
32 M 51 RA Infliximab Microbial eczema 11 months New onset33 M 57 AS Infliximab Microbial eczema 5 months New onset
34 M 64 AS Infliximab Tinea 10 months New onset35 F 40 RA Adalimumab Tinea 13 months New onset
AS, ankylosing spondylitis; NC, nonclassified; PsA, psoriatic arthritis; RA, rheumatoid arthritis.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp486–491
Side-effects of TNF-a antagonists in rheumatic disease, H-H. Lee et al. 487
worsening of pre-existing chronic inflammatory and infectious
skin diseases (Fig. 1). An association between a particular
anti-TNF-a treatment and a particular skin disease was not
observed. Twenty of 35 patients had RA (14 women, six
men), 13 of 35 patients had AS (four women, nine men),
one man had PsA and one man had a nonclassified rheumatic
disease (Fig. 2).
Treatment modalities
The anti-TNF-a treatment modalities used to treat the patients
with rheumatic diseases are summarized in Table 2. Seventeen
patients received infliximab, two of these in combination with
methotrexate (MTX) and one in combination with lefluno-
mide (LEF). Twelve patients were treated with adalimumab,
one of these in combination with MTX, one with LEF, and
two with MTX and LEF together. The other six patients
received etanercept, one of these in combination with LEF.
Frequent occurrence of chronic inflammatory skin
diseases
A total of 16 of 35 patients presented with inflammatory skin
eruptions, with eight of 16 showing psoriasis-like skin lesions
(Fig. 3a) and eight of 16 showing eczema either as atopic der-
matitis (AD)-like eruptions or as eczematous skin lesions
(Fig. 3b).
Of the eight patients with psoriasis-like skin lesions, five
had the typical clinical pattern of psoriasis vulgaris and three
exhibited palmoplantar pustular psoriasis. Two of these eight
patients had a history of psoriasis vulgaris, and six had lesions
for the first time.
Of the eight patients with eczema, the skin lesions were
located primarily on the extremities within the antecubital and
popliteal fossae, but also in the face or trunk area. A moderate
to severe itch was always a concomitant symptom in these
patients.
Infectious skin diseases
Of the 35 patients, 13 presented with a new onset of an
infectious skin disease during anti-TNF-a antibody treat-
ment. Three of the 13 patients showed typical skin eruptions
16
13
6
0
4
8
12
16
20
Chronic inflammatoryskin disease
Infectious skin disease Other skin disease
Nu
mb
er o
f pat
ien
ts
Fig 1. Distribution of skin diseases (n ¼ 35). Chronic inflammatory
skin disease: psoriasis-like lesions (n ¼ 8), atopic dermatitis-like
eruptions (n ¼ 8). Infectious skin disease: pityriasis versicolor (n ¼ 3),
tinea corporis (n ¼ 3), microbial eczema (n ¼ 5), herpes simplex
virus infection (n ¼ 1), acute cutaneous staphylococcal superinfection
(n ¼ 1). Other skin disease (n ¼ 1, respectively): dermatitis
herpetiformis, pityriasis rosea, dermatitis sicca, alopecia areata,
androgenetic alopecia, leucocytoclastic vasculitis.
14
46
9
110
4
8
12
16
20
RA AS PsA NK
Nu
mb
er o
f pat
ien
ts
f m
Fig 2. Diagnosis and gender distribution in the study population
(n ¼ 35). RA, rheumatoid arthritits; AS, ankylosing spondylitis;
PsA, psoriatic arthritis; NK, nonclassified rheumatic disease.
Table 2 Dosage of tumour necrosis factor (TNF)-a antagonists as recommended by the manufacturer and current treatment of rheumatic diseaseof the study population (n ¼ 35)
TNF-a antagonist
Number and sex of patients with rheumatic disease
RA (n ¼ 20) AS (n ¼ 13) PsA (n ¼ 1) NC (n ¼ 1)
M F M F M F M F
Infliximab (Remicade�) 3 mg kg)1 i.v. (8 weeks) 2 1a 9 4b 1b – – –
Adalimumab (Humira�) 40 mg s.c. (2 weeks) 2c 9a,b – – – – 1c –Etanercept (Enbrel�) 2 · 25 mg s.c. (1 week) 2a 4 – – – – – –
AS, ankylosing spondylitis; i.v., intravenous; NC, nonclassified; PsA, psoriatic arthritis; RA, rheumatoid arthritis; s.c., subcutaneous.aOne patient in combination with leflunomide (LEF). bOne patient in combination with methotrexate (MTX). cOne patient in combinationwith MTX and LEF.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp486–491
488 Side-effects of TNF-a antagonists in rheumatic disease, H-H. Lee et al.
resulting from the yeast Pityrosporum ovale, causing pityriasis
versicolor in all patients (Fig. 3c), whereas tinea corporis was
diagnosed in three of 13 patients. One of the 13 patients had
a reactivation with herpes simplex virus that appeared clinic-
ally as small vesicles on the right-upper abdomen. Five of the
13 patients developed a bacterial infection of the skin present-
ing clinically as microbial eczema, and one patient had an
acute cutaneous staphylococcal superinfection with acute
erythematous swelling of the right ear.
Other skin diseases
Six of the 35 patients showed various other skin eruptions.
One 39-year-old woman developed androgenetic alopecia and
telogen hair loss 8 months after commencing adalimumab
treatment. A 23-year-old woman developed alopecia areata
immediately after beginning LEF monotherapy; after the first
injection of adalimumab 1 year later, the disease activity
increased dramatically. A 62-year-old woman had dermatitis
herpetiformis, which was diagnosed many years before the
anti-TNF-a treatment but flared 6 months after initiation of
the treatment with adalimumab. The development of a leuco-
cytoclastic vasculitis was observed in a 49-year-old woman,
and a 76-year-old woman developed pityriasis rosea. Finally, a
63-year-old man had extremely dry skin that presented clinic-
ally as dermatitis sicca.
Discussion
TNF-a is a pleiotropic proinflammatory cytokine and plays,
among others, a central role in the pathogenesis of rheumatic
diseases. High levels of TNF-a have been found in the syn-
ovial fluid of patients with RA and AS,17–19 but also in those
with PsA and in psoriasis skin lesions.20
In this report we present 35 of 150 patients with rheumatic
disease receiving anti-TNF-a-based treatment experiencing the
onset of cutaneous lesions. The data show a broad spectrum
of skin eruptions. Patients with lesions typical of chronic
inflammatory skin diseases represent the majority, but infec-
tions and several other skin manifestations were also observed.
No allergic or toxic skin reaction occurred in the patients, but
instead a diverse clinical pattern that might be related to a
genetic predisposition.
Psoriasis and AD are common chronic inflammatory skin
disorders that are characterized by the infiltration of leuco-
cytes, the activation of skin-resident cells and an increased
production of numerous cytokines, chemokines and inflamma-
tory molecules. Nearly half (16 of 35) of the patients with
rheumatic diseases and skin eruptions developed either AD- or
psoriasis-like lesions.
There is strong evidence that psoriasis is an immune-related
disease.21 Many studies have argued for a key role of T cells
in the pathogenesis of psoriasis, but recent data also point to a
potential role of innate inflammatory triggers. Lymphocytes in
the lesions are mainly skin-homing CD4+ and CD8+ T cells
with an activated, memory/effector type-1 immunopheno-
type.22
Activated dendritic cells (DCs) and infiltrating T cells deter-
mine a type-1 inflammatory pathway that links the activation
of multiple inflammatory genes to the release of interferon
(IFN)-c from T cells, interleukin (IL)-12 from the activated
DCs and, as described recently, IL-23 from keratinocytes.23
(a)
(b)
(c)
Fig 3. (a) Patient with psoriasis-like skin lesions. (b) Patient with
eczematous eruptions in both popliteal fossae. (c) Patient with an
infectious skin disease (pityriasis versicolor) during treatment with
antitumour necrosis factor-a biologics.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp486–491
Side-effects of TNF-a antagonists in rheumatic disease, H-H. Lee et al. 489
TNF-a can stimulate inflammation in the type-1 pathway
through activating effects on DCs or by increasing transcrip-
tion of type-1 genes.
AD is a multifactorial and polygenetic skin disease charac-
terized by T helper 2 cells, mast cells and eosinophils produ-
cing IL-4, IL-5, IL-10 and IL-13.24 Acute eczematous skin
lesions show an early infiltration of CD4+, but also CD8+,
T cells in the dermis producing IL-4. In the late stage of
AD, IFN-c is the major cytokine triggering the chronic inflam-
mation.
The genetic background of psoriasis and AD has been exten-
sively studied and several susceptibility genes have been des-
cribed.25 Recent genome-wide linkage scans have identified
multiple loci linked to each other. An overlap between psori-
asis and AD susceptibility loci on chromosomes 1q21, 3q21,
17q25 and 20p12 suggest that some susceptibility factors are
found within genes or gene families with common effects on
epithelial immunity. However, many genes from these loci are
not yet identified or tested.26 Genes linked to psoriasis include
HLA-C, SLC9A3R1, NAT9, RAPTOR and SLC12A8; those linked to
AD include SPINK5, MS4A2 and PHF11.27
The identification of RUNX1 as an RA-associated gene has
been published recently.28 RUNX1 is a haematological tran-
scriptional regulator responsible for acute myelogenic leukae-
mia. On the other hand, one susceptibility factor for psoriasis
has been suggested to be the defective regulation of SLC9A3R1
(a phosphoprotein implicated, in association with other mole-
cules, in the regulation of membrane dynamism for synapse
formation and T-cell activation) or NAT9 (a new member
of the N-acetyl-transferase superfamily encoded by a still
unknown gene) by RUNX1. Thus, RUNX1 might establish a
genetic relationship between RA and psoriasis.
The other large group of patients (13 of 35) identified in
this study is characterized by the onset of cutaneous infectious
diseases during anti-TNF-a treatment. This observation is rela-
ted to the role of TNF-a in direct defence against infections.
Interestingly, fungal and viral skin infections were most fre-
quently observed in this study. However, these infectious skin
eruptions were mild and easily controlled by the appropriate
pharmacological treatment.
Recently, the use of TNF-a antagonists has been associated
with new cases of active tuberculosis worldwide.29,30 It is
known that TNF-a plays a central role in the cellular defence
mechanisms by monocytes/macrophages against infection
caused by Mycobacterium tuberculosis. Treatment with anti-TNF-aagents suggests they pose a threat of increased risk of onset of
not only tuberculosis but also of other (opportunistic) infec-
tions. However, owing to the different mode of action and
the specific pharmacodynamics of the TNF-a antagonists, the
role of the actual risk regarding infectious diseases may be not
as expected. This assumption has to be proven by future clin-
ical studies.
The other skin diseases developed by six of 35 patients
appeared only as single cases. We documented one case each
of dermatitis herpetiformis, pityriasis rosea, dermatitis sicca,
leucocytoclastic vasculitis, androgenetic alopecia and alopecia
areata, respectively. In regard to alopecia areata, it is known
that TNF-a induces the loss of hair; therefore, the paradoxical
side-effect of development of alopecia areata following anti-
TNF-a treatment is still a matter of discussion.31
Several other reports have also referred to adverse skin reac-
tions during treatment with anti-TNF-a antagonists. Interest-
ingly, all cases occurred during the application of infliximab.
Devos et al. described six patients receiving infliximab for RA
or Crohn’s disease, four of whom developed leucocytoclastic
vasculitis, lichenoid drug reaction, acute folliculitis or superfi-
cial granuloma, respectively, and two developed perniosis-like
eruptions.15 Vergara et al. reported four patients (three with
RA, one with AS) with an interface dermatitis pattern.13 In
addition, a positive patch test with infliximab suggested
T-cell-specific sensitization. AD-like eruptions were noticed by
Wright:14 in two patients, the eczematous skin lesions
occurred with accentuation in the antecubital fossae and were
assessed as nonallergic reactions to the infliximab therapy.
Chan et al. reported one patient with acute pustular dermatitis
following infliximab infusions, accompanied with necrosis of
the subcutaneous tissue, a systemic group A b-haemolytic
streptococcal infection and other severe systemic illness.11 The
diagnosis of necrotizing fasciitis was confirmed. Furthermore,
Kent et al. referred to one patient with RA who developed bul-
lous skin lesions that were neither typical of pemphigus vul-
garis nor of bullous pemphigoid despite a common clinical
pattern.12 Finally, Sfikakis et al. recently reported five patients
(three of them with a rheumatic disease) who developed pso-
riasis induced by anti-TNF-a treatment.31
Taken together, our data show that a wide range of differ-
ent skin lesions might occur during anti-TNF-a treatment.
These are based on either the exacerbation of a pre-existing
skin disease or indicate the new onset of the skin disease.
Chronic inflammatory or infectious skin diseases were
most frequently observed. However, these diseases are also
frequent in the general population. If a patient receiving
anti-TNF-a treatment is referred to a dermatologist, any skin
disease should be considered and a thorough work-up is
necessary to determine the exact diagnosis. In none of our
patients was a discontinuation or termination of the anti-
TNF-a treatment necessary and all skin diseases were treated
regularly.
Finally, our data indicate the need for long-term observa-
tions of patients treated with anti-TNF-a agents, for instance
through the introduction of nationwide registries.
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Side-effects of TNF-a antagonists in rheumatic disease, H-H. Lee et al. 491
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07685.x
Changes in skin barrier function following long-termtreatment with moisturizers, a randomized controlled trialI. Buraczewska,*� B. Berne,* M. Lindberg,� H. Torma* and M. Loden�
*Department of Medical Sciences, Dermatology and Venereology, University Hospital, Uppsala, Sweden
�ACO HUD NORDIC AB, Box 622, SE-194 26 Upplands Vasby, Sweden
�Department of Medicine, Occupational and Environmental Dermatology, Karolinska Institute and Occupational and Environmental Health, Stockholm Centre for
Public Health, Stockholm County Council, Stockholm, Sweden
CorrespondenceIzabela Buraczewska.
E-mail: izabela.buraczewska@acohud.se
Accepted for publication2 October 2006
Key wordscapacitance, epidermis, long-term treatment,
moisturizers, skin barrier function, transepidermal
water loss
Conflicts of interestI.B. and M.L. are employed by ACO HUD
NORDIC AB, which provided funding for the
research presented in this article. No other authors
have anything to declare.
Summary
Background Moisturizers are commonly used by patients with dry skin conditionsas well as people with healthy skin. Previous studies on short-term treatmenthave shown that moisturizers can weaken or strengthen skin barrier function andalso influence skin barrier recovery. However, knowledge of the effects on skinbarrier function of long-term treatment with moisturizers is still scarce.Objectives To investigate the impact of long-term treatment with moisturizers onthe barrier function of normal skin, as measured by transepidermal water loss(TEWL) and susceptibility to an irritant, and to relate those effects to the compo-sition of the designed experimental moisturizers.Methods Volunteers (n ¼ 78) were randomized into five groups. Each group treat-ed one volar forearm for 7 weeks with one of the following preparations:(i) one of three simplified creams, containing only a few ingredients in order tominimize the complexity of the system; (ii) a lipid-free gel; (iii) one ordinarycream, containing 5% urea, which has previously been shown to decrease TEWL.The lipids in the simplified creams were either hydrocarbons or vegetable trigly-ceride oil, and one of them also contained 5% urea. After 7 weeks, treated andcontrol forearms were exposed for 24 h to sodium lauryl sulfate (SLS) using apatch test. TEWL, blood flow and skin capacitance of both SLS-exposed andundamaged skin were evaluated 24 h after removal of patches. Additionally, a24-h irritancy patch test of all test preparations was performed on 11 volunteersin order to check their possible acute irritancy potential.Results Changes were found in the barrier function of normal skin after 7 weeksof treatment with the test preparations. The simplified creams and the lipid-freegel increased TEWL and skin response to SLS, while the ordinary cream had theopposite effect. One of the simplified creams also decreased skin capacitance. Alltest preparations were shown to be nonirritant, both by short-term irritancypatch test and by measurement of blood flow after long-term treatment.Conclusions Moisturizers influence the skin barrier function of normal skin, asmeasured by TEWL and susceptibility to SLS. Moreover, the effect on skin barrierfunction is determined by the composition of the moisturizer. The ingredientswhich influence the skin barrier function need to be identified, and the mechan-ism clarified at the molecular level.
Moisturizers are commonly used by patients with dry skin
conditions, and their application is also a widespread habit
among people with healthy skin. However, there is still a
lack of knowledge about the effects of moisturizers on skin
barrier function, especially after long-term treatment. Studies
conducted so far, both in healthy and diseased skin, show
that some moisturizers may weaken skin barrier func-
tion—observed as changes in transepidermal water loss
(TEWL) and susceptibility to irritants—while others may
strengthen it.1–6 Moisturizers can also influence skin barrier
recovery.1,7 It can be assumed that differences in composi-
tion account for the differences in effect. Lipid content and
� 2007 The Authors
492 Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
natural moisturizing factors such as urea have been suggested
to be most important.
The aim of the present study was to investigate the impact
of long-term treatment with moisturizers on the barrier func-
tion of normal skin, as measured by TEWL, and susceptibility
to an irritant, and to relate those effects to the composition of
the moisturizers used. The moisturizers chosen for the study
were one ordinary cream, which has previously been shown
to decrease TEWL,1 and three simplified creams containing
only a few ingredients in order to minimize the complexity of
the system.
The lipids used in the simplified creams were either pure
hydrophobic hydrocarbons derived from mineral oil (paraffin
and isohexadecane) or a more polar vegetable oil consisting of
triglycerides and sterols (canola oil), in order to see the differ-
ence between two contrasting types of lipids. Urea was added
to one composition.
The duration of the treatment was chosen to be 7 weeks,
which is longer than the application time in the majority of
similar studies performed to date. An irritancy patch test of all
test preparations was also performed in order to check their
possible acute irritancy potential.
Materials and methods
Test preparations
Five test preparations were used in the study: one ordinary
cream, hereafter called Complex cream; three simplified
creams emulsified with a long-chain polymer, with a lipid
phase consisting of either the hydrocarbons isohexadecane and
paraffin (Hydrocarbon cream) or a vegetable triglyceride oil,
canola oil (Canola cream and Canola/urea cream); and one
lipid-free gel consisting of the polymer used in simplified
creams (Polymer gel). The polymeric emulsifier in the simpli-
fied creams and the Polymer gel was acrylates/C10-30 alkyl
acrylate crosspolymer. The creams were oil-in-water (o/w)
emulsion, and all test preparations had their pH adjusted to
� 5. The Canola/urea cream also contained urea. Table 1 pre-
sents detailed compositions along with the number of volun-
teers testing each preparation.
Participants and experimental design of the long-term
treatment study and the irritancy test
The study consisted of two parts: a long-term treatment study
with the test preparations and an irritancy test.
Volunteers (n ¼ 78; 58 women and 20 men, aged 25–
60 years) participated in the long-term treatment study, and
11 volunteers (eight women and three men, aged 27–
46 years) in the irritancy test. Both studies were double-blind
and randomized. Informed consent was obtained from all
volunteers and the study was approved by the Regional Ethi-
cal Review Board (Uppsala, Sweden). Volunteers were not
allowed to use any skin care products on their forearms, nei-
ther during the test period nor in the three preceding days.
In the long-term treatment study, volunteers treated one
volar forearm twice daily for 7 weeks with one test prepar-
ation, leaving the other forearm untreated to serve as the con-
trol. The treatment side was randomized. After 7 weeks, on
day –1, both volar forearms, treated and control, were exposed
to sodium lauryl sulfate (SLS) using a patch test: 50 lL of 1%
aqueous solution of SLS (purity ‡ 99%, Sigma-Aldrich, St
Louis, MO, U.S.A.) in large Finn chambers, Ø 12 mm (Epitest
Ltd OY, Tuusula, Finland).8 The chambers were fixed to the
skin on the volar aspect of the forearm with adhesive tape
Table 1 Composition of test preparations and numbers of volunteers testing them
Test preparation Lipids Emulsifiers Urea Other ingredients pHNo. ofvolunteers
Complex creama 20%: Capric/caprylic
triglyceride, canola oil,cetearyl alcohol,
paraffin, glycerylstearate
1Æ3%: PEG-100 stearate,
carbomer, polysorbate 60
5% Water, propylene glycol,
glyceryl polymethacrylate,dimethicone, sodium
lactate, methylparaben,propylparaben,
lactic acid, citric acid
5 15
Hydrocarbon
cream
40%: Isohexadecaneb
(20%), paraffinc (20%)
0Æ4%: Acrylates/C10-30 alkyl
acrylate crosspolymerd0% Water 5 16
Canola cream 40%: Canola oile 0Æ4%: Acrylates/C10-30 alkyl
acrylate crosspolymerd0% Water 5 15
Canola/urea
cream
40%: Canola oild 0Æ4%: Acrylates/C10-30 alkyl
acrylate crosspolymerd5% Water 5 16
Polymer gel 0% 0Æ4%: Acrylates/C10-30 alkyl
acrylate crosspolymerd0% Water, methylparabenf 5 16
aCanoderm� kram 5%, ACO HUD NORDIC AB, Stockholm, Sweden; bArlamol HD, Uniqema, Gouda, the Netherlands; cMerkur White OilPharma, Merkur Vaseline, Hamburg, Germany; dPemulen TR-2, Noveon Inc., Cleveland, OH, U.S.A.; eAkorex L, Karlshamns AB, Karlshamn,
Sweden; fNipagin M, Clariant, Clariant International, Pontypridd, U.K.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
Long-term treatment with moisturizers, I. Buraczewska et al. 493
(Scanpore, Norgeplaster, Oslo, Norway). They were removed
after 24 h, on day 0. On removal of the patches, the skin was
rinsed gently with water and patted dry with a paper towel.
Evaluations were performed on treated and control forearms
24 h after removal of patches (day 1) on the SLS-exposed skin
area and on an area of undamaged (non-SLS-exposed) skin a
few centimetres below it. SLS-exposed skin and undamaged
skin areas were assessed for transepidermal water loss (TEWL)
and blood flow, and undamaged skin was also evaluated for
skin capacitance.
Volunteers participating in the irritancy test had the five test
preparations, water, and 1% aqueous solution of SLS (pur-
ity ‡ 99%, Sigma-Aldrich) applied on the skin on the volar
aspect of forearms for 24 h to assess their acute irritancy
potential using a patch test: 50 lL in large Finn chambers,
Ø 12 mm (Epitest Ltd OY).8 The chambers were fixed to the
skin with adhesive tape (Scanpore, Norgeplaster). They were
removed after 24 h, and the skin was then rinsed gently with
soap and water and patted dry with a paper towel.
Visual scoring was performed 1 h and 24 h after removal
of patches, and TEWL was measured 24 h after removal of
patches.
Evaluations
TEWL was quantified using DermaLab (Cortex Technology,
Hadsund, Denmark) according to published guidelines for
TEWL measurements.9 Skin electrical capacitance was meas-
ured with a Corneometer 820 (Courage and Khazaka GmbH,
Cologne, Germany). Cutaneous blood flow was measured with
a laser Doppler flowmeter (Periflux PF1d, Perimed, Stockholm,
Sweden)10 equipped with a special multifibre probe (PF 113
integrating probe, Perimed).
Visual scoring ranged from 0 to 4 according to the Euro-
pean Society of Contact Dermatitis (ESCD) guidelines on clin-
ical scoring of acute SLS irritant reactions,8 where erythema,
oedema and scaling were taken into consideration. The scale
was as follows: 0 ¼ negative: no damage; 0Æ5 ¼ doubtful:
very weak erythema or minute scaling; 1 ¼ weak: weak ery-
thema, slight oedema, slight scaling and/or slight roughness;
2 ¼ moderate: moderate degree of erythema, oedema, scaling
and/or roughness; 3 ¼ strong: marked degree of erythema,
oedema, scaling, roughness; 4 ¼ very strong/caustic: as 3,
with necrotic areas.
Calculations and statistics
The results are shown using boxplots (Figs 1–3). The box is
defined by the upper (Q3) and lower (Q1) quartiles and the
median is marked by a subdivision of the box. The whiskers
are the lines that extend from the top and bottom of the box
to the lowest and highest observations that are inside the
region defined by the following limits: lower limit, Q1 – 1Æ5(Q3–Q1); upper limit, Q3 + 1Æ5 (Q3–Q1). Outliers are points
outside of the lower and upper limits and are plotted with
asterisks.
In long-term treatment with moisturizers, results from
measurements of TEWL, skin capacitance and blood flow of
treated forearms are presented as a percentage ratio to the cor-
responding values obtained from control forearms, which
serve as 100%.
Moreover, relative increases of TEWL in SLS-exposed skin
area in comparison with TEWL in undamaged skin on the
same forearm were calculated for treated and untreated fore-
arms to estimate the difference in skin reactivity to SLS after
treatment, according to the following formula: relative increase
(%) ¼ (vSLS/vUND) · 100, where vSLS represents the value
measured on SLS-exposed skin and vUND represents the corres-
ponding value from undamaged skin on the same forearm.
In the irritancy test, results from visual scoring are
tabulated as frequencies. The percentage of total possible
score (% TPS) was calculated as follows: % TPS ¼ (0 · n0 +
0Æ5 · n0Æ5 + 1 · n1 + 2 · n2 + 3 · n3 + 4 · n4)/[4 · (n0 +
n0Æ5 + n1 + n2 + n3 + n4)] · 100, where ni represents the
Complex creamPolymer gelCanola/urea creamCanola creamHydrocarbon cream
200
175
150
125
100
75
50
TEWL
Capacitance
P =
0·0
18
P =
0·0
11
P =
0·0
07
P =
0·4
14
P =
0·0
16
P =
0·5
72
P =
0·0
17
P =
0·4
53
P =
0·0
06
P =
0·1
67
Fig 1. TEWL and skin capacitance of
undamaged skin treated with test preparations
for 7 weeks. Values are presented as
percentage ratio of the values obtained from
the corresponding control areas, which serve
as 100%. P-values relate to differences from
control areas. The results are presented as box
plots with the median value as a line across
the box and the first quartile value at the
bottom (Q1) and the third at the top (Q3).
The whiskers are lines that extend from the
top and bottom of the box to the lowest and
the highest observation within a defined
region, with outliers plotted as asterisks
outside this region. Reference line is given at
100%. n ¼ 15 for Canola cream and Complex
cream and 16 for Canola/urea cream,
Hydrocarbon cream and Polymer gel.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
494 Long-term treatment with moisturizers, I. Buraczewska et al.
number of volunteers with a visual score of i. TEWL is presen-
ted as a percentage ratio of the corresponding value obtained
from water.
An explorative data analysis was performed using a Wil-
coxon signed rank test on paired data to analyse differences
between results of TEWL, visual scoring, skin capacitance and
blood flow for all test preparations. A Kruskal–Wallis test of
equality of medians was used to investigate the differences
between the simplified creams.
Minitab� statistical software, Release 14 for Windows
(Minitab Inc., State College, PA, U.S.A.), was used for calcula-
tions and plots. The level of significance was set at P < 0Æ05.
Results
Treatment of normal skin for 7 weeks with Hydrocarbon
cream, Canola cream, Canola/urea cream and Polymer gel
increased TEWL in comparison with the untreated controls,
while treatment with Complex cream, conversely, decreased
TEWL. In addition, skin capacitance was decreased in skin
treated with Hydrocarbon cream, in comparison with the con-
trol, while no difference was found for the remaining test
preparations (Fig. 1).
No signs of irritation measured as changes in blood flow
were found on the areas treated with the test preparations in
comparison with controls (data not shown).
After exposure to SLS, skin treated with Hydrocarbon
cream, Canola cream, Canola/urea cream and Polymer gel had
significantly higher TEWL than SLS-exposed skin on untreated
forearms, while skin treated with Complex cream had lower
TEWL than control (Fig. 2). However, the relative increase in
TEWL after SLS exposure was the same in skin treated with
Hydrocarbon cream, Canola cream, Polymer gel and Complex
cream as in untreated control skin. Skin treated with Canola/
Complex creamPolymer gelCanola/urea creamCanola creamHydrocarbon cream
1000
900
800
700
600
500
400
300
200
100
Treated
Control
P = 0·394 P = 0·478 P = 0·002 P = 0·587 P = 0·094
Fig 3. Relative increase of TEWL after SLS
exposure in comparison with TEWL of
undamaged skin area on the same forearm,
calculated for the skin areas treated with test
preparations and untreated control areas. For
explanation of the boxplots, see Figure 1.
Complex creamPolymer gelCanola/urea creamCanola creamHydrocarbon cream
350
300
250
200
150
100
50
0
TEWL
Blood flow
P =
0·0
09
P =
0·0
36
P =
0·0
21
P =
0·0
33
P =
0·7
12
P =
0·0
01
P =
0·0
01
P =
0·0
45
P =
0·0
44
P =
0·0
34
Fig 2. Transepidermal water loss (TEWL) and
blood flow of sodium lauryl sulfate (SLS)-
exposed skin treated with test preparations,
in relation to control areas (not treated with
moisturizers but also exposed to SLS). For
explanation of the graph, see Figure 1.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
Long-term treatment with moisturizers, I. Buraczewska et al. 495
urea cream had a higher relative increase of TEWL than the
untreated control skin (Fig. 3).
Blood flow closely followed the pattern of TEWL, except in
skin treated with Polymer gel, where blood flow did not differ
significantly from the control. In contrast to other test prepa-
rations, skin treated with Complex cream had lower blood
flow after SLS exposure than the corresponding untreated fore-
arm (Fig. 2).
The Kruskal–Wallis test revealed no differences between the
simplified creams (Hydrocarbon cream, Canola cream and
Canola/urea cream) in their effects on TEWL of undamaged
skin (P ¼ 0Æ685), TEWL of SLS-exposed skin (P ¼ 0Æ687) and
blood flow of SLS-exposed skin (P ¼ 0Æ833). However, there
was a difference in impact on skin capacitance (P ¼ 0Æ017).
The occlusive irritancy patch test showed that Canola cream,
Canola/urea cream, Hydrocarbon cream and Polymer gel were
no more irritant than water, evaluated both by visual scoring
and TEWL measurements. Hydrocarbon cream produced elevat-
ed visual scoring 24 h after removal of patches, but not after
1 h. Moreover, Complex cream was less irritant than water in
visual scoring 1 h after removal of patches, and showed no
difference from water at 24 h, either in visual scoring or
TEWL. SLS, used as a reference, gave significantly higher
results than water (Table 2).
Discussion
In this study we found changes in the barrier function of nor-
mal skin after 7 weeks of treatment with test preparations, as
observed by changes in TEWL and skin response to SLS.
Simplified creams with different types of lipids (Hydro-
carbon cream, Canola cream and Canola/urea cream), as well
as Polymer gel, increased TEWL after long-term treatment.
After exposure to SLS, skin treated with these test preparations
had higher TEWL compared with untreated control skin.
Blood flow was also elevated, except for Polymer gel. There
were no differences between the three simplified creams in
impact on TEWL and blood flow. However, looking at the
relative increase of TEWL after SLS exposure in treated skin, a
significant increase was found only for Canola/urea cream. In
accordance with previous findings, treatment with Complex
cream had the opposite effect, with a decreased TEWL and
skin response to SLS.1 However, there was no difference from
control in the relative increase of TEWL in comparison with
untreated skin after SLS exposure.
Skin capacitance was decreased after usage of Hydrocarbon
cream, and remained unchanged, compared with controls, for
all other test preparations. This result suggests that the
increased TEWL is caused by impairment of the skin barrier
and not by elevated hydration of the stratum corneum.11 Test
preparations were shown to be not irritant, both by short-
term irritancy patch test and by measurement of blood flow
after long-term treatment.
Our findings are in accordance with previously performed
studies in which 2- or 3-week treatments with moistur-
izers influenced skin barrier function. Held et al.3 showed Tab
le2
Irrita
tion
pote
ntia
lof
test
prep
arat
ions
,gi
ven
asre
sults
ofvi
sual
scor
ing
and
tran
sepi
derm
alw
ater
loss
(TEW
L).Visua
lsc
orin
gha
sa
cate
gory
scale
from
0to
4.N
umbe
rof
volu
ntee
rsw
ith
the
resp
ective
scor
ean
dpe
rcen
tage
tota
lpo
ssib
lesc
ore
(TPS
)is
give
n.TEW
Lat
the
24-h
read
ing
ispr
esen
ted
asa
perc
enta
gera
tio
tore
sults
obta
ined
for
wat
er.M
edian
valu
ean
dfirs
tan
dth
ird
quar
tile
sar
egi
ven
(n¼
11).
P-va
lues
relate
todi
ffer
ence
from
wat
er.
Scor
e
Wat
erH
ydro
carb
oncr
eam
Can
ola
crea
mCan
ola/
urea
crea
mCom
plex
crea
mPo
lym
erge
l1%
SLS
1h
24h
1h
24h
1h
24h
1h
24h
1h
24h
1h
24h
1h
24h
02
104
43
117
1111
113
110
0
0Æ5
61
17
40
20
00
40
42
13
06
04
02
00
04
01
62
00
00
00
00
00
00
63
30
00
00
00
00
00
00
04
00
00
00
00
00
00
00
TPS
(%)
13Æ6
1Æ1
14Æ8
8Æ0
13Æ6
06Æ
80
00
13Æ6
034
Æ129
Æ5P-
valu
e0Æ
800
0Æ03
61Æ
000
1Æ00
00Æ
091
1Æ00
00Æ
009
1Æ00
01Æ
000
1Æ00
00Æ
033
0Æ00
4
TEW
L,%
(Med
ian,
Q1–
Q3)
–10
0–
97Æ6
(92Æ
7–10
2Æ2)
–10
1Æ4
(89Æ
0–11
2Æ3)
–94
Æ4(9
2Æ9–
102Æ
7)–
98Æ2
(91Æ
4–10
5Æ2)
–10
3Æ1
(90Æ
3–11
3Æ1)
–46
3Æ1
(298
Æ7–6
07Æ7
)
P-va
lue
––
–0Æ
398
–0Æ
760
–0Æ
307
–0Æ
683
–0Æ
563
–0Æ
004
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
496 Long-term treatment with moisturizers, I. Buraczewska et al.
impairment of skin barrier function by a cream with a high
lipid content (70%); 4 weeks of treatment increased skin sus-
ceptibility to SLS but did not change TEWL of undamaged
skin. The same cream also increased susceptibility to SLS after
only 5 days of treatment,12 and to nickel in nickel-allergic vol-
unteers after 7 days of treatment.13 The opposite results were
found by Loden et al.,1,2,4,5 who showed that 2- or 3-week
treatments of normal or atopic skin with moisturizers contain-
ing urea either decreased TEWL and susceptibility to SLS or
had no effect, but did not impair the skin barrier function.
The ordinary cream with 20% lipids was chosen as it has
been shown to decrease TEWL in short-term studies.1 A num-
ber of studies have shown that moisturizers with high lipid
content can affect barrier function.3,12,13 In the simplified
creams, we used as high a quantity of lipids as possible in the
formulations; with the emulsifier chosen, the maximum
amount turned out to be 40%. Two kinds of lipid were used
in the simplified creams, either pure hydrophobic hydrocar-
bons derived from mineral oil (paraffin and isohexadecane) or
a more polar vegetable oil consisting of triglycerides and
sterols (canola oil), in order to compare the effects of two
contrasting types of lipid. These creams were stabilized with a
polymeric emulsifier, which is believed not to penetrate and
influence skin barrier function due to its large molecular
size.14 In contrast, the Complex cream contained a mixture of
various fatty substances, of both vegetable and mineral origin,
and was emulsified with a combination of polymers and low-
molecular-weight emulsifiers. It also contained urea, which
was shown in a recent placebo-controlled study to reduce
TEWL and decrease skin susceptibility to SLS.2 Because the
Complex cream itself decreased TEWL,1 the same amount of
urea was incorporated into one of the simplified creams. In
addition, the polymeric emulsifier in a gel formulation was
used as a reference.
In the present study, the test preparations interfered with
the normal function or structure of the skin. The mechanism
of their action is not known, but there are several possibilities
to consider, and they can have both direct and indirect effects
on the skin barrier, stratum corneum.
Increased TEWL and dryness can be caused by exposure to
irritants. However, the patch test of the test preparations did
not reveal increased irritation compared with water. Moreover,
there were no signs of irritation, as measured with blood
flow, after 7 weeks of usage.
Water is the main component in the majority of moisturiz-
ers, and water itself may contribute to dryness and increased
TEWL. Prolonged contact with water disrupts the intercellular
lipid lamellae structure in the stratum corneum.15 Although
water evaporates quickly after application,16,17 the effect on
the skin barrier of 7 weeks of a twice-daily exposure is
unknown.
Models of the skin barrier (for review see18) emphasize that
barrier function depends on the structural organization of the
stratum corneum. Lipids in moisturizers may remain on the
skin surface or enter the skin,19 and more physiological lipids
may penetrate into the epidermis and affect skin barrier struc-
ture and recovery.20–22 The three simplified creams investi-
gated in this study, all of which had a negative effect on the
skin barrier, contained high proportions of lipids, 40%, but
the type of lipid was not of importance for the effect. At the
same time, Complex cream with a lower level of lipids, 20%,
produced the opposite result. The results for the simplified
creams may thus be explained by the impact of lipids, but it
does not explain the impairment of the skin barrier obtained
after treatment with Polymer gel.
Several emulsifiers have structures similar to the intercellular
bilayer lipids, and it is conceivable that such emulsifiers might
influence the organization of these lipids.14 As the impairment
of the skin barrier was also seen after application of the Poly-
mer gel, this negative effect could well be due to the emulsi-
fying polymer used in the simplified creams and the Polymer
gel, acrylates/C10-30 alkyl acrylate crosspolymer. The possible
impact of monomers, which may be present in low concentra-
tion, on lipid structures must be taken under consideration.
However, the Complex cream also contained a polymeric
emulsifier, carbomer, which is similar in structure to acry-
lates/C10-30 alkyl acrylate crosspolymer, and emulsifiers of
lower molecular weight—cetearyl alcohol, polysorbate 60,
glyceryl stearate and PEG-100 stearate—without producing a
negative impact on the skin barrier.
Humectants such as urea, glycerine and propylene glycol
may penetrate the skin.23,24 It has been suggested that the
decreased TEWL and lower response to SLS after treatment
with the Complex cream is due to its urea content.1,5 How-
ever, the addition of urea to the Canola/urea cream did not
improve skin barrier function, and even made the skin more
susceptible to SLS. The urea might have favoured the absorp-
tion of potentially damaging ingredients in the formulation or
allowed for increased penetration of SLS during the 24-h
exposure. Other studies with moisturizers containing urea
also showed various effects on skin barrier (reviewed in Loden
et al.5,25,26).
Formation of the constituents of the skin barrier, as well as
its regulation, is controlled by epidermal enzymes. Among
these, special attention is drawn towards lipid-processing
enzymes, enzymes taking part in the formation of the corni-
fied envelope of corneocytes, and enzymes which are involved
in the process of desquamation.27–31 If moisturizers or their
ingredients affected the activity or turnover of these enzymes,
they could influence skin barrier function.
Even though changes in TEWL and susceptibility to irritants
after use of moisturizers have been shown by a number of
studies,1–6 this subject has not yet been investigated thor-
oughly. To our knowledge, our study is the first to examine
the effect of moisturizers on normal skin after such a long
period of application. Moreover, our test preparations were
designed to minimize complexity and thus facilitate analysis.
We have shown that moisturizers influence the skin barrier
function of normal skin, as measured by TEWL and suscepti-
bility to SLS. Moreover, that influence depends on the compo-
sition of the moisturizer. A complex interaction of lipids and
other components of the cream with the structure of the
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
Long-term treatment with moisturizers, I. Buraczewska et al. 497
stratum corneum, and effects on keratinocytes, can be
assumed. Changes in TEWL following SLS exposure are prob-
ably due to an increased (or decreased) bioavailability of SLS
due to impaired barrier function.
These results raise many questions about the nature of the
observed influence. More knowledge about the effect of mois-
turizers on skin and its barrier function is needed, in order to
improve treatment of various skin disorders and assist in the
design of preparations to target skin problems efficiently and
safely.
Acknowledgments
This study was supported by ACO HUD NORDIC AB, Stock-
holm, Sweden. We would like to thank Carl Wessman and
Gudrun Artz for the great help they have provided.
References
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Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp492–498
498 Long-term treatment with moisturizers, I. Buraczewska et al.
CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2006.07695.x
Oral treatment with probiotic Lactobacillus johnsoniiNCC533 (La1) for a specific part of the weaning periodprevents the development of atopic dermatitis inducedafter maturation in model mice, NC/NgaR. Inoue, A. Nishio, Y. Fukushima* and K. Ushida
Laboratory of Animal Science, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan
*Nutrition Business Group, Nestle Japan Manufacturing, Ltd, Shinagawa, Tokyo 140-0002, Japan
CorrespondenceDr Kazunari Ushida.
E-mail: k_ushida@kpu.ac.jp
Accepted for publication4 August 2006
Key wordsatopic dermatitis prevention, faecal IgA,
immunomodulation, Lactobacillus johnsonii
NCC533, probiotics
Conflicts of interestNone declared.
Summary
Background The inhibitory effect of probiotic bacteria on atopic dermatitis has beenshown in human infants, but the mechanism is still unclear.Objective This study aimed to show the effects of the administration of a probioticduring the weaning period in mouse models on production of the intestinalsecretory IgA (sIgA) and on the development of atopic dermatitis (AD) in laterlife.Methods The effects of the administration of Lactobacillus johnsonii NCC533 (La1) dur-ing weaning were evaluated using a mouse model (Balb/c). The weaning periodof mice was divided into three phases according to the evolution of faecal IgA.La1 was administered in each phase and the evolution of the faecal IgA was esti-mated. In the next experiment, the effect of the administration of La1 in phase 2on host immunity after maturation was further assessed by using the modelNC/Nga mouse for human AD.Results Administration of La1 in each phase showed a distinct effect on the pro-duction of sIgA. But sIgA production was only positively stimulated when La1was administrated in phase 2. The development of AD induced by mite antigenfrom 6 weeks old was significantly prevented by the primary administration ofLa1 in phase 2. AD-like lesions were significantly milder than those of the con-trol mice, and histological observations showed an almost normal appearance ofthe epidermis and upper dermis of the mice treated with La1.Conclusion This study suggested that the primary administration of La1 in a specificpart of the weaning period is effective in preventing or inhibiting the develop-ment of AD after maturation by modulating or accelerating the gut immuneresponse.
The intestinal microbiota of monogastric animals is comprised
of more than 500 distinct bacterial species, which play an im-
portant role in host nutrition and health by promoting the
nutrient supply, and shaping and maintaining normal mucosal
immunity.1 Evidence that the intestinal microbiota contributes
significantly to host physiological functions, in particular,
immunological functions, has been derived from studies on
germ-free and conventional animals. The gut immune system
of germ-free mice is certainly maldeveloped compared with
that of conventional mice.2,3
The intestinal microbiota is quantitatively the most import-
ant source of microbial stimulation on the mucosal immune
system and presumably provides a primary signal for driving
the postnatal maturation of the immune system;4 thus, it is
suggested that the intestinal microbiota is significantly associ-
ated with the development of both food allergies and other
immune diseases.5 The bacterial stimulation derived from the
colonization of intestinal commensal bacteria is certainly
required for driving Th2-type skewed neonatal immunity
towards the Th1-type and it is a crucial step in the develop-
ment of immunity for the prevention of allergies.6 However,
bacterial transmission and colonization have been highly
reduced as a result of modern high standards for hygiene with
regard to the neonate, and the high prevalence of allergies in
developed countries has been attributed to this phenomenon
for several years (hygiene hypothesis).7
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509 499
In support of the significance of the intestinal microbiota in
regard to allergies, Sudo et al.8 reported that germ-free mice
failed to establish oral tolerance, whereas Bifidobacterium infantis
mono-associated mice developed oral tolerance normally. They
also reported the interesting finding that the reconstitution of
the intestinal microbiota of germ-free mice by B. infantis was
effective only in the early life of mice but not in old age. This
suggested that early life is extremely important for the intro-
duction of bacterial antigen stimulation in order to shape neo-
natal immunity to a correct balance of cytokines.
In a previous study, our findings agreed with this concept,
indicating that there is a period around weaning that is pre-
sumably critical for the maturation of the host immune sys-
tem in terms of immune deviation.9 In this period, the
amount of luminal secretory IgA (sIgA), which mediates the
primary immunological defence line in the mucosal immune
system, decreased drastically and remained at a low level for
several days. On the other hand, the diversity of the intesti-
nal microbiota increased remarkably in this period, as if
responding to the decrease of amount of the sIgA. In this
period, a very large number of bacterial antigens are pre-
sumably transported to immune inductive sites, such as
Peyer’s patches and thus the mucosal immune system may
be actively stimulated.
From these observations, we proposed that this period
could provide an opportunity for the development of toler-
ance against allergies by stimulating and/or modulating the
host immune system via the gut immune system. The oral
administration of the probiotic Lactobacillus johnsonii NCC533
(La1) was selected for this purpose. A stronger immunomod-
ulatory effect was expected from this strain than from other
probiotic strains because, unlike most other probiotics,
this strain has the ability to attach to intestinal epithelial cells
by supporting lipoteichoic acid and the elongation factor
Tu (EF-Tu), both of which strongly stimulate immune
functions.10–12
One objective of this study was to find an efficient way to
stimulate the immune system and/or to modulate it with the
administration of the probiotic La1 in early life using a mouse
model; the evolution of faecal IgA was used as a reference
marker in the development of gut immune systems. Another
objective was to estimate the efficacy of La1 administration in
early life as a means to prevent allergies in maturity. In
experiment 1, an effort was made to determine the most effi-
cient time during the weaning period to administer La1. The
NC/Nga mouse, the model mouse for atopic dermatitis
(AD),13 was used in experiment 2 to estimate the effects of
the administration of La1 on the prevention or inhibition of
AD.
Materials and methods
Experiment 1
Definition of the experimental phases for the administration. From the
evolution of faecal IgA in mice, which was demonstrated in a
previous study,9 three experimental phases for the administra-
tion of La1 were defined (Fig. 1a). A period when the mice
are aged 18–20 days represented phase 1. Mice from 20–
22 days old and from 22–24 days old were in phase 2 and
phase 3, respectively.
Animals. Six pregnant BALB/c mice were obtained from a
commercial supplier (Japan Clea Co., Ltd., Osaka, Japan).
Pups delivered from these mice were used as the donors of
fresh faeces. Five pups were selected randomly from each
litter, and a total of 30 pups were divided into six experi-
mental groups (five pups in each group). They were
housed under conventional circumstances in our facilities
and not separated until weaning at 21 days of age. After
weaning, male and female mice were housed separately and
fed on a standard pellet diet (CE-2, Japan Clea Co. Ltd).
Preparation of Lactobacillus johnsonii NCC533. Lactobacillus johnsonii
NCC533 (La1) was obtained from the Nestle Japan Co., Ltd.
(Tokyo, Japan). La1 was precultured overnight at 37 �C with
5 mL of anaerobic GAM broth (Nissui, Tokyo, Japan) supple-
mented with 1% glucose. Then, 200 lL of the precultured
La1 was transferred into the fresh medium, incubated at
37 �C for 20 h, and centrifuged at 10 000 g and 4 �C for
1 min. The bacterial pellet of La1 was washed twice with
phosphate-buffered saline (PBS) and collected by centrifuga-
tion in the same manner as described above. The pellet was
resuspended in 1 mL of PBS and used immediately for admin-
istration. This procedure was repeated every day during the
administration period.
Administration of Lactobacillus johnsonii NCC533. Five pups from
one of the six experimental groups were gavaged with
100 lL each of resuspended La1 (1Æ5 · 1011 cfu) on all
3 days of phase 1 and designated as the P1-La1 group
(Fig. 1a). Five other pups from the remaining experimental
groups were administered PBS in phase 1 and designated as
the P1-control group. This method was repeated in phases
2 and 3, and the P2-La1, P2-control, P3-La1 and P3-control
groups were established. The dose was always administered
at 10 a.m.
Sample collection and measurement of total IgA. Fresh faeces were col-
lected from all 30 pups at ages 18–24, 27, 30, 33, 36 and
40 days (total 360 samples). The IgA concentration in these
samples was measured as described elsewhere.8 Briefly, the
faeces were homogenized in PBS and their supernatants
were subjected to an enzyme-linked immunosorbent assay
(ELISA) using a mouse IgA ELISA Quantitation kit (Bethyl
Laboratories, Montgomery, TX, U.S.A.).
Induction of allergic diarrhoea. At 6 weeks of age, all the mice in
this experiment were systemically primed by ovalbumin
(OVA) for the induction of allergic diarrhoea as described in
Kweon et al.14 From the 7th to 10th intubation, the incidence
of diarrhoea was evaluated for 2 h after intubation.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
500 Primary probiotic La1 for AD prevention, R. Inoue et al.
Experiment 2
Animals and administration of Lactobacillus johnsonii NCC533. As the
administration in phase 2 showed a particular effect on
the faecal IgA evolution in experiment 1, the administra-
tions were performed in phase 2 in experiment 2. Six preg-
nant NC/Nga mice were obtained from a commercial
supplier (Japan SLC, Shizuoka, Japan). Pups delivered from
these mice were used in this experiment. Pups were divided
into two groups and administered either La1 or PBS in
phase 2 as described in experiment 1 (Fig. 1b). They were
housed and reared as described in experiment 1 but were
fed another commercial mouse chow different from experi-
ment 1 (Labo MR stock; Nihon Nosan Kogyo, Tokyo,
Japan).
Collection of faeces and measurement of faecal IgA. The collection of
fresh faeces and measurement of faecal IgA were performed as
described in experiment 1.
Induction of human atopic dermatitis-like lesion. At 6 weeks of age, all
mice were shaved to remove the hair from the upper back.
Twenty micrograms of antigen from human mites (Mite
Extract Dermatophagoides farinae, LSL, Tokyo, Japan) diluted in
200 lL of PBS was swabbed onto the shaved area three times
a week using a cotton swab (Fig. 1b). Shaving the hair from
the region where the antigen was applied was performed dur-
ing each of the applications. This method is according to
Matsuoka et al.15 The values estimated by the following meth-
ods were evaluated for males and females separately because
Matsuoka et al.15 demonstrated that there was a sex difference
in the symptoms of the AD-like lesion.
Evaluation of skin lesions. In a preliminary experiment, it was esti-
mated that, in the conditions under which the animals were
housed in this experiment, the symptoms of AD-like lesions
began to develop from the 7th to the 9th application of the
mite antigen. Therefore, the severity of the induced skin
lesions was scored from the 7th application of the mite antigen.
20 22
Weaning
0 21 40
Faecal sample collection period
Phase 1
Phase 2
Phase 3
Day after birth 18 20
PBS
La1
20 22
PBS
La1
P1-control
P1-La1
0 21 4018
0 21 4018 22 24
PBS
La1
Weaning
Weaning
P2-control
P2-La1
P3-control
P3-La1
Control
La1
0 21 4018
PBS
La1
42 87
Weaning
Faecal sample collection period Mite antigen application period(three times per week)
Experiment 1
Experiment 2
Day after birth
dissection
(a)
(b)
Fig 1. Experimental schedule for
(a) experiment 1 and (b) experiment 2. The
administration period for each phase is shown
with a heavy line, and the shaded region
indicates the weaning period (from days 18
to 24 after birth). The deltas indicate the
application of mite-extracted antigen on the
back of NC/Nga mice. Each experimental
group was treated with either PBS or La1
(1Æ5 · 1011 cfu), as indicated with arrows.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
Primary probiotic La1 for AD prevention, R. Inoue et al. 501
Scoring was performed as described in Matsuoka et al.15 by a
volunteer who was unaware of the experiment details.
Scratching behaviour. Scratching behaviour was observed after
each application of the mite antigen. It was noted from the
7th application as well as the scoring of skin lesions. Points
were scored as described in Matsuoka et al.15
Dissection and sample collection. The mite antigen was applied for
5 weeks (a total of 15 times). At the time when a 16th application
would have been administered, no mite antigen was applied;
instead the mice were anaesthetized, killed and dissected. Our pre-
liminary study indicated that the AD-like lesions were the most
severe at the time of the 15th application of the mite antigen.
Each mouse was anaesthetized by an intraperitoneal injec-
tion of 50 lL of pentobarbital sodium (Schering-Plough,
Osaka, Japan). The region to which antigen had been applied
was photographed using a Nikon Cool Pix 990 camera
(Nikon, Tokyo, Japan). The skin from that region was then
collected and fixed with 10% neutral formalin for the histo-
logical analysis. Venous blood was collected from the inferior
vena cava with a 21-gauge needle and a 1-mL syringe.
Measurement of total IgE in serum. The concentration of total IgE in
serum was measured using a YAMASA IgE EIA Kit (YAMASA,
Tokyo, Japan) according to the manufacturer’s instructions.
Estimation of the IgG1/IgG2a ratio in serum. The concentrations of
total IgG1 and IgG2a in serum were measured using the
Mouse IgG1 and IgG2a ELISA kit (Bethyl Laboratories) accord-
ing to the manufacturer’s instruction.
Histological analyses of antigen-applied skin. The fixed skin was
embedded in paraffin, and 3-lm thick serial paraffin sections
were prepared. Sections were stained with either haematoxylin
and eosin or toluidine blue. The number of mast cells stained
with toluidine blue was counted from the upper dermis to the
dermal muscle in a field of 2 · 2 mm under a microscope.
Eight fields were randomly selected for counting the mast
cells, and the average density mm)2 was estimated.
Statistical analyses
A normality test and F-test were performed to assess the nor-
mality of the data of faecal IgA on each experimental day
using Statcel (Seiunsha Inc., Tokyo, Japan) with Excel 2003
for Windows (Microsoft, Seattle, U.S.A.). When significance
was detected by either test, the Mann–Whitney U-test was per-
formed (control vs. the group that received La1 on each day)
with Statview for Windows (SAS Institute Inc., Cary, NC,
U.S.A.). When normality of the faecal IgA data was confirmed,
a Student’s t-test was performed with Excel 2003.
Skin scores, scratching scores and the IgG1/G2a ratio were
analysed with the Mann–Whitney U-test (control vs. the group
receiving La1 on each day). The number of mast cells was ana-
lysed by the Student’s t-test (control vs. the group receiving La1).
Results
Experiment 1
Effect of the administration of La1 in the three phases during weaning on the
evolution of faecal IgA. The P1-La1 group had a lower amount of
faecal IgA throughout the experimental period after the admin-
istrations (Fig. 2a). On day 19, the amount of faecal IgA in P1-
La1 was significantly lower than that in the P1-control
(0Æ395 ± 0Æ013 vs. 0Æ584 ± 0Æ087 mg g)1 faeces; P < 0Æ01).
Afterwards, the amount in each group decreased rapidly until
weaning. The faecal IgA in the P1-control group showed a
minimum amount on day 22 (0Æ042 ± 0Æ011 mg g)1 faeces),
and that in P1-La1 on day 23 was (0Æ044 ± 0Æ017 mg g)1 fae-
ces). After showing the minimum amount, the amount of faecal
IgA began to recover in both groups. However, the recovery of
the faecal IgA amount occurred more rapidly in the P1-control
group than in the P1-La1 group. The faecal IgA amount in the
P1-control was significantly higher from day 27 onward until
the end of the experimental period (0Æ704 ± 0Æ032 vs.
0Æ34 ± 0Æ099 mg g)1 faeces on day 40; P < 0Æ01).
The evolution of faecal IgA in the P2-La1 group was different
from that in the other seven groups, including the P2-control
(Fig. 2b). Although the P2-control showed a significantly
higher amount of faecal IgA than P2-La1 on day 21, the faecal
IgA amount in P2-La1 was higher than that in the P2-control
for 4 days after the last La1 administration and was signifi-
cantly higher on days 22, 23 and 27. Unlike the other seven
groups, the evolution of the faecal IgA in the P2-La1 group
did not show a rapid decrease and recovery during the wean-
ing period.
Both the P3-La1 and control groups showed a similar
faecal IgA evolution during the experimental period
(Fig. 2c). The P3-La1 group tended to show a slightly higher
faecal IgA amount than the P3-control group after the admin-
istrations, but a significant difference was detected only on
day 40 (1Æ613 ± 0Æ108 vs. 1Æ086 ± 0Æ133 mg g)1 faeces;
P < 0Æ05).
Induction of allergic diarrhoea. Despite the high incidence of allergic
diarrhoea reported by Kweon et al.,14 whose study showed
100% induction by the 9th intubation, diarrhoea after 10
intubations of OVA was induced on only five of all 30 mice
in this experiment. Only two of those five mice showed severe
symptoms similar to those described by Kweon et al.14 The
shape of the distal colon contents was evaluated by dissection,
as in Kweon et al.;14 however, all mice that did not exhibit
symptoms maintained a healthy shape of their intestinal con-
tents, as did untreated mice (images not shown).
Experiment 2
The evolution of faecal IgA. Figure 3 shows the evolution of faecal
IgA in NC/Nga mice. Male mice in the La1 group tended to
have a higher amount of faecal IgA from days 27 to 33 than
those in the control group, and it was significantly higher on
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
502 Primary probiotic La1 for AD prevention, R. Inoue et al.
day 27 (0Æ145 ± 0Æ075 vs. 0Æ031 ± 0Æ020 mg g)1 faeces;
P < 0Æ01). Female mice of the La1 group showed a very simi-
lar evolution of faecal IgA to that of P2-La1. In the female La1
group, the decrease of faecal IgA stopped after the first admin-
istration, while the faecal IgA of the control group kept
decreasing until day 22. The amount of faecal IgA in the
female La 1 group tended to be higher than that of the control
female group throughout the experimental period, and signi-
ficant differences were detected on days 22 and 27
(0Æ037 ± 0Æ039 vs. 0Æ001 ± 0Æ001 mg g)1 faeces on day 22,
P < 0Æ05; 0Æ076 ± 0Æ064 vs. 0Æ028 ± 0Æ065 mg g)1 faeces on
day 27, P < 0Æ05).
Appearance of an atopic dermatitis-like lesion. Almost all male and
female control mice had symptoms of AD-like lesions, which
were represented as skin scores (Fig. 4). Excoriation and
haemorrhage were seen on the region to which antigen was
applied in six of eight male mice (75%) and eight of nine
female mice (89%) in the control group but on two of 11
male (18%) and two of five female mice (40%) in the La1
0
18 20 22 24 26 28 30 32 34 36 38 40
18 20 22 24 26 28 30 32 34 36 38 40
18 20 22 24 26 28 30 32 34 36 38 40
50
100
150
200
250
300(a)
(b)
(c)
0
50
100
150
200
250
300
0
50
100
150
200
250
300
Day after birth
*
*
* * * * *
***
*
*
Fig 2. Effect of oral La1 administration on the evolution of faecal
IgA from suckling to maturity. (s), La1-administered mice (n ¼ 5).
(d), PBS-administered mice (n ¼ 5). The oral administration was
conducted in (a) phase 1, (b) phase 2, and (c) phase 3. Data are shown
by the percentage mean value for the average IgA amount on day 18.
Bars represent the standard deviation (*P < 0Æ05, La1 vs. control).
0
50
100
150
200
250
300
0
50
100
150
200
250
300
*
*
Male
Female
*
*
Day after birth
18 20 22 24 26 28 30 32 34 36 38 40
18 20 22 24 26 28 30 32 34 36 38 40
Fig 3. Effect of oral La1 administration on the evolution of
faecal IgA in male and female NC/Nga mice from suckling to
maturity. (s), La1-treated mice (male: n ¼ 8, female: n ¼ 9).
(d), PBS-administered mice (male: n ¼ 11, female: n ¼ 5). The oral
administration was conducted in phase 2. Data are shown by the
percentage mean value for the average IgA amount on day 18. Bars
represent the standard deviation (*P < 0Æ05, La1 vs. control).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
Primary probiotic La1 for AD prevention, R. Inoue et al. 503
group, respectively (Fig. 4). Only one male mouse in the La1-
administered group showed lesions as severe as those of mice
in the control group. The skin scores of the La1 group were
clearly lower than those of the control group on every day
they were estimated, except for the score of male mice on the
10th day of application.
Scratching behaviour. Scratching scores were evaluated to clarify
the frequency of scratching behaviour. Male mice of the La1
group had lower scratching scores than those of the control
group throughout the evaluation period, except for the scores
on the 8th day of application (from the 7th to the 15th appli-
cations; data not shown). The scratching score of the control
(b) Male Lal
(c) Female control
(d) Female La1
(a) Male control
Fig 4. Skin features of the region treated with
antigen in NC/Nga mice after 15 applications.
(a) Control male mice; the male mice
administered PBS in phase 2 (days 20–22
after birth), (b) La1 male mice; the male
mice administered La1 in phase 2, (c) Control
female mice; the female mice administered
PBS in phase 2, (d) La1 female mice; the
female mice administered La1 in phase 2.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
504 Primary probiotic La1 for AD prevention, R. Inoue et al.
male mice increased gradually and peaked at the 12th applica-
tion (26Æ5 ± 17Æ7). It then decreased to 17Æ8 ± 18Æ3 at the
13th application and remained around 18 afterwards. The
scratching score of La1 male mice remained around 15
throughout the evaluation period, except at the 7th, 9th and
15th applications (7th: 7Æ5 ± 5Æ7, 9th: 7Æ8 ± 9Æ1, 15th:
10Æ8 ± 11Æ9).
The scratching scores of La1 female mice were slightly
higher than those of control female mice from the 7th to
10th applications (Fig. 5). However, the scratching scores of
control female mice drastically increased from the 10th to the
11th applications (from 6Æ7 ± 4Æ2 to 21Æ9 ± 10Æ0). They con-
tinued to increase afterwards, although a slight decrease was
observed at the 15th application. As for the La1 female mice,
the scratching score increased gradually, but it was lower than
that of the control female mice from the 10th application to
the end of the evaluation.
IgE concentration and IgG1/G2a ratio in serum. IgE in some serum
samples, especially the samples from the La1 group, was not
detectable even when serum diluted 10-fold less was used for
the assay (Table 1). The concentration of serum IgE detected
tended to be higher in the control group than in the La1
group, although its value varied widely from mouse to
mouse. IgE was detected in five of eight mice (62%), and the
mean value of the concentration was 447Æ5 ± 612Æ2 ng mL)1
except for undetectable samples. In contrast to this, it was
detected in only two of 11 mice (18%) of the La1 group
(71Æ1 and 15Æ0 ng mL)1). In female mice, serum IgE was
detected in almost all control mice (eight of nine mice;
89%), and the mean value of the concentration was
391Æ6 ± 342Æ2 ng mL)1 except for one undetected sample.
The concentrations of serum IgE from La1 female mice,
which were detected in two of five mice (40Æ0%), were 84Æ8and 56Æ8 ng mL)1.
The IgG1/G2a ratio in male mice of both control and La1
showed very similar values (2Æ37 ± 1Æ45 vs. 2Æ30 ± 1Æ54).
However, the IgG1/G2a ratio in female mice was significantly
different between the control and the La1 group. Female mice
in the control group showed a very high IgG1/G2a ratio
(5Æ68 ± 6Æ62), whereas the ratio of the La1 female mice was
1Æ82 ± 0Æ62 (P < 0Æ05).
Histological analysis of the skin treated with antigen. Figure 6 shows
the histological status of antigen-treated skin of representative
individuals from each experimental group. A thickened upper
dermis and separation of the epidermis layer were observed in
both male and female mice in the control group. However,
the treated skin of almost all mice in the La1 group had a
practically normal histological status. There was no specific
sign of AD on the treated skin of the La1 group and the
untreated NC/Nga mice.
Mast cell density of the skin to which antigen was applied. The number
of mast cells in the treated region was counted using toluidine
blue staining, and the densities of mast cells in each group
were estimated. From the male mice in the control group,
97Æ2 ± 33Æ2 cells mm)2 of treated skin were detected. In the
case of the La1 male mice, a lower number of mast cells than
in the control males were detected (88Æ0 ± 29Æ0 cells mm)2,
P > 0Æ05 vs. control male mice. The La1 female mice had a
significantly lower density of mast cells than did the female
control mice. The mast cell density of La1 female mice was
91Æ2 ± 19Æ7 cells mm)2, while that of female mice in the
control group was 145Æ9 ± 49Æ7 cells mm)2 (P < 0Æ05; con-
trol vs. La1 group).
0
10
20
30
40
50
60
7 8 9 10 11 12 13 14 15
*
Antigen application
e r o c s g n i h c t a r c S
Fig 5. Scratching scores of NC/Nga female mice from the 7th to the
15th mite antigen application. The scratching scores were evaluated
according to Matsuoka et al.15 Data are shown by the mean value, and
bars represent the standard deviation. (s), La1 treated mice. (d), PBS
treated mice, see Fig. 3 (*P < 0Æ05, La1 vs. control).
Table 1 Serum total IgE concentration of NC/Nga mice after 15 miteantigen applications (ng mL)1)
Control La1
Male Female Male Female
1511Æ3 940Æ0 71Æ1 84Æ8427Æ5 677Æ5 15Æ0 56Æ8134Æ8 673Æ8 ND ND115Æ0 452Æ5 ND ND
48Æ8 134Æ8 ND NDND 127Æ5 ND ND
ND 98Æ8 NDND 27Æ5 ND
ND NDND
ND
Control mice were treated with PBS in phase 2 (days 20–22after birth), and La1 mice were treated with La1 during the
same period. Total IgE was estimated with a YAMASA IgE EIAKit (YAMASA), and 10-times diluted serum was assayed. ND,
not detectable.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
Primary probiotic La1 for AD prevention, R. Inoue et al. 505
(a)
(b)
(c)
(d)
Non-treated NC mice
Female Lal
Female control
Male Lal
Male control
Fig 6. Histological status of the treated skin in NC/Nga mice after 15 antigen applications. Images of the skin from the same region of nontreated
age-matched NC/Nga mice are shown as a reference. The bar on the right side of each image represents the thickness of upper dermis and arrows
in the images indicate the separation of the epidermis layer. (a) Control male mice, (b) La1 male mice, (c) Control female mice, (d) La1 female,
see Fig. 4.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
506 Primary probiotic La1 for AD prevention, R. Inoue et al.
Discussion
Unlike systemic immunity, the mucosal immune system and
gut immune system, especially, play critical roles in the sup-
pression of the immune responses to nonhostile antigens.16
Thus, it has been suggested that the stimulation and/or modu-
lation of the gut immune system by the administration of pro-
biotics are effective for the alleviation of food allergies as well
as atopic diseases.17 This study focused on the administration
of probiotics during the developmental process, and the effects
were assessed using a mouse model because the immune
system during the developmental process is supposed to be
more sensitive to probiotics than a mature system.
In experiment 1 of this study, the probiotic L. johnsonii
NCC533 (La1) was administered in the developmental process
of the mouse model, and the most effective period for the
administration was estimated. The La1 administration in phase
2 had a positive effect on the production of sIgA. It has been
suggested that the oral administration of probiotic bacteria
stimulates IgA production not only in the intestine but also in
serum. However, most studies demonstrated a significant
increase of the pathogen-specific sIgA in either the intestine or
serum.18–20 Moreover, some studies have reported that the
oral administration of probiotic did not affect the amount of
total faecal IgA.21,22 A few studies have demonstrated that
there was a significant increase of the total faecal IgA as a
result of the administration of probiotic bacteria in humans
and in mice,23,24 but those increases of the faecal IgA were
also achieved in early life as in this study. These facts indicate
that the stimulation of oral La1 administration in phase 2, the
period around weaning, was effective for the production of
sIgA in the intestine. In this period, the intestine of pups must
be highly vulnerable to foreign antigens as the supplemen-
tation of the maternal defensive molecules is stopped by
weaning, as indicated by the drastic decrease of faecal IgA.9
Furthermore, quite a few of the self-sIgA are produced in the
intestine of pups.9 Thus, the administered La1 should have
easily reached the immune inductive site and then enhanced
the development of the gut immune systems, including self
sIgA production. The immune cells for IgA production seem
to be already deployed in Peyer’s patches at this period,25
although they are not producing a large quantity of sIgA
because there is less antigen stimulation by food or bacteria
until weaning.5,26
In contrast with this, the administration in phase 1 surpris-
ingly showed negative effects on the production of sIgA after
administration. This effect was probably caused specifically in
this period. At this time, the pup intestines were still protected
by the maternal defensive molecules.9,27,28 Thus, the adminis-
tered La1 must have been prevented from reaching the
immune inductive site either partly or mostly by maternal
defensive molecules, such as sIgA and lactoferrin. Further-
more, an insufficient number of immune cells for proper
immune responses, such as CD4+, are deployed in the intes-
tine during this period.29 Therefore, immune responses might
have been improperly induced by some La1 that reached the
immune inductive site while escaping from the maternal
defensive molecules. The La1 administration in phase 3 did
not show much positive effect on IgA production. Increasing
sIgA in this period9 may have affected the viability of the
administrated La1 because self-secreted sIgA might play a role
in the prevention of bacterial colonization.9
Early life is quite important for the immune development
of T-helper cells.8 It is, therefore, quite likely that these differ-
ent immunomodulatory effects of La1 administration in each
phase have respective effects on the development of immune
diseases, such as allergies and autoimmune diseases, after
maturation. However, nearly all of the mice unexpectedly
showed tolerance to the induced allergic diarrhoea. The toler-
ance is probably not caused by La1 administration but, rather,
is characteristic of the mice used in this experiment. These
mice were completely conventional whereas the mice in the
reference study were SPF.14 The mice in this experiment cer-
tainly had more antigenic stimulation to their gut immune
system from early life than did the SPF mice. Thus, their
immune system skewed more towards the Th1 type than that
of the SPF mice, regardless of the La1 administration. The Th1
type skewed the immune system of the mice in this experi-
ment and probably inhibited the development of allergic diar-
rhoea. This result implicitly supported the hygiene hypothesis
proposed by Strachan.7
In experiment 2, we proposed that the immunomodulatory
effect of the La1 administration in early life is applicable for
the prevention and/or inhibition of AD because the mucosal
immune system is deeply connected with systemic immunity.5
In fact, Kalliomaki et al.,30,31 in a randomized placebo-
controlled trial, suggested that probiotic therapy might be effi-
cient for the treatment of atopic dermatitis. An NC/Nga
mouse, which is a model for atopic diseases, was used in
experiment 2. This mouse develops a human AD-like lesion
triggered by a mite antigen under conventional circum-
stances.13 The La1 administration was done in phase 2 in this
experiment because administration in this phase had the most
significant effect on the sIgA production in experiment 1.
In fact, the administration of La1 in phase 2 also showed a
positive effect on the production of IgA in NC/Nga mice in
both male and female mice (Fig. 3). The induction of an
AD-like lesion by the mite antigen application was successful
(Figs 4, 6), and remarkable inhibitory effects on the lesion
were observed as a result of the primary administration of La1
in phase 2. The antigen-treated skin of nearly all of the mice
in the La1 group showed a practically normal appearance and
histological status. Involvement of serum IgE level and mast
cell infiltration into antigen-applied skin also suggested that
the mice in the control group had more severe allergic symp-
toms than that of the mice in La1 group.
The sex difference of the inhibitory effect by the La1
administration was observed on the IgG1/G2a ratio and
density of mast cells. The sex difference of lesion severity in
this mouse model has been reported in several studies13,15,32
although the precise reason for it is still unknown. Female
mice tend to show more severe symptoms when lesions are
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp499–509
Primary probiotic La1 for AD prevention, R. Inoue et al. 507
induced as they were in this experiment.15 The difference
in lesion severity may be attributed to the fact that the
inhibitory effect in male mice was smaller than that in
females. As in the study by Matsuoka et al.,15 our results
showed that the symptoms of AD-like lesions in female
mice, the scratching scores, and the density of mast cells,
for instance, were more severe than those in male mice.
The higher inhibitory effects in female mice than in male
mice, therefore, were presumably achieved by the primary
administration of La1.
This study strongly suggested a strong link between the gut
immune system and AD and proposed a preventive method
for AD using a lactic acid bacterium that possesses the ability
to adhere to intestinal mucosa. The inhibitory effect of the
primary administration of La1 on AD was derived from the
efficient stimulation and/or modulation of the gut immune
systems, as represented by the increase of sIgA production.
Although the precise relation between gut and systemic
immune system has not yet been revealed, an immunomodu-
lation of the gut immune system by oral administered antigen
can certainly be manifested in the systemic immune system.33
For instance, it has been demonstrated by several studies that
tolerance to orally administered antigen is established not only
in the mucosal but also in the systemic immune system.34,35
Thus, the immune system skewed towards Th1 type cells or
anergy to nonharmful antigens established firstly in gut by
La1 administration may have also been achieved in the sys-
temic immune system through this immunological relation.
Dendritic cells or/and T-regulatory cells may play a role in
this immunological relation because they seem to possess the
ability to establish a tolerance both in mucosal and systemic
immunity.36–38
Acknowledgments
The authors thank Dr Keizo Nakayama and Dr Takamitsu
Tsukahara for sharing their knowledge of histopathology. A
part of this study was supported by the Applied Science Board
for Lactic Acid Bacteria (Tokyo, Japan).
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CONTACT DERMATITIS AND ALLERGY DOI 10.1111/j .1365-2133.2006.07674.x
Compositae dermatitis from airborne parthenolideE. Paulsen, L.P. Christensen* and K.E. Andersen
Department of Dermatology, Odense University Hospital, Sdr. Boulevard 29, DK-5000 Odense C, Denmark
*Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Aarslev, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark
CorrespondenceEvy Paulsen
E-mail: evy.paulsen@ouh.fyns-amt.dk
Accepted for publication10 September 2006
Key wordsairborne, allergic contact dermatitis, Compositae,
parthenolide, plants, Tanacetum parthenium
Conflicts of interestNone declared.
The studies were carried out at the Department of
Food Science, Danish Institute of Agricultural
Sciences, Research Centre Aarslev and the
Department of Dermatology, Odense University
Hospital.
Summary
Background Compositae dermatitis confined to exposed skin has often been consid-ered on clinical grounds to be airborne. Although anecdotal clinical and plantchemical reports suggest true airborne allergy, no proof has been procured.Feverfew (Tanacetum parthenium) is a European Compositae plant suspected of caus-ing airborne contact allergy, and its most important allergen is the sesquiterpenelactone (SQL) parthenolide (PHL).Objectives The aims of this study were to (i) assess the allergenicity of feverfew-derived monoterpenes and sesquiterpenes and their oxidized products in fever-few-allergic patients and (ii) re-assess the role of PHL and other SQLs in airbornecontact allergy.Patients and methods Feverfew-allergic patients were patch tested with extracts andfractions containing volatile monoterpenes and sesquiterpenes as well as extractsof airborne particles from flowering feverfew plants, obtained by fractionation ofether extracts, dynamic headspace and high-volume air sampler (HIVAS) tech-nique, respectively.Results Among 12 feverfew-allergic patients, eight had positive patch-test reactionsto a HIVAS filter extract, while two tested positive to a headspace extract. Subse-quent analysis of the HIVAS extract by gas chromatography and mass spectro-metry detected PHL in a concentration of 510 ng mL)1 in the HIVAS extract.Testing with a dilution series of PHL showed positive reactions down to 8Æ1 ngin selected patients. None of the 12 patients tested positive to monoterpenes orsesquiterpenes, whether they were oxidized or not.Conclusions The clinical results have proved that some feverfew-allergic patients aresensitive to airborne particles released from the plant, and isolation of PHL fromthe particle-containing HIVAS extract in allergenic amounts is strong evidence ofPHL as the responsible allergen.
The classical form of Compositae dermatitis is an airborne pat-
tern eczema, first described in the U.S.A. as weed dermatitis
in the 1930s, and 30–40 years later in India as Parthenium
dermatitis and in Australia as Australian bush dermatitis.1–3
The hypothesis of airborne allergens has been much debated
since then, and although the clinical features, the allergenicity
of dried plant parts and positive patch-test reactions to a filter
from a high-volume air sampler (HIVAS) as well as to air-dried
clothing are suggestive of true airborne contact allergy, no def-
inite proof has been established.1–7 Furthermore, a European
equivalent to airborne Compositae dermatitis has been ques-
tioned because of the humid, colder, climatic conditions.8
Feverfew [Tanacetum parthenium (L.) Schultz-Bip.] is a frequent
sensitizer in European Compositae-allergic patients.9,10 On the
basis of case reports, this plant, which is native to south east
Europe and partly naturalized from gardens in Northern Eur-
ope, has been considered to be a cause of airborne contact
dermatitis (ABCD).11,12 Its main allergenic sesquiterpene lac-
tone (SQL) is parthenolide (PHL, Fig. 1),11 which may consti-
tute up to 0Æ9% of dry weight in aerial parts of the plant.13,14
However, experimental studies using HIVAS and dynamic
headspace (DHE) techniques around feverfew plants failed to
detect both particle-bound and volatile PHL.14 The volatiles
emitted from feverfew plants were mainly monoterpenes, but
as only a minority of feverfew-allergic patients tested positive
to these, their contribution to airborne Compositae dermatitis
seemed to be of small significance.15
On the other hand, it has been established, notably in fra-
grance research, that oxidized monoterpenes are often stronger
allergens than the nonoxidized substances.16 Therefore, the aims
� 2007 The Authors
510 Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp510–515
of this study on airborne Compositae dermatitis were to
(i) assess the allergenicity of feverfew-derived monoterpenes
and sesquiterpenes and their oxidized products in feverfew-
allergic patients and (ii) re-assess the possible role of PHL and
other SQLs in view of more accurate and sensitive gas chromato-
graphy–mass spectrometry (GC-MS) methods available with
lower detection and quantification thresholds compared with
GC-MS techniques used previously for detection of airborne
SQLs.14
Patients, materials and methods
Patients
Adults, who by previous patch testing in our clinic had had at
least 2+ reactions to feverfew extract 1% petrolatum (pet.)
and PHL 0Æ1% pet., were invited to participate in the study.
Exclusion criteria were pregnancy, treatment with systemic
steroid or other immunosuppressive or -modulating drugs as
well as recent ultraviolet (UV) radiation therapy.
Patch-test materials were applied to the patient’s back for
48 h using Finn Chambers� (Epitest Ltd, Helsinki, Finland)
on Scanpor� (Scanpor Alpharma AS, Oslo, Norway), and read-
ings were taken on day 3 according to the criteria of the
International Contact Dermatitis Research Group.17
All participants gave their written, informed consent, and in
general the study followed the Declaration of Helsinki proto-
cols and was approved by the local ethics committee (case no.
20040123).
Plant material and parthenolide content
Twelve specimens of T. parthenium (L.) Schulz Bip. were grown
in a greenhouse at 17–23 �C in 20-L pots and watered daily.
Plants were grown under natural light conditions. The concen-
tration of PHL (Fig. 1) in the aerial parts of the feverfew
plants was �0Æ2% of dry weight as determined by analytical
reversed-phase high performance liquid chromatography using
acetonitrile–water as mobile phase.13,14
Analysis for parthenolide in high-volume air sampler
filters
A HIVAS (Air sampler #15000, The Science Source, Waldo-
boro, ME, U.S.A.) was placed in a closed chamber of
1Æ5 · 1Æ5 · 1Æ5 m in a greenhouse together with eight flower-
ing feverfew plants at a distance of 15–20 cm from the plants.
The sampler was mounted with filter disks capturing plant
particles down to 4 lm in size, and equipped with a cap pro-
tecting the filter from direct contact with the plants so only
airborne particles released from the plants were collected. The
air sampler collected airborne particles 8 h a day over a 16-h
time period (from 06Æ00 to 22Æ00) with a 30-min pause each
hour in order to avoid overheating. The sampling of airborne
particles was performed at 18–20 �C over a 6-day period. The
average flow rate through the air sampler was measured to
215 L min)1. The HIVAS filter was changed once a day and
kept in a 250-mL blue-cap flask at )24 �C until analysis. In
total six HIVAS filters were obtained, which were randomly
divided into two groups with four (group I) and two (group
II) HIVAS filters, respectively.
The HIVAS filters in groups I and II were extracted with
50 mL 96% ethanol, filtered, and the eluates combined and
concentrated in vacuo (30 �C) to 50 mL each for testing as is on
Compositae-sensitive patients and/or analysis. Only the HIVAS
extract obtained from the group I filters was tested on patients
and after testing, both the group I and II filter extracts were
analysed by GC-MS for PHL and other SQLs. GC-MS analysis
was performed on a Trace DSQ single quadrupole mass spec-
trometer operated at 70 eV and directly coupled to a Trace GC
Ultra gas chromatograph (both, Thermo Finnigan, Austin, TX,
U.S.A.) equipped with a split/splitless PTV injector (split flow
10 mL min)1, 200 �C) and a Zebron capillary column
(30 m · 0Æ25 mm internal diameter, film thickness df ¼0Æ25 lm, ZB-1, Phenomenex, Torrance, CA, U.S.A.). Helium
was the carrier gas at a constant column head pressure of
10 psi. The temperature of the column and transfer line was
200 �C. GC-MS was performed on 2 lL HIVAS extract. Com-
pounds in the HIVAS extract were separated by the following
oven temperature gradient: 32 �C for 1 min, programmed to
260 �C at 10 �C min)1 followed by constant temperature for
20 min. The mass spectrometer was operated in full scan
mode over a mass range from 39 to 650 amu (1 scan g)1)
and analysed for PHL and other SQLs. PHL was the only SQL
detected in the HIVAS extracts, and in order to improve the
sensitivity for detection of PHL by GC-MS a selected ion mon-
itoring (SIM) acquisition method was developed. PHL was
detected by GC-MS SIM by setting the mass selective detector
to repeatedly scan for the following ions m/z 145, 190 and
248 (molecular ion). PHL was repeatedly identified in the
HIVAS extracts in full and SIM scan modes by comparison
with an authentic standard of PHL (Sigma-Aldrich, Steinheim,
Germany).
Quantification of PHL in the HIVAS extracts was per-
formed in SIM mode using PHL as external standard. The
calibration curve was determined to be linear in the concen-
tration range 50–1000 ng mL)1 (r2 ¼ 0Æ988). The detection
limit of PHL was determined from the calibration curve to
be �20 ng mL)1 [Signal to noise (S/N) ¼ 10] in SIM
scan mode and the quantification limit to be 50 ng mL)1
(S/N ¼ 3).
O
O
Parthenolide (PHL)
O
Fig 1. Chemical structure of the sesquiterpene lactone parthenolide
(PHL) present in the aerial parts of Tanacetum parthenium (feverfew).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp510–515
Dermatitis from airborne parthenolide, E Paulsen et al. 511
Isolation of volatiles from feverfew
Volatiles were isolated from aerial parts of feverfew plants by
DHE and column chromatography (CC), respectively, for test-
ing. Volatiles emitted from feverfew were collected from the
closed chamber mentioned above by DHE technique as
described previously with a few alterations.14 Volatiles were
collected directly from the chamber in a 100-mL pipette
equipped with 50 g Porapak Q 50–80 mesh (Waters, Milford,
MA, U.S.A.), and eluted with diethyl ether, concentrated to a
final volume of 300 lL14 and dissolved in 5 mL 96% ethanol.
Four DHE collections were made and the extracts combined to
one headspace extract (20 mL), which was used for testing
and for oxidation experiments. The combined headspace
extract was analysed by GC and GC-MS as described previous-
ly.14 The total concentration of the volatiles in the ethanol
headspace extract was > 1% and used for testing as is.
Volatiles were extracted with 2 L of diethyl ether from
800 g aerial parts of feverfew plants for 60 min. The diethyl
ether extract was concentrated in vacuo (30 �C) to 10 mL. Vola-
tiles were isolated from the extract by silica gel CC using an
n-hexane-diethyl ether gradient. Fractions containing volatiles
only and no PHL were combined, the solvent removed in vacuo
(30 �C), and the residue dissolved in 40 mL 96% ethanol for
testing and oxidation.
Oxidation by ultraviolet radiation of volatiles isolated by
dynamic headspace and column chromatography
The combined DHE extract and the extract obtained by CC
were purged with O2 and oxidized for 30 min using a UV-
immersion lamp reactor (Sigma-Aldrich) equipped with a
400-mL reaction vessel made of borosilicate glass. The photon
source was provided with a TQ 150 high-pressure Hg lamp
with linkage and condenser made of silica (Sigma-Aldrich).
Analysis of the oxidized extracts by GC-MS revealed quantita-
tive, but no qualitative, differences in the volatile profile com-
pared with the nonoxidized extracts.
Results
Clinical studies
Twelve people (11 female, one male, mean age 54 years) with
contact sensitization from feverfew and PHL were recalled for
patch testing with extracts and fractions containing volatile
monoterpenes and sesquiterpenes as well as HIVAS filter
extracts of airborne particles from feverfew plants. The results
of the initial patch testing, including repeated testing with the
crude feverfew extract and PHL, are summarized in Table 1.
Contrary to former results, the subjects were mainly positive
to airborne fractions: eight of 12 tested positive to an extract
from HIVAS filters, and two of 12 had positive reactions to an
extract collected by the DHE technique. On the other hand, no
one tested positive to the monoterpene and sesquiterpene
fraction. Ten consecutive eczema patients tested negative to
the HIVAS filter extract as well as to the oxidized and nonoxi-
dized headspace extracts. After PHL had been detected in the
extract from the HIVAS filters in minute amounts, it was
decided to retest some of the eight people with positive reac-
tions to the crude extract in order to determine the threshold
elicitation concentration of PHL. Two dilution series of PHL,
incorporated in a dry film using the TRUE Test� technology,
were kindly provided by Mekos Laboratories AS (Hillerød,
Table 1 Results of patch testing with oxidized and nonoxidized extracts from feverfew, collected by HIVAS, DHE or direct extraction, in 12
feverfew-allergic patients
Patient no. 1 2 3 4 5 6 7 8 9 10 11 12
Sex/age (years) F/48 F/55 F/42 F/59 F/53 F/48 M/79 F/47 F/61 F/55 F/52 F/59
AllergenFeverfew 1% pet. ++ ++ ++ +++ ++ +++ ++ ++ ++ +++ ++ ++
PHL 0Æ1% pet. +++ ++ ++ +++ +++ +++ ++ +++ ++ +++ +++ +++l-Carvone 1% pet. – – – – – – 0 + – 0 foll. – 0 foll.
l-Carvone 5% pet. 0 foll. 0 0 – – – 0 0 – ++ 0 0Nonoxidized monoterpenes and sesquiterpenes – – 0 – – – – – 0 – – –
Oxidized monoterpenes and sesquiterpenes – – 0 – – – 0 – 0 0 foll. – –HIVAS filter extract ++ + ++ – – + ++ – ++ ++ 0 ++
Nonoxidized DHE extract – – 0 foll. – – – ++ – – + – –Oxidized DHE extract – – 0 foll. – – – + 0 – – – 0 foll.
HIVAS filter under Scanpor� – – – – – – – – – – – –HIVAS filter in FC under Scanpor� 0 – – – – – – – – – – –
Colophonium 1500 lg cm)2 + – – – – – ++ + – – – –FM I 450 lg cm)2 ++ – – ++ ++ ++ + – – ++ ++ –
Feverfew p.s. No No No Yes Yes No Yes ? No Yes No YesABCD suspected by patient No Yes No No Yes No Yes No Yes Yes Yes Yes
F, female; M, male; PHL, parthenolide; HIVAS, high-volume air sampler; DHE, dynamic headspace; FC, Finn Chamber; FM I, fragrance mix
I; p.s., primary sensitizer; ABCD, airborne contact dermatitis; foll., follicular reaction; 0, doubtful positive reaction.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp510–515
512 Dermatitis from airborne parthenolide, E Paulsen et al.
Denmark). The results are shown in Table 2: two of five per-
sons tested positive to 30 and 10 ng PHL, corresponding to
24Æ3 and 8Æ1 ng per patch as a TRUE Test� allergen occupies
0Æ81 cm2.
Six patients were sensitized by gardening, and three of these
reported feverfew to be one of the primary sensitizers,
whereas four were occupationally sensitized as gardeners/
greenhouse workers or sandwich makers. The last two pati-
ents were both privately and occupationally sensitized by
gardening.
Chemical studies
The particles and volatiles collected by HIVAS and DHE tech-
niques, respectively, were derived from several feverfew plants
in a greenhouse instead of single specimens to imitate the nat-
ural outdoor conditions and ensure a sufficient amount of any
volatile allergen for patch testing.
PHL was detected using GC-MS SIM by repeatedly scanning
for the following ions m/z 145, 190 and 248 (molecular ion),
which are some of the characteristic and/or major ions in the
mass spectrum of PHL. The retention time for PHL was
23Æ9 min and was repeatedly identified in the combined
HIVAS extracts in full and SIM scan mode by comparison with
an authentic standard of PHL as illustrated in Figure 2.
PHL in the combined HIVAS extracts used for testing
(group I) was determined to be 510 ng mL)1 HIVAS extract
corresponding to an average release of �800 ng PHL h)1 or
100 ng PHL h)1 per feverfew plant. The presence of PHL was
also detected in the group II HIVAS extracts that were not
used for testing. The concentration of PHL in this combined
HIVAS extract corresponded to an average release of �310 ng
PHL h)1 or 40 ng PHL h)1 per feverfew plant, thus confirm-
ing that PHL was released over the whole collection period,
but, as expected, not released uniformly.
Table 2 Results of patch testing with two dilution series ofparthenolide
Patient no. 2 3 7 9 12
Sex/age (years) F/55 F/42 M/79 F/61 F/59PHL concentration
0Æ010 mg cm)2 NT NT NT NT +++0Æ0033 mg cm)2 ++ ++ NT ++ ++
0Æ0011 mg cm)2 ++ ++ NT ++ +++0Æ00037 mg cm)2 0 foll. + foll. NT ++ ++
0Æ00012 mg cm)2 0 foll. – NT ++ ++
0Æ060 lg cm)2 – – + foll. ++ ++0Æ030 lg cm)2 – 0 foll. 0 + ++
0Æ010 lg cm)2 – – – 0 +0Æ0030 lg cm)2 – – – – 0 foll.
0Æ0010 lg cm)2 – – – – –
PHL, parthenolide; F, female; M, male; 0, doubtful positivereaction; foll., follicular reaction; NT, not tested.
0 5 10 15 20 25 30 35 40
0 5 10 15 20 25 30 35 40
Retention time (min)
Rel
ativ
e ab
unda
nce
Rel
ativ
e ab
unda
nce
PHL
PHL
Parthenolide (PHL) standard (500 ng ml–1)
High-volume air sampler (HIVAS) extract
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Fig 2. Gas chromatography–mass
spectrometry (GC-MS) selected ion
monitoring (SIM) chromatogram of a
standard sample of parthenolide (PHL) in a
concentration of 500 ng mL)1 and the
combined high-volume air sampler (HIVAS)
extract, respectively. PHL was detected at
23Æ9 min in SIM and full scan mode (m/z
39–650) in both samples. The selected ions in
the GC-MS SIM acquisition method were m/z
145, 190 and 248 [molecular ion (M)+].
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp510–515
Dermatitis from airborne parthenolide, E Paulsen et al. 513
Analysis of the combined headspace extract by GC-MS
revealed the monoterpenes camphene, p-cymene, camphor,
trans-chrysanthenol and trans-chrysanthenyl acetate as the major
volatiles, and no PHL was detected in the extracts in accord-
ance with previous investigations.14
The major terpenes in the monoterpene and sesquiterpene
extract were camphor and trans-chrysanthenyl acetate as
shown by GC-MS,14 although the concentrations of oxygen-
ated monoterpenes such as bornyl acetate were higher in this
extract compared with the headspace extract. The total con-
centration of the volatiles in this ethanol extract was > 2%
and tested as is.
Discussion
Although Hjorth et al.,4 in their frequently quoted paper on
Compositae dermatitis in Danish patients, emphasized the air-
borne pattern, later studies have shown a greater diversity of
clinical features in European Compositae-allergic patients,
which is probably why the existence of airborne Compositae
dermatitis in Europe has been doubted.8,18,19
Concerning feverfew, anecdotal reports have been suggest-
ive of the plant as a cause of ABCD based on clinical features
and information on exposure.11,20
However, this is the first study where several feverfew-aller-
gic patients consistently showed positive patch-test reactions
to airborne fractions that were subsequently proved to contain
PHL. This allergen is not volatile, so its presence must be due
to plant particles containing traces of PHL.14 PHL is probably
synthesized in glandular trichomes on the leaf surface as it has
been reported in other allergenic Compositae species.21 The
concentration of PHL in the HIVAS extract of 510 ng mL)1
would allow an amount of �8 ng per Finn Chamber�, pro-
viding that the chamber contained 15 lL. However, the parti-
cle-bound PHL was probably unevenly distributed, and
furthermore, the extract was not applied by a micropipette,
but by capillary tubes, which would lead to a higher concen-
tration of PHL in the Finn Chamber�.
The fact that some of the subjects were very sensitive to
the allergen, reacting positively down to 8Æ1 ng, is strong
evidence of PHL as the allergen causing the reactions to the
HIVAS extract. The average amount of PHL released from a
single test plant in this study was 100 ng h)1, meaning that
a border of feverfew plants may release milligrams of the
allergen, which would be sufficient to elicit reactions in
moderately sensitive persons: among the five subjects tested
with the PHL dilution series, the least sensitive tested posi-
tive to 0Æ0011 mg cm)2 corresponding to < 1 lg per test
area. Furthermore, the feverfew plants used in this study
contained 0Æ2% PHL of dry weight in the aerial parts, but
concentrations of up to 0Æ9% PHL of dry weight in leaves
has been reported previously as well as concentrations
exceeding 1Æ2% PHL of dry weight in leaves of post-flower-
ing feverfew plants.13 So the release of PHL under real-life
conditions may be even higher as those reported in the pre-
sent study.
The fact that PHL elicits patch-test reaction on normal skin
on the back of some PHL-sensitive patients in a dose of 10 ng
in a TRUE Test� patch makes it one of the most powerful
contact allergens in the environment.22 Dermatitic skin on
exposed areas may be even more sensitive. PHL has been isol-
ated from a number of other Compositae plants such as, e.g.
tansy (T. vulgare), Anthemis cretica, Inula aschersoniana, Ambrosia and
Arctotis spp. as well as members of the Magnoliaceae family,23
and furthermore there is evidence of true cross-reactivity
between PHL and the allergens of the popular Chrysanthemum
indicum L. [Dendranthema indicum (L.) Desm.].24 The implications
are that it may be quite difficult to avoid this strong allergen
and closely related compounds.
The degree of sensitivity to PHL varied in the five subjects
tested with the dilution series (Table 2): the most sensitive
patient (no. 12), who was diagnosed with Compositae allergy
in 2004, reported feverfew as a primary sensitizer and still
had problems with summer-exacerbated eczema. On the other
hand, the second most sensitive person (patient no. 9) was
diagnosed with Compositae allergy in 2003, probably sensiti-
zed by gardening, but not feverfew, and her hand eczema was
well controlled now.
The relation between the patients’ suspicion of ABCD and a
positive reaction to the HIVAS extract was not unambiguous,
but in five of the eight patients the two parameters were coin-
cident. Feverfew as a primary sensitizer was not associated
with positive reactions to the HIVAS extract.
As expected, PHL was not detected in the headspace extracts
and neither were other SQLs. The positive reactions to nonoxi-
dized/oxidized extracts in two patients thus could not be
explained by their SQL allergy, but patient no. 7 had a colo-
phonium and patient no. 10 a fragrance allergy, and it is
possible that their headspace extract reactions are due to cross-
reactivity between volatile terpenes from feverfew and
terpenes in colophonium and fragrance mix I. Cross-reactivity
between fragrance terpenes, colophonium and Compositae
plant extracts has been reported before, although it is not
invariably seen in all patients as documented in this study.25
Likewise, there was no association between positive reactions
to fragrance mix/colophonium and the extract from the
HIVAS filters. Also, all people in this study tested negative to
the monoterpene and sesquiterpene fraction isolated directly
from feverfew plants, whether the fraction was oxidized or
not, emphasizing that these are not primary allergens.
Carvone was included in the patch-test series due to a pre-
viously reported association between sensitization to this mon-
oterpene and Compositae extracts.26 Two of the 12 subjects
tested positive (Table 1), and the allergy was of old relevance
in one and unknown relevance in the other, making true
cross-reactivity a possibility in the latter.
In conclusion, the clinical part of this study has proved
that feverfew-allergic patients are sensitive to airborne
and—to a lesser degree—volatile fractions from feverfew,
and the subsequent isolation of the SQL PHL from the parti-
cle-containing airborne fraction in amounts sufficient to elicit
positive patch-test reactions in the same patients is strong
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp510–515
514 Dermatitis from airborne parthenolide, E Paulsen et al.
evidence of PHL as the allergen responsible for ABCD in
feverfew-allergic patients. In accordance with previous stud-
ies, monoterpenes and sesquiterpenes seem to be of minor
importance.14,15
Acknowledgments
Kgl. Hofbundtmager Aage Bangs Fond is gratefully acknow-
ledged for financial support and Mekos Laboratories AS, Hil-
lerød, Denmark for providing the parthenolide dilution series.
References
1 Shelmire B. Contact dermatitis from vegetation. Patch testing and
treatment with plant oleoresins. J S Med Assoc 1940; 33:337–46.2 Lonkar A, Jog MK. ‘Epidemic’ contact dermatitis due to Parthenium
hysterophorus (Compositae family of plants). Report of 350 cases.Contact Derm Newsl 1972; 11:291–2.
3 Burry JN, Kuchel R, Reid JG, Kirk J. Australian bush dermatitis:Compositae dermatitis in South Australia. Med J Aust 1973;
1:110–16.4 Hjorth N, Roed-Petersen J, Thomsen K. Airborne contact dermatitis
from Compositae oleoresins simulating photodermatitis. Br J Derma-tol 1976; 95:613–20.
5 Hausen BM. Kompositenallergie. Ein Beitrag zur Identifizierung derKontaktallergene, atiologischen Diagnostik und aktuellen Bedeu-
tung. Allergologie 1979; 2:143–7.6 Schumacher MJ, Silvis NG. Airborne contact dermatitis from
Ambrosia deltoidea (triangle-leaf bursage). Contact Derm 2003; 48:212–
16.7 Srinivas CR. Transmission of Parthenium dermatitis by clothing.
Arch Dermatol 2005; 141:1605.8 Schmidt RJ. Compositae. Clin Dermatol 1986; 4:46–61.
9 Geier J, Hausen BM. Epikutantestung mit dem Kompositen-Mix.Allergologie 2000; 23:334–41.
10 Paulsen E, Andersen KE, Hausen BM. Sensitization and cross-reac-tion patterns in Danish Compositae-allergic patients. Contact Derm
2001; 45:197–204.11 Mensing H, Kimmig W, Hausen BM. Airborne contact dermatitis.
Hautarzt 1985; 36:398–402.12 Hausen BM, Vieluf IK. Allergiepflanzen/Pflanzenallergene. Handbuch und Atlas
der allergieinduzierenden Wild-und Kulturpflanzen, 2nd edn. Landsberg/Mun-chen: Ecomed Verlagsgesellschaft mbH, 1997.
13 Awang DVC, Dawson BA, Kindack DG. Parthenolide content offeverfew (Tanacetum parthenium) assessed by HPLC and 1H-NMR
spectroscopy. J Nat Prod 1991; 54:1516–21.14 Christensen LP, Jakobsen HB, Paulsen E et al. Airborne Compositae
dermatitis: monoterpenes and no parthenolide are released fromflowering Tanacetum parthenium (feverfew) plants. Arch Dermatol Res
1999; 291:425–31.15 Paulsen E, Christensen LP, Andersen KE. Do monoterpenes released
from feverfew (Tanacetum parthenium) plants cause airborne Composi-tae dermatitis? Contact Derm 2002; 47:14–18.
16 Matura M, Skold M, Borje A et al. Selected oxidized fragrance ter-
penes are common contact allergens. Contact Derm 2005; 52:320–8.17 Wahlberg JE. Patch testing. In: Textbook of Contact Dermatitis (Rycroft
RJG, Menne T, Frosch PJ, Lepoittevin J-P, eds), 3rd edn. Berlin andHeidelberg: Springer Verlag, 2001:435–68.
18 English J, Norris P, White I, Cronin E. Variability in the clinicalpatterns of Compositae dermatitis. Br J Dermatol 1989; 121 (Suppl.
34): 27.19 Paulsen E, Andersen KE. Compositae dermatitis in a Danish derma-
tology in 1 year (II). Clinical features in patients with Compositaecontact allergy. Contact Derm 1993; 29:195–201.
20 Diepgen TL, Haberle M, Baurle G. Fallstricke in der Berufsderma-tologie: Das aerogene Kontaktekzem auf Pflanzen. Dermatosen 1989;
37:23–5.21 Hausen BM, Spring O. Sunflower allergy. On the constituents of
the trichomes of Helianthus annuus L (Compositae). Contact Derm 1989;20:326–34.
22 Johansen JD, Andersen KE, Svedman C et al. Chloroatranol, anextremely potent allergen hidden in perfumes: a dose–response
elicitation study. Contact Derm 2003; 49:180–4.23 Fischer NH, Olivier EJ, Fischer HD. The biogenesis and chemistry
of sesquiterpene lactones. III. Germacranolides. In: Progress in theChemistry of Organic Natural Products 38 (Herz W, Grisebach H, Kirby
GW, eds). Wien: Springer-Verlag, 1979.24 Schulz KH, Hausen BM, Wallhofer L, Schmidt-Loffler P. Chrysanth-
emen-Allergie. 2. Experimentelle Untersuchungen zur Identifizie-rung der Allergene. Arch Derm Forsch 1975; 251:235–44.
25 Paulsen E, Andersen KE. Colophonium and Compositae mix asmarkers of fragrance allergy: cross-reactivity between fragrance ter-
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26 Paulsen E, Andersen KE, Carlsen L, Egsgaard H. Carvone: an over-looked contact allergen cross-reacting with sesquiterpene lactones?
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� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp510–515
Dermatitis from airborne parthenolide, E Paulsen et al. 515
DERMATOLOGICAL SURGERY AND LASERS DOI 10.1111/j .1365-2133.2006.07648.x
Comparative study of photodynamic therapy vs. CO2 laservaporization in treatment of condylomata acuminata,a randomized clinical trialK. Chen, B.Z. Chang, M. Ju, X.H. Zhang and H. Gu
Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, 14 Jiangwangmiao Road, Nanjing 210042, China
CorrespondenceHeng Gu.
E-mail: guhengy@yahoo.com.cn
Accepted for publication30 August 2006
Key words5-aminolaevulinic acid, condylomata acuminata,
photodynamic therapy, randomized clinical trial
Conflicts of interestNone declared.
Summary
Background Most conventional therapies for condylomata acuminata (CA) are trau-matic and have high recurrence rates.Objectives To investigate the efficacy and safety of topical application of 5-amino-laevulinic acid (ALA) photodynamic therapy (PDT) for the treatment of CA.Methods Sixty-five patients with CA were allocated into the treatment (ALA-PDT)group and treated with 20% ALA solution under occlusive dressing for 3 hfollowed by irradiation with the helium–neon laser at a dose of 100 J cm)2
and a power of 100 mW. Another 21 CA patients were allocated into the con-trol group and treated with the CO2 laser. The treatment was to be repeated1 week later if the lesion was not completely removed after the firsttreatment.Results After one treatment, the complete removal rate was 95% in the ALA-PDTgroup and 100% in the control group. After two treatments with ALA-PDT, thecomplete removal rate in the treatment group was 100%. The recurrence rate forALA-PDT group was 6Æ3% which was significantly lower than that in control group(19Æ1%, P < 0Æ05). Moreover, the proportion of patients with adverse effects in theALA-PDT group (13Æ9%) was also significantly lower than that in control group(100%, P < 0Æ05). The side-effects in patients treated with ALA-PDT mainlyincluded mild burning and/or stinging restricted to the illuminated area.Conclusions The present study shows that topical application of ALA-PDT is a simp-ler, more effective and safer therapy with a lower recurrence for treatment of CAcompared with conventional CO2 laser therapy.
Condylomata acuminata (CA) are caused by human papilloma-
virus (HPV). Based on the national sexually transmitted disease
(STD) surveillance system, it has been ranked as the third
most prevalent STD in China. More and more attention has
been paid to this medical condition mainly because it has
been believed to be related to genital carcinogenesis. Until
now, there is no medicine that exerts direct anti-HPV effects.
Conventional treatment of CA usually includes excision, phys-
ical therapies, topical medication treatment and immunothera-
pies. However, most conventional treatment strategies are
traumatic, need treatment of long duration and have a high
rate of relapsing episodes.1
Topical application of photodynamic therapy (PDT) with
5-aminolaevulinic acid (ALA) for the treatment of cutaneous
cancer was first described by Kennedy et al.2 Since then, the
application of ALA-PDT in clinical practice has become more
and more popular, and it can be used not only for treatment
of cutaneous cancers and/or precancerous lesions but also for
the treatment of nonmalignant skin disorders, such as psoria-
sis, viral infections and diseases of epidermal appendages.3,4
Recently, studies have showed that ALA-PDT is an attractive
technique in the treatment of CA.5–8 In order to explore
further the efficacy and safety of ALA-PDT on CA, a clinical
trial to evaluate the topical application of ALA-PDT was con-
ducted among 86 patients with CA from December 2004 to
December 2005.
Subjects and methods
Subjects
The patients attending the Institute of Dermatology, Chinese
Academy of Medical Sciences, during the period from Decem-
ber 2004 to June 2005 were invited to participate in the
� 2006 The Authors
516 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp516–520
study. All patients provided written informed consent. The
diagnosis of CA was made based on clinical examination in
addition to the acetowhitening test. The age range of the
patients was 18–50 years. All of the wart lesions were located
on the external genitals or distal urethra. The patients were
excluded from the study if they had been treated with any
systemic immunotherapies within the preceding 4 weeks and/
or any topical antiviral drugs within the preceding 2 weeks,
had other STDs, or were pregnant or breastfeeding. Patients
presenting with vaginal and/or cervical condylomata were also
excluded from the study. The eligible patients were randomly
allocated into the ALA-PDT group or CO2 laser (control)
group in an allocation ratio of 3 : 1.
5-aminolaevulinic acid-photodynamic therapy
A 20% ALA (Fudan Zhangjiang Bio-Pharm Co. Ltd, Shanghai,
China) solution (w/v) was prepared by dissolving ALA in
sterile 0Æ9% NaCl immediately prior to its application. The
external genital skin around the lesions was washed with an
aqueous 0Æ4% chlorhexidine solution. A thin cotton swab or
an absorbent ball was soaked in the solution and was gently
inserted into the distal urethra or put on the external genitals,
to be in contact with and cover the warts and the adjacent
normal skin (5-mm border). Then small amounts of 20% ALA
were dropped on to the surface of the absorbent ball every
30 min. The lesions were occluded with food-grade cling film
and covered with thick gauze for light protection. Patients
were asked to lie still until illumination was performed. The
time interval between drug application and illumination was
3 h. Light irradiation of 100 J cm)2 at 100 mW was applied
to the lesion and the adjacent normal skin (5-mm border)
using a cylindrical helium–neon laser fibre (Yage Laser Appar-
atus Co. Ltd, Wuhan, China) emitting a 632Æ8-nm laser light.
In patients with multiple lesions, the warts were irradiated
one by one. If the warts were not totally removed after
1 week, a repeat treatment was given once a week for max-
imum of 3 weeks. The lesions were evaluated after each ALA-
PDT treatment session. The follow-up was conducted at 4, 8
and 12 weeks after the treatment finished.
CO2 laser treatment
After routine sterilization, the lesion was given topical anaes-
thesia with lidocaine. The lesions and their adjacent normal
skin (5-mm border) were vaporized with CO2 laser (Shanghai
Laser Institute, Shanghai, China) one by one until all the
lesions were completely removed. If the lesions were still
there after the first treatment, a repeat treatment was given
once a week for a maximum of 3 weeks. The follow-up was
conducted at 4, 8 and 12 weeks after the treatment finished.
Assessment
Laboratory-based evaluation was conducted for all the patients
for routine check of blood and urine, liver function and renal
function before and after the treatment. The number, location
and area of the warts and the adverse effects were evaluated
for each patient at each follow-up.
Statistical analysis
The indicators of efficacy were the clearance rate of wart le-
sions and the recurrence rate in those patients whose lesions
had completely cleared. Lesions were designated ‘complete
response’ if all (100%) of the lesions disappeared. A recur-
rence was defined as occurrence of the lesions after a complete
response. The clearance rate ¼ (the number of the warts
which had been cleared completely/the number of the
warts) · 100%.
The number of the warts in the two groups before treat-
ment was evaluated with the Wilcoxon matched pairs test.
The number of the warts after treatment and the adverse
effects in the two groups were evaluated with the v2 test.
Results
General characteristics
A total of 86 patients were enrolled into the study in which 65
patients were allocated into the ALA-PDT group and 21 patients
into the control group. Among the ALA-PDT group, 44 (67Æ7%)
were males and 21 (32Æ3%) females with a mean age of
35Æ2 years (SD ¼ 10Æ4 years). The duration of disease was
91Æ9 ± 90Æ5 (mean ± SD) days, and the number of the warts
was 1Æ5 ± 0Æ7 (mean ± SD). For this study, the locations of
warts were chosen as those found in the distal urethra in 17
(26Æ2%) patients and on the external genitalia in 48 (73Æ9%)
patients. Twenty-one patients (15 men and six women) with a
mean age of 35Æ9 (SD ¼ 9Æ3) years were allocated into the CO2
laser (control) group. The mean duration of disease in this
group was 89Æ9 ± 80Æ3 (mean ± SD) days. The average number
of warts was 1Æ9 ± 0Æ9 (mean ± SD). Five (23Æ8%) patients had
lesions located in the distal urethra and 16 (76Æ2%) in the exter-
nal genitalia rather than the distal urethra.
Clinical efficacy
In the ALA treatment group, a complete response was
observed among 15 of 17 (88Æ2%, 89Æ5% of the total warts)
patients with urethral CA (Fig. 1a,b) and in 45 of 48 (93Æ8%,
96Æ3% of the total warts) patients with lesions located in other
areas 1 week after the first treatment. All 65 patients achieved
complete response after two PDT treatments. In the CO2 laser
treatment group, a complete response was found in all 21
patients after one treatment (Table 1).
Follow-up for patients with complete response lasted for
12 weeks. Of these patients, four relapsing patients were
found in each group, resulting in a recurrence rate of 6Æ3%
and 19Æ1% for the ALA-PDT and control groups, respectively,
which was significantly different between these two groups
(P < 0Æ05).
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp516–520
PDT vs. CO2 laser vaporization in treatment of CA, K. Chen et al. 517
Safety evaluation
In all patients with ALA-PDT treatment, excellent tissue heal-
ing was achieved without the occurrence of any infection,
ulcers, scarring or urethral malformations. The mean time of
healing ranged from c. 7 to c. 10 days. The rate of adverse
reaction was 13Æ9% in the ALA-PDT group. Most patients
experienced a mild burning and/or stinging sensation during
irradiation, and a few patients experienced erythema, mild
oedema and erosion. The rate of adverse reaction was 100%
in the CO2 laser group. The side-effects mainly included pain,
erosion, ulcer, hyperpigmentation and hypopigmentation.
Urethral malformations occurred in two of five patients with
urethral CA. The rate of adverse reaction was significantly
higher in the CO2 laser group than in the ALA-PDT group
(P < 0Æ05).
Discussion
The conventional treatment of CA is usually targeted for
removing the macroscopic lesions and for avoiding their
recurrence. The commonly used modalities for CA recently
include three categories: topical remedies (0Æ5% podophyllo-
toxin, 5% fluorouracil); physical therapies (CO2 laser therapy,
cryotherapy, electrocautery) and/or surgical excision; immu-
notherapy, including systemic or topical immunoregulants.
Most conventional therapies are likely to be painful and lead
to topical tissue damage or scarring. Urethral malformations
can occur with laser or cryotherapy in patients with urethral
CA. In addition, it is troublesome that most conventional
treatments are associated with a high recurrence rate.1
ALA-PDT is a new technique introduced in the 1990s. The
mechanisms of cellular damage mediated by ALA-PDT include
a variety of biochemical and molecular reactions, creating a
complex process that leads to tissue destruction at both macro-
structural and microstructural levels. ALA is selectively
absorbed by tumour cells and rapidly proliferating cells, and
transformed to endogenous protoporphyrin IX (PpIX) after
the exogenous application of ALA. The PpIX is then activated
by red light, leading to the formation of singlet oxygen,
which leads to the killing or destruction of tumour cells and
proliferative cells.9 ALA-PDT is a well-established treatment
regimen for superficial epithelial skin tumours, but it is evi-
dent that inflammatory diseases of the skin and virus-induced
lesions can also profit from PDT. Some basic investigations
proved that ALA-PDT could inactivate the active viral particles.
In the study by Smetana et al.,10 ALA was added to cells that
Fig 1. Urethral condylomata acuminata. Clinical appearance (a)
before and (b) 1 week after photodynamic therapy using
5-aminolaevulinic acid.
Table 1 Response rates in patients with condylomata acuminata after one or two cycles of ALA-PDT or CO2 laser therapy
Number oftreatments
Location of lesions
TotalUrethral orifice Other area
ALA-PDT
(np ¼ 17, nw ¼ 19)
CO2 laser
(np ¼ 5, nw ¼ 7)
ALA-PDT
(np ¼ 48, nw ¼ 81)
CO2 laser
(np ¼ 16, nw ¼ 34)
ALA-PDT
(np ¼ 65, nw ¼ 100)
CO2 laser
(np ¼ 21, nw ¼ 41)
1 17 (89Æ47%) 7 (100%) 78 (96Æ30%) 34 (100%) 95 (95%) 41 (100%)2 2 (10Æ53%) – 3 (3Æ70%) – 5 (5%) –
Total 19 (100%) 7 (100%) 81 (100%) 34 (100%) 100 (100%) 41 (100%)
The numbers of the warts in the two groups before treatment were evaluated with the Wilcoxon matched pairs test. The numbers of thewarts after treatment in the two groups were evaluated with v2 test. In both the ALA-PDT and CO2 laser therapy groups, a similar complete
response was achieved for the warts in different location after completing all the treatments (P > 0Æ05). ALA-PDT, 5-aminolaevulinic acid-photodynamic therapy; np, number of patients; nw, number of wart lesions.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp516–520
518 PDT vs. CO2 laser vaporization in treatment of CA, K. Chen et al.
were infected with the human immunodeficiency virus. Subse-
quent exposure to red light PDT drastically reduced the virus
titre. In guinea pigs infected with herpes simplex virus (HSV),
administration of ALA and exposure of the lesions to red light
shortened the duration of the appearance of vesicles from
more than a week to a few days and reduced the HSV titre in
the lesions.
In recent years, a few studies on ALA-PDT treatment for CA
have been conducted. A report by Wang et al.5 describes 164
patients with urethral CA who were treated with ALA-PDT.
The complete response rate was 95%, and the recurrence rate
was 5% after 6–24 months of follow-up. Stefanaki et al.6 used
ALA-PDT in the treatment of 12 male patients with CA.
Lesions were treated with 20% ALA and irradiated at the opti-
mal time with a dose of 70 J cm)2 or 100 J cm)2 light. The
overall cure rate was 72Æ9%, 12 months after treatment. Fehr
et al.7 and Uhlir et al.8 also treated the patients with CA with
ALA-PDT and achieved a satisfactory efficacy. CA, a benign
epidermal hyperplasia, is an infectious disease caused by HPV
infection.
ALA-PDT for the treatment of CA aims directly at two
points. Firstly, a large amount of ALA can permeate into the
warts through an abnormal stratum corneum, which is then
distributed and accumulated selectively in the hyperplastic tis-
sue.11–13 Subsequently, the ALA-PDT can cause the necrosis
and apoptosis of the keratinocytes infected by HPV selectively
and finally cleans up the warts.14 Secondly, in addition, ALA-
PDT may have some direct antiviral activity. The photosensitizer
can bind to virion surface glycoproteins, and thus impede the
early steps of the infection cycle.15
In this study, ALA-PDT for the treatment of CA also
showed a satisfactory therapeutic effect: the complete
response rate was 95% after one ALA-PDT treatment. Among
them, the complete response rates of urethral CA and the
external genitalia CA were 89Æ5% and 96Æ3%, respectively.
All patients (100%) achieved complete response after two
ALA-PDT treatments. The therapeutic effect of ALA-PDT was
as effective as the CO2 laser. Determination of the reasonable
ALA density, the optimal laser exposure time and the proper
total amount of laser irradiation is the key to obtaining the
optimal therapeutic results of ALA-PDT. It was reported that
patients with urethral CA were given topical 10% ALA fol-
lowed by PDT once a week, and the complete response rates
were 27% and 73%, respectively, after one and two ALA-
PDT treatments.5 In our study using 20% ALA-PDT for the
treatment of 17 patients with urethral CA, the complete
response rates were 89Æ5% and 100%, respectively, after one
and two ALA-PDT treatments. This prompts us to consider
that it is possible to improve the therapeutic effect with the
increase of ALA density. Ideally, irradiation should occur
after sufficient accumulation of intracellular PpIX to destroy
the lesions and to be relatively sparing of the adjacent nor-
mal skin. In this study, in accordance with the previous in-
vestigation (the optimal treatment time was between 30 min
and 3 h after ALA application),16 20% ALA was applied for
3 h followed by light exposure. In the study by Frank and
Bos,17 the lesions of seven patients with CA were treated
with 20% ALA for 14 h followed by irradiation with a dose
of 100 J cm)2. Only four patients obtained overall cure
3 months after treatment. The low therapeutic effect may
correlate to irradiation at an unsuitable time. A light over-
dosage often led to increased tissue damage18 and a light un-
derdosage could not achieve a satisfactory clinical therapeutic
effect. The irradiation in topical ALA-PDT was applied at a
light dose of between 60 and 250 J cm)2.19 In addition, the
exposed degree of the lesion during the therapeutic process
is also one of the factors influencing the therapeutic effect.
In this study, the complete response rate was 89Æ5% and
96Æ3% in the patients with urethral CA and in those with CA
in other areas, respectively, after one PDT treatment. Com-
pared with the latter, the former had an inferior therapeutic
effect. Such a lower effect can be correlated to the difficulty
of exposure of the lesion to treatment.
Conventional treatment usually targets only visible wart le-
sions, and high recurrence rates (10–65%) result partly from
the location of HPV as far as 1 cm from the clinical border of
the warts.12 In the study, the recurrence rate in ALA-PDT
group (6Æ3%) was much lower than that in CO2 laser treat-
ment group. The reason for the lower recurrence rate after
ALA-PDT may be related to simultaneous treatment of micro-
scale and subclinical lesions.7
Conventional treatments are often painful, and sometimes
lead to traumatic infection and scarring. In this study, urethral
malformations occurred in two of five patients with urethral
CA in the CO2 laser group. Compared with conventional treat-
ment, topical ALA-PDT is a safe new treatment for CA. The
most frequent side-effect is only a mild burning and/or sting-
ing sensation during irradiation.
Based on the present study, it can be concluded that topical
application of ALA-PDT is a simpler, more effective and safer
therapy with a lower recurrence for treatment of CA compared
with conventional CO2 laser therapy.
References
1 Krogh GV. Clinical relevance and evaluation of genitoanal
papilloma virus infection in the male. Semin Dermatol 1992; 11:229–40.
2 Kennedy JC, Pottier RH, Pross DC. Photodynamic therapy withendogenous protoporphyrin IX: basic principles and present
clinical experience. J Photochem Photobiol B Biol 1990; 6:143–8.3 Lui H, Anderson RR. Photodynamic therapy in dermatology: recent
developments. Dermatol Clin 1993; 11:1–13.4 Nelson JS, Jeffes EWB, Weinstein JL et al. Topical 5-aminolevulinic
acid for the photodynamic therapy of psoriasis and actinic keratos-es. Lasers Surg Med Suppl 1995; 7:43.
5 Wang XL, Wang HW, Wang HS et al. Topical 5-aminolaevulinicacid-photodynamic therapy for the treatment of urethral condylo-
mata acuminata. Br J Dermatol 2004; 151:880–5.6 Stefanaki IM, Georgiou S, Themelis GC et al. In vivo fluorescence
kinetics and photodynamic therapy in condylomata acuminata. Br JDermatol 2003; 149:972–6.
7 Fehr MK, Hornung R, Degen A et al. Photodynamic therapy ofvulvar and vaginal condyloma and intraepithelial neoplasia using
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topically applied 5-aminolevulinic acid. Lasers Surg Med 2002; 30:273–9.
8 Uhlir M, Jirsa M, Otcenasek M et al. Photodynamic therapy ofbenign viral vulvar lesions. Ceska Gynekol 2002; 67:320–3.
9 Loh CS, MacRobert AJ, Bedwell J et al. Oral vs. intravenous admin-istration of 5-aminolaevulinic acid for photodynamic therapy. Br J
Cancer 1993; 68:41–51.10 Smetana Z, Malik Z, Orenstein A et al. Treatment of viral infections
with 5-aminolevulinic acid and light. Lasers Surg Med 1997; 21:351–8.
11 Svaasand LO. Dosimetry model for photodynamic therapy with
topically administered photosensitizers. Lasers Surg Med 1996;18:139–49.
12 Schultz RE, Miller JW, MacDonald GR et al. Clinical and molecularevaluation of acetowhite genital lesions in men. J Urol 1990;
143:920–3.13 Barry B. Properties that influence percutaneous absorption. In:
Dermatological Formulations: Percutaneous Absorption (Barry B, ed.). NewYork: Marcel Dekker, 1983; 145–6.
14 Oleinick NL, Morris RL, Belichenko I. The role of apoptosis inresponse to photodynamic therapy: what, where, why and how.
Photochem Photobiol Sci 2002; 1:1–21.15 North J, Neyndorff H, King D, Levy JG. Viral inactivation in blood
and red cell concentrations with benzoporphyrin derivative. BloodCells 1992; 18:129–40.
16 Ross EV, Romero R, Kollias N et al. Selectivity of protoporphyrin IXfluorescence for condylomata after topical application of 5-amino-
laevulinic acid: implications for photodynamic treatment. Br J Der-matol 1997; 137:736–42.
17 Frank RG, Bos JD. Photodynamic therapy for condylomata acumi-
nata with local application of 5-aminolevulinic acid. Genitourin Med1996; 72:70–1.
18 McCaughan JS, Guy JT, Hicks W et al. Photodynamic therapy forcutaneous and subcutaneous malignant neoplasms. Arch Surg 1989;
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EPIDEMIOLOGY AND HEALTH SERVICES RESEARCH DOI 10.1111/j .1365-2133.2006.07608.x
Teledermatology in the U.K.: lessons in service innovationT.L. Finch, F.S. Mair* and C.R. May
Centre for Health Services Research, University of Newcastle upon Tyne, 21 Claremont Place, Newcastle upon Tyne NE2 4AA, U.K.
*Department of General Practice and Primary Care, University of Glasgow, 1 Horselethill Road, Glasgow G12 9LX, U.K.
CorrespondenceTracy Finch.
E-mail: Tracy.Finch@ncl.ac.uk
Accepted for publication27 June 2006
Key wordsinnovation, teledermatology, telemedicine
Conflicts of interestNone declared.
Summary
Background Teledermatology has the potential to revolutionize the delivery ofdermatology services by facilitating access to specialist services at a distance. Inthe U.K. over the previous decade there have been numerous attempts at introdu-cing and using teledermatology; however, the development of teledermatology asroutine service provision remains limited.Objectives To identify factors that promote successful use of teledermatology as apart of routine service provision.Methods A longitudinal qualitative study of teledermatology, drawing on data fromin-depth semistructured interviews; observations of systems in practice; and pub-lic meetings. Data were analysed collectively by the research team using estab-lished qualitative analytical techniques to identify key thematic categories. Thesample consisted of teledermatology services within the U.K. (n ¼ 12) studiedover 8 years (1997–2005). Individual participants (n ¼ 68 interviews) were con-sultant dermatologists, researchers, teledermatology nurses, administrators,patient advocates, general practitioners and technologists.Results The analysis compared services that did or did not become part of routinehealthcare practice to identify features that supported the normalization of tele-dermatology. Requirements for using and integrating teledermatology into prac-tice included: political support; perceived benefit and relative commitment thatoutweighs effort; pragmatic approaches to proving efficacy and safety; perceptionof risk as being ‘manageable’ on the basis of professional judgement; high levelsof flexibility in practice (in terms of individuals, technology and organization);and reconceptualizing professional roles.Conclusions Successful implementation of teledermatology as a routine servicerequires greater understanding of and attention to the interplay between socialand technical aspects of teledermatology, and how this is accommodated both byhealthcare professionals and the organizations in which they work.
Teledermatology is the delivery of dermatology services ‘at a
distance’ through the use of information and communication
technology.1 It represents part of a general shift in medicine
towards the increasing use of technology to address problems
of inefficiencies in healthcare provision in the context of in-
creasing demand for services, and to overcome inequities in
access to services across geographical regions.2 In the U.K.
over the past decade the potential of telehealthcare has gener-
ated much interest among dermatology service providers, who
have been grappling with the problem of lengthy waiting lists
for routine dermatological appointments.3
Dermatology has, from the outset, been hailed as a medical
specialty particularly well suited to telehealthcare, for it relies
to a large extent on visual information for diagnosis.1,4 Tele-
dermatology has been practised both in real time through the
use of live interactive videoconferencing (synchronous), and
as store-and-forward systems involving the transmission of
still images and textual information for remote assessment and
subsequent review at a later point in time (asynchronous).
There has been considerable research globally5–7 and within
the U.K.8,9 to demonstrate and evaluate the potential of tele-
dermatology. In the U.K., efforts were initially focused on real-
time teledermatology; however, store-and-forward systems
have become increasingly favoured as being more cost-
efficient and convenient for healthcare providers.8,10 Teleder-
matology has also been used for various purposes, including
triage,11 diagnostic and management services,5,9,12 and
advice/opinion for primary care practitioners.3 It has been set
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up in a number of ways, including as direct referral from pri-
mary care using images and clinical history sent to secondary
care dermatology services for assessment/opinion and for
triage of referrals; and as facilitating community-based clinics
led by nurses or general practitioners (GPs) with a special
interest in dermatology (using teledermatology for consultant
support). Teledermatology has generally demonstrated high
levels of concordance in diagnosis and management plans
compared with face-to-face consultations.4,7,13,14 Studies asses-
sing patients’ views of teledermatology tend to report positive
results,15–19 although many such studies lack analysis of the
needs of different dermatology patient groups.20 The derma-
tology community itself has responded variably to the idea of
teledermatology, ranging from pockets of enthusiasm to resist-
ance and concerns about perceived threats to the professional
practice of dermatology and the doctor–patient relationship.21
Teledermatology, despite its potential for facilitating service
provision, does raise important questions for professional
practice and about the way in which services must be recon-
figured to accommodate new ways of working. In telehealth-
care generally, there is a problem of pilot and demonstration
projects that fail to become a normal part of healthcare deliv-
ery.22 Introducing a new technical system in itself raises prob-
lems and tensions around new technologies, professional roles
and identities, and integrating old and new approaches to
healthcare provision, because they invariably change the ways
that work is organized and undertaken.23,24 Where service de-
velopments based on the introduction of new technology are
combined with efforts at conducting evaluation research, mak-
ing telehealthcare ‘work’ in clinical practice becomes addition-
ally complicated.25
This paper aims to further research on teledermatology
beyond evaluation studies to provide an understanding of fac-
tors that contribute to the success of teledermatology in practice,
through a large-scale qualitative study of teledermatology in
the U.K. that spans the last 8 years.
Materials and methods
Participants and settings
Between 1997 and 2005, we have undertaken a longitudinal
qualitative study of teledermatology services, both ‘real’ and
‘experimental’, and their development in depth, and over
time. The data collected covers representative instances of
National Health Service (NHS) provision of teledermatology in
the U.K., consisting of 12 known services dating back from
1994 to the present: one government sponsored (T1); a com-
mercial provider (no longer in operation) (T4); research stud-
ies that were discontinued (T2, T3, T5, T6); GP-based
systems still in operation (T7, T8, T10); and nurse-led sys-
tems still in operation (T9, T11, T12). The services that were
developed as research studies included one that was based on
real-time videoconferencing, and three that used store-and-
forward. All of the research studies aimed to assess diagnostic
concordance and the utility of teledermatology for patient
management. The teledermatology services that remain in op-
eration are all based on store-and-forward systems, and
include some that operate as nurse and/or GP with special
interest-led clinics with a store-and-forward link for consultant
review and/or triage, and others that are set up for use by
GPs either to access consultant opinion/advice or for triage
purposes. We cannot provide further details of the telederma-
tology services because Multicentre Research Ethics Committee
(MREC) approval of the study, and consent provided by indi-
vidual participants, was granted on the condition that we do
not disclose any details about the sites and services that might
identify individual respondents. Given the relatively small
number of services in operation, even small details (such as
locations, dates etc.) are likely to identify respondents and as
such have had to be omitted.
For the purpose of the analysis, the services we have stud-
ied are referred to throughout the Results section as ‘normal-
ized’ or ‘non-normalized’. In this context, ‘normalized’ is
used to refer to services that have become a routine compon-
ent of dermatology services in a particular area, even if this is
on a relatively small scale26 in relation to the total number of
patients managed. ‘Non-normalized’ refers to pilot projects or
research studies that were discontinued after a brief period of
time.
Interview respondents for the study were selected on the
basis of known experience in teledermatology or for their
patient advocacy roles in relation to dermatology. Appropriate
ethical and research governance approval was obtained, and
stringent procedures were followed to ensure the anonymity
of individual participants and the services to which they are
linked.
Data collected included key informant interviews (n ¼ 68);
observations of public meetings (n ¼ 2); observation; and tex-
tual analysis of project documents. All interviews were audio-
taped, transcribed, and then submitted for data analysis.
Interviewees included consultant dermatologists (19),
researchers (17), teledermatology nurses (11), service admin-
istrators (8), patient advocates (4), technologists (6) and GPs
(3). These figures represent total interviews rather than partici-
pants, as some provided multiple interviews over time.
Two full public meetings were also included: British Associ-
ation of Dermatologists Conference about teledermatology
(June 2001); and ‘How to Improve Dermatology Services:
Lessons from Action on Dermatology’ (April 2004). Partici-
pants were interviewed up to four times (typically once or
twice). Interviews were conducted either in-person, at a site
convenient for the respondent, or over the telephone. A semi-
structured interview guide was used for all interviews, but
was varied according to the specific role of interviewee and
context of interview. Across all interviews, however, we
explored: aspects of the development of the service (from idea
through to practice); organizational context and clarification
of roles of key participants; purpose of the particular service
development and evaluation (if appropriate); and experiences
of service development and utilization (both positive and
negative).
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522 Teledermatology in the U.K., T.L. Finch et al.
Ethical approval for this study was granted by the Northern
and Yorkshire MREC, and all employing NHS Trusts of indi-
vidual clinical respondents, as required by the Department of
Health Research Governance Framework for Health and Social
Care (2001, revised 2005).
Data analysis
Data for analysis consisted of interview transcripts. However,
contemporaneous field notes and project documentation were
used to support and strengthen the development and interpret-
ation of the interview data. Data collection and analysis pro-
ceeded in an iterative fashion and a constant comparative
approach to data analysis was used.27 All authors participated
in ‘data clinics’, during which transcripts were coded and key
themes were identified and agreed.
Results
The results of this study reveal the degree of modifications in
practice that are required in order to make teledermatology
operate successfully. Key themes in the process of normaliza-
tion included: the policy context; perceived benefit and related
commitment; evidence gathering and both proving safety and
managing risk; reorganizing services; and issues surrounding
professional roles and boundary crossing. These themes and
some of the associated factors that emerge as promoting or
inhibiting successful innovation in the context of teledermato-
logy services are illustrated below.
Policy context
New services are not promoted in a vacuum. The policy context
in which new services are introduced may either facilitate or
inhibit new initiatives. This is certainly true of the teledermatol-
ogy services we studied, as most of these were conceived
through high-level central or local health policy support (see
Table 1). However, where such support was not sustained, and
thus not translated into resources, services failed to normalize. It
has been suggested that such policy directives may increase an
organization’s willingness to adopt new health technologies but
may still do little to aid the organization’s ability to integrate
new technological systems into existing practices successfully.28
Perceived benefit and related commitment
The introduction of teledermatology services was often chal-
lenging (see Table 2), and the full impact of these services on
working practices only became apparent to participants once
they started working with the system in practice, and began
the process of identifying and resolving problems in order to
make the teledermatology system work. The technical systems
thus required new ways of working: unless the objectives of
teledermatology were clear, respondents questioned whether
the additional effort needed was worth it (Table 2). The tele-
dermatology services that became normalized were those for
which the perceived benefits, for example saving patients’
and/or health professionals’ travelling time and costs, or redu-
cing waiting times, clearly outweighed the effort and commit-
ment required to make the system work in the local setting.
The issue of the need for widespread and ongoing support by
all of those contributing to such service reconfigurations was a
recurring theme.
Table 1 Policy background
‘I mean this policy, I presume it was from the top, Downing
Street or wherever these politics come from. I suppose it musthave been because I had this commissioner geezer come into
my office and say you have got to do some [teledermatology],but that’s effectively what he said…’ (Consultant Dermatologist
– T11)‘I have been trying to locate regional policy leads in England to
talk about telemedicine and then I found out that they’ve alldisappeared because it’s all been completely reorganized.’
(Telemedicine Service Manager – T1)
Table 2 Perceived benefit and related commitment
Normalized Non-normalized
‘Oh definitely I had many tears… at the beginning because I feltvery isolated too. Here I was trying to, my IT knowledge was very
poor, I’d hardly touched a computer [it all] seemed such a newand foreign system to me as well as trying to get to grips with
dermatology. I did feel a bit isolated because I didn’t actually haveanyone to run to next door to help me and my IT problems and
things tended to have to be sorted out over the phone and some-times would take hours. So it was really quite scary at the begin-
ning, and, but amazingly, I mean at times I felt, I thought I can’tdo this, I’m going to pack this in, I can’t do it, but after I would
say about six months, as I got more familiar with it and obviouslymy knowledge increased it became much easier and now one can
see the benefits of the service, it’s great, it’s really good.’ (Tele-dermatology Nurse – T9)
Interviewer: Well that all comes down to that old chestnut about what was it for,that was never quite clear…‘No, it’s never been defined as to ‘‘this service is specifically forthis’’. It hasn’t, it’s just been ‘‘we’ve got some money, we’ll do
this and see how it goes, get the waiting list down’’. Some areasof the project were wanting to get waiting lists down and that
really I think is all we were looking at, others were looking atpatients, others were looking at fulfilling the fact that we should
be advancing the technology and I found it very difficult to say‘‘we’re doing this teledermatology because…’’. In the end I didn’t
know why we were doing it.’ (Teledermatology Nurse – T5)
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Teledermatology in the U.K., T.L. Finch et al. 523
Evidence gathering, ‘proving’ safety and managing risk
With an increasing emphasis on evidence-based practice,
those developing teledermatology services appeared acutely
aware of the need to produce some level of ‘evidence’ to sup-
port their continued activities and to demonstrate that their
system was a safe mode of practice. Innovations in healthcare,
because they require sometimes quite profound change and
adaptation in existing practices, are perceived as posing new
risks and presenting fresh dilemmas for clinicians, thus much
time and effort was expended in trying to gather evidence to
prove the safety and utility of the services we studied. Formal
evaluation studies tended to be perceived as time consuming
and/or difficult to conduct within a service setting, and some
of those involved clearly felt the effort spent on such evalu-
ations was not entirely justifiable, or impeded the achieve-
ments of service objectives (Table 3). Integration of
experimental and real NHS services was a problem.25 How-
ever, those developing teledermatology services were focused
on the potential risks and responsibilities posed by such new
ways of working, and this was something that participants
often built into the design of the system (technically or soci-
ally), or addressed through ongoing modification of the
system (or participants’ use of it) in practice. The telederma-
tology services that were sustained in practice tended to be
those for which potential risk was acknowledged and
addressed by building safeguards into the system (along with
deference to professional judgement and responsibility). The
providers of such services also emphasized ‘audit’ in terms of
close monitoring of the effects of using the system on patient
outcomes, rather than formal (scientific) ‘evaluation’ (see
Table 3). Subsequently, as such services came into regular use
alongside existing services, the potential risks were accorded
less importance and teledermatology was seen to be little dif-
ferent from existing dermatology practices.
Reconfiguring services
In order for teledermatology services to become accepted in
practice, there was a need for acknowledgment of an ongoing
process of modification (see Table 4). However, as evident
particularly in the earlier teledermatology services we studied,
the hardware and software were frequently overemphasized
(especially image quality) to the neglect of the crucially im-
portant issue of how the service needed to be adapted by its
users to make it workable in practice. If equipment was sim-
ply ‘parachuted’ into an existing clinical context, without ade-
quate and ongoing support, the chances for successful
introduction and integration of the teledermatology system
into practice were severely compromised.
Professional roles and boundary crossing
The introduction of technological systems within clinical prac-
tice can promote profound changes in professional roles, and
affect the organization of professional knowledge and practice.
The introduction of such systems requires the acknowledg-
ment of frequently invisible investment in ‘workability’ by
health professionals. The results of this study reveal how this
process of achieving workability in teledermatology required
changes in professional roles and boundaries of responsibility.
At the individual level, professionals such as nurses and con-
Table 3 Evidence gathering, proving safety and managing risk
Normalized Non-normalized
‘[…] we are all slightly obsessed by, well in theory we’re obsessedwith evidence-based medicine and actually in practice, we just get
on and do it [yeah], if treatment works we just go and use it […].We have evaluated our project, sort of, nowhere near as thoroughly
or in any great academic way [as others, we] never received anyfunding and did not have the time to do it, so we did set out certain
criteria however before we started the project, we would see XXXpatients minimum in the first year, we wanted to see waiting lists
come down and so on, and all these things have happened […] wehave seen a drop in the number of patients being referred up to the
hospital so I think it is working.’ (Consultant Dermatologist – T11)
‘… going back to the ethics bit in relation to the GPs is the morecomplicated the research procedures become the more difficult it’s
going to be for actual practising practitioners to be involved. Allthe actual procedures interfere with the actual technology, you
could have argued with what we’re doing that if you hadn’t hadto make the allowances for informed consent the whole thing
could have been done in a different way again, the delays wouldhave been less, so there’s a delay related to that allowing people
to consider the whole thing and take away the written informa-tion and talk to people about it.’ (Project Lead – T6)
‘Yes, to start with, I reviewed all the patients but we very rapidly
found that most patients were better by the time I saw them, so wewere able to show that it was possible not only to make a diagnosis,
to provide a management and follow-up plan, arrange investiga-tions, which would be available by the time I saw them. We were
also able rapidly to see that it was possible to make an accuratediagnosis or differential diagnosis very quickly.’ (Consultant Derma-
tologist – T9)
‘I don’t know about [colleague] but I suspect there’s a learningcurve, in dermatology a lot of what you learn is spot diagnosis,
there are lots of books on this, is a picture a little bit of informa-tion, what’s the diagnosis, we all can do the spot diagnosis but
when it comes to actually deciding when it’s a real patient and it’snot just to get 10/10 or 2/10 in a test, it might be a bit different.
But I am certainly viewing it a bit like that, it’s going to be spotdiagnosis and working out management plans in a way you would
for that sort of test, whether it be a test that you mark your owntest that you personally do for yourself I don’t know, about how
we can cope with volume.’ (Teledermatology Consultant – T5)
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524 Teledermatology in the U.K., T.L. Finch et al.
sultants needed to make changes or adjustments to their (tra-
ditionally perceived) professional roles in order to make the
new systems ‘work’, in terms of achieving the best possible
outcomes for patients. Normalization of the service was asso-
ciated with acknowledgment and acceptance of such changes
in responsibility, as illustrated in Table 5. However, concerns
were sometimes raised about whether teledermatology services –
and telehealthcare services more generally – were attempting
to shift responsibility for care from secondary to the primary
care sector (Table 5). The capacity of primary care to absorb
and deal effectively with the potential added burdens associ-
ated with the introduction of teledermatology was an issue
raised by some participants.
Discussion
In this study, we have been able to observe closely, and over
time, how different approaches to innovating the delivery of
Table 5 Professional roles and boundary crossing
Normalized Non-normalized
‘I think because really it’s a nurse-led service, and is it working […]
it’s helping us to keep our waiting list manageable, patients like it,and the nurses are there to nurse, the doctors diagnose and pre-
scribe whereas nurses are increasingly prescribing but don’t diag-nose, they nurse people and make people better, they tell them how
to put the creams on and so on, so they do spend time with them,so I think it is working from that point of view, in that’s what the
patients need […] what it’s done it’s increased dermatologicalexpertise throughout the [region], that’s what it was about is in-
creasing our capacity to help patients with dermatological condi-tions, and to some extent it doesn’t really matter what their
qualifications are, providing they are able to do that […]. I thinkmost people accept that dermatological nursing is, in it’s own right,
a very satisfactory way of dealing with people with skin diseases.’(Consultant Dermatologist – T11)
‘If telemedicine works in one area how many more areas will
there become […] and are they [GPs] really as equipped to dealwith this addition to their, to their [existing] role, that may be
something which some GPs might consider to be just one step toofar you know. Telemedicine for dermatology one minute then
ENT then, then you know. And [then it’s] ‘‘what’s the hospitaldoing then?’’ ’ (Teledermatology Project Manager – AOD pilot,
non-normalized)
‘I think it’s [my role] still limited in teledermatology, because wecan’t, if you’re in a clinic with a doctor and a doctor says to a
patient you have got eczema, you have got psoriasis, here’s a pre-scription, I can then go in to the patient and say here’s your pre-
scription, you’ve got eczema and I can spend ten minutes talkingto the patient, going through the treatment saying this is how you
should use your treatments. When you’ve got a patient in teleder-
matology I know has got eczema, but the doctor hasn’t made adiagnosis, so I’m in a way more confined because I can’t give def-
inite treatment advice even though I know it would benefit thepatient while they were waiting. I could say to a patient, ‘‘Go and
see a GP and see if he’ll give you some Ulavate,’’ but I had to stopmyself. I felt as if I wanted to say so much more, wanted to do so
much more but I had to be so careful in case I gave wrong infor-mation, only because it hadn’t been given that seal of approval as
that is what the patient’s got.’ (Teledermatology Nurse – T5)
Table 4 Reconfiguring services
Normalized Non-normalized
‘In my honest opinion I do feel that [organizational issues are]
going to be a problem for anybody who sets [teledermatology] upbecause face-to-face consultations are a must and you should have
side by side the technology and investment in technology andhuman resources as well to do all these consultations...’ (Consultant
Dermatologist – T7)
‘No, we purchased [the teledermatology system] off the shelf,through a tendering process, but we worked with a fairly small,
flexible organization who were willing to try and adapt the system,they couldn’t adapt it completely to our own needs but they were
very good at listening to what we wanted because we were […]quite a large pilot for them. We had a very, really useful two-way
communication system set up and they took on board an awful lotof the suggestions we made in order to make the system work bet-
ter in the way that we particularly used it…’ (Teledermatology Pro-
ject Manager – T12)
‘[…] the equipment was a bit of a pain sometimes when it broke
down. I do know that initially, they were very attentive to startwith.’
Interviewer: Well, it was in development, wasn’t it?
‘But from about two thirds of the way through the interest had
waned so that the girls would be stuck there with nothing work-ing and the patient still there, and then to have to say to patients,
‘‘I’m terribly sorry,’’ so that perhaps wasn’t good, we want com-mitment of the people properly to the maintenance of the equip-
ment and everything else.’ (Teledermatology Nurse Manager – T5)
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Teledermatology in the U.K., T.L. Finch et al. 525
dermatology services through the introduction of telederma-
tology have succeeded or failed and the contributing factors
underlying these outcomes. The study is unique (not only in
relation to teledermatology but for telehealthcare generally) in
that it analyses the development of most of the known tele-
dermatology services on a national level, over a significant
period of time (8 years), thus providing a sound analysis of
the differences between success and failure in implementation
efforts in this context.
Various factors promoting the successful implementation
of teledermatology were identified, and include: (i) a per-
ception of the benefits of new services that outweighed the
effort and commitment required to make such services
work; (ii) a willingness to rely on demonstrated utility in
practice rather than formal scientific evidence; (iii) allowing
health professionals to make modifications to the technical
system, rather than imposing new practices around fixed
technological systems; (iv) accommodating uncertainty and
allowing risk management through professional judgement
rather than protocol; and (v) flexibility in division of labour
and responsibility across nursing and medical domains.
‘Successful’ teledermatology services are based on working
relationships (nurses, consultants, GPs) underpinned by trust
and confidence in professional judgement, combined with
flexibility within the service to allow for modifications as
required to achieve follow-up and continuity of care for
patients. The original ‘policy’ vision of how teledermatology
would be utilized, as a technological fix for long waiting
lists and consultant shortages, failed to be realized. Never-
theless, several teledermatology services have survived and
proved successful, occasionally playing a major role in ser-
vice delivery, while at other times serving as a useful
adjunct to traditional services. It is also important to note
that while teledermatology was at the outset often viewed
as a diagnostic service it appears to have gained increasing
acknowledgment as a triage and management service.3 Flexi-
bility with regard to how such services are deployed and
utilized, based on local requirements and circumstances, and
perceived benefits relative to the necessary commitment to
making such services work, has proven essential for teleder-
matology to have a role in service provision. Cross-sector
and professional support for such initiatives was also a cru-
cial factor.
This study demonstrates that, when introducing telederma-
tology services, the ways in which services become reconfig-
ured must be given careful thought and consideration, in
terms of understanding not only what is required of the
technological system to enable professionals to use it, but
also acceptance on the part of administrators that the service
will need to be redesigned and modified in practice to fit in
with the local context of service provision, and to meet the
particular needs of the local community. The lessons learnt
from following the history and development of teledermatol-
ogy services merit consideration by those involved in the de-
velopment and implementation of other telehealthcare
innovations.
Acknowledgments
We wish to acknowledge the material and intellectual contri-
bution of Dr Maggie Mort to the ongoing programme of
research on which this paper is based, and for her comments
on the original manuscript. We thank all of our study partici-
pants for giving their time to participate in this study, and to
Ms D. Mukadam and Ms Cheryl Wiscombe for administrative
support. Funding was supplied by Department of Health
(grant ICT/032) and Economic and Social Research Council
(grants L218 25 2067 and RES 000270084).
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EPIDEMIOLOGY AND HEALTH SERVICES RESEARCH DOI 10.1111/j .1365-2133.2006.07617.x
The Family Dermatology Life Quality Index: measuring thesecondary impact of skin diseaseM.K.A. Basra, R. Sue-Ho* and A.Y. Finlay
Department of Dermatology, Cardiff University, Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, U.K.
*Department of Microbiology, Wycombe General Hospital, Queen Alexandra Road, High Wycombe, Bucks, HP11 2TT, U.K.
CorrespondenceM.K.A. Basra.
E-mail: drkhurshid69@hotmail.com;
FinlayAY@cf.ac.uk
Accepted for publication31 July 2006
Key wordsdermatology, family, health-related quality of life,
quality of life, secondary impact
Conflicts of interestA.Y.F. is joint copyright owner of the
Dermatology Life Quality Index (DLQI) and
Family Dermatology Life Quality Index (FDLQI).
A.Y.F.’s department gains some income from the
use of the DLQI. A.Y.F. gives paid consultancy
advice to several pharmaceutical companies but this
study has not involved any pharmaceutical
company and has not received any external
funding. M.K.A.B. is a joint copyright owner of
the FDLQI.
This study was conducted at the University
Hospital of Wales, Cardiff, U.K.
Summary
Background Skin diseases are known to have a major impact on the lives of patientsand their families. Many instruments are available to measure the health-relatedquality of life (HRQoL) of patients but no measure has been developed so far toquantify the secondary impact on family members of the patients.Objectives To develop and validate a dermatology-specific instrument to measurethe adverse impact on the HRQoL of family members of patients with skindisease.Methods Detailed semi-structured interviews were conducted with family membersof patients to identify different aspects of HRQoL affected. An initial draft versionof the questionnaire based on the main topic areas was pilot tested to assess theface and content validity. A 10-item questionnaire, the Family Dermatology LifeQuality Index (FDLQI), was finalized after modifications to the draft question-naire based on feedback from families and dermatology professionals and onitem reduction. Psychometric evaluation was conducted on a new cohort of fam-ily members (n ¼ 132) who completed the FDLQI and the patients (n ¼ 109)who completed the Dermatology Life Quality Index (DLQI).Results Fifty-nine different aspects of family members’ HRQoL were identifiedfrom the analysis of the interviews, which were categorized into main topicareas. Factor analysis of 10 items of the final questionnaire revealed two factorsand together these explained 60% of the common variance. The FDLQI demon-strated high internal consistency (Cronbach’s a ¼ 0Æ88) and test–retest (intraclasscorrelation coefficient ¼ 0Æ94) reliabilities. The responsiveness of the instrumentto change was shown by significant change in the family members’ FDLQI scoresin cases where patients’ clinical condition either improved or worsened. Con-struct validity was assessed by testing a number of a priori hypotheses. A strongcorrelation was seen between the family members’ FDLQI scores and patients’DLQI scores (r ¼ 0Æ69), a significantly higher FDLQI score was seen for inflam-matory skin diseases compared with noninflammatory diseases/isolated lesions(P < 0Æ0001), and there was a positive relationship between the family members’FDLQI scores and patients’ disease severity (r ¼ 0Æ49).Conclusions The FDLQI is simple and practical and seems to have the potential tobe used as an additional outcome measure in clinical practice and evaluationresearch.
Skin disease can have a major impact on patients’ lives1,2 and
a number of patient-completed instruments3–6 have been
described to measure this impact. Quality of life (QoL) of
individuals is closely related to the QoL of those around them
such as partners and in some situations the QoL of a partner
may be more impaired than the patient.7 Family caregivers
may experience a major impact on their lives such as physical
and mental exhaustion, social disruption, marital problems
and financial implications.7 Efforts to improve the QoL of
individuals should therefore also address the QoL of the fam-
ilies or caregivers.8 The concept of Family QoL9 has been
described as being a situation where ‘the family needs are met
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528 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp528–538
and family members enjoy their life together as a family and
have the chance to do things that are important to them’.
Family QoL is considered to be an important outcome of ser-
vices and policies,10 and should be analysed independently
and additionally to the QoL of the patient.11 In some specialit-
ies, for example paediatrics, the QoL of the family is regarded
as a part of the patient’s health.12 However, in dermatology,
the awareness of the importance of this impact of disease on a
patient’s family is relatively recent.
Because the lives of some people with skin disorders are
severely disrupted and because family members are involved
in caregiving, it would be expected that families’ lives would
also be affected.13 The families of children with eczema have
lower QoL than families of healthy children14,15 and taking
care of a child with moderate to severe atopic eczema has
been shown to be more stressful than caring for a child with
insulin-dependent diabetes mellitus.16 Family QoL is related to
the severity of eczema17 and can actually improve following
specialist clinical care.18 The impact of a patient’s skin disease
on family members has been studied in only a small number
of dermatological conditions19–22 with most attention focused
on atopic eczema.
Disease-specific instruments to measure the secondary
impact of skin disease on the family have been described for
atopic eczema.15,23,24 Although disease-specific instruments are
good for tapping specific aspects of a certain disease, they do
not allow generalizability.25 However, a generic instrument is
useful not only for comparisons across different dermatoses
and monitoring interventions, but also in meta-analysis
research. In the same way that dermatology-specific measures
such as the Dermatology Life Quality Index (DLQI)5 or Skin-
dex6 can be used across a wide range of skin diseases, there is
a need for a generic dermatology-specific instrument that could
be used to measure the secondary impact on health-related
QoL (HRQoL) of families across different skin diseases. If it
were possible to quantify this secondary impact of skin disease,
the awareness of the secondary impact would be enhanced,
encouraging a clinical focus on addressing these problems. The
existence of such a tool would allow its use as an appropriate
outcome measure in clinical and health service research.
This paper describes the development and evaluation of an
instrument to quantify the impact of a patient’s skin disease
on family members—the Family Dermatology Life Quality
Index (FDLQI). The aim was to construct and validate a gen-
eric dermatology-specific tool for the family members of
patients, applicable to a wide range of skin diseases, which
should be simple and user-friendly for clinical use and could
serve as an additional outcome measure in evaluation research
or clinical trials.
Materials and methods
This open prospective study was approved by the South East
Wales Local Research Ethics Committee and completed in a
number of stages. Written informed consent was given by
participants at all stages.
Study sample
The main study participants were the immediate family mem-
bers accompanying patients attending the dermatology outpa-
tient clinic of the University Hospital of Wales, Cardiff.
Inclusion criteria for the family members were age above
18 years, ability to understand and read the English language,
having a close relationship with the patient and living in the
same household. Family members were excluded if they or
the patients had any other significant illness or disability that
impaired their quality of life, in order to avoid confusion of
that impact with dermatology-related HRQoL.
Interviews and item generation
Detailed semi-structured interviews were conducted with 50
family members or partners of patients with different skin dis-
eases.26 Participants were encouraged to describe all the ways
that their lives had been affected by living with a patient with
skin disease. Enquiries were then made into those aspects of
the family member’s QoL27 that were not mentioned by the
participant but were considered important by the interviewer,
using a checklist of possible HRQoL domains. From the con-
tent analysis of interview transcripts, 59 aspects of family
members’ affected HRQoL were identified. These were categ-
orized into main topic areas by a consensus of the investiga-
tors (Fig. 1). A preliminary draft questionnaire based on the
main topic areas of family members’ HRQoL was formulated
which contained 19 items, each given a four-point response
and score format, i.e. not at all/not relevant ¼ 0, a little ¼ 1,
quite a lot ¼ 2 and very much ¼ 3. All questions asked about
the QoL impact over the last 1 month.
Pilot test
The purpose of the pilot test was to test the content and face
validity of the draft questionnaire and to select appropriate
items for the final questionnaire. A new cohort of 20 subjects
who were immediate family members of patients attending
the dermatology clinic was recruited. Each participant was
given the draft questionnaire to complete and then asked to
comment on the clarity, understandability and relevance of in-
dividual items and also to identify those aspects of family
impact that were not covered in the questionnaire. At the
same time, opinion was sought from 14 dermatology staff
members regarding the practicality and face validity of the
questionnaire.
Finalization of the questionnaire
The final selection of the items was based on the results of the
pilot test and initial statistical analysis of the responses to
assess the internal consistency of the items. Some of the items
were rewritten or revised and some were amalgamated to
enhance relevance and practicality and reduce ambiguity. The
criteria adopted for item removal was that either the specific
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The Family Dermatology Life Quality Index, M.K.A. Basra et al. 529
item was endorsed by very few respondents or had low item–
total correlation (< 0Æ20).28 Because our aim was to develop
an instrument that would be both evaluative and potentially
sensitive to change, the items that were scored high by the
majority of the subjects were retained for the final question-
naire.
The present version of the questionnaire (the FDLQI) is
self-administered, contains 10 items (see Appendix) and can
be completed in 2–3 min. Each question inquires about the
family member’s perception of a certain specific impact on
his/her HRQoL over the last 1 month, a time consistent with
the episodic nature of many dermatological conditions, and is
scored on a four-point scale (0–3). The scores of individual
items are summed to generate a total scale score with a range
of 0–30; higher total FDLQI scores indicate greater impair-
ment of the family member’s HRQoL.
Psychometric evaluation of the Family Dermatology Life
Quality Index
The technical properties of the FDLQI were assessed on a new
cohort of family members and patients with various skin con-
ditions. Eligible family members/partners were given a copy
of the FDLQI to complete while the patients (only those older
than 16 years) were requested to complete the DLQI.5,29 The
DLQI is a self-administered questionnaire and consists of 10
questions, each scored on a four-point scale. Scores are added
to yield a total score; higher scores mean greater impairment
of patient’s HRQoL. A global question (GQ) was included at
the end of both the FDLQI and DLQI which asked the
respondent about his/her subjective assessment of the patient’s
disease severity over the last month on a 0–10 visual analogue
scale; 0 indicating ‘clear’ skin and 10 ‘worst possible’. The
medical record of the patient was used to ascertain the clinical
diagnosis. Enrolled participants were also given an envelope
containing the FDLQI with the GQ to self-assess disease sever-
ity, to complete after 1 week and return by post, but only if
they thought that the patient’s skin condition had remained
stable during this period. A subset of subjects was approached
3–6 months later at one of their routine follow-up visits by
one of the investigators (M.K.A.B.) and asked whether the
patients’ skin condition had changed (improved or deterior-
ated) since the last administration of the questionnaire. If it
had, the family members/partners were given the FDLQI with
the GQ about the patient’s disease severity to complete again.
At each stage questionnaires with more than one missing item
were excluded from analysis.
Factor analysis
For an instrument to be able to use a summed total score, it is
necessary to demonstrate that the instrument shows either
strict or essential unidimensionality.30 Strict unidimensionality
indicates the presence of a single common factor whereas
essential unidimensionality shows the presence of a reasonably
dominant common factor along with other secondary minor
factors. Exploratory factor analysis31 was performed on the 10
items to identify the factor structure underlying the FDLQI
items and to determine whether expected essential unidimen-
sionality was present to support the use of a total FDLQI score.
Because we expected that the factors would be interrelated,
we used the oblique rotation produced by the Oblimin
method followed by Kaiser normalization.32
Reliability
The FDLQI was evaluated in terms of internal consistency
and test–retest reliability. Internal consistency was assessed by
means of Cronbach’s alpha (a) coefficient,33 which expresses
the degree of consistency of the items. It has been suggested
that a should be above 0Æ7034 and not more than 0Æ90.28
a coefficient values below 0Æ70 are regarded as indicative of
0 10 20 30 40 50
Psychological distress
Burden of care
Effect on social life
Effect on holidays
Effect on work load
Effect on job
Effect on routine expenditure
Effect on leisure activities
Effect on physical well-being
Effect on sleep
Need to avoid favourite activities
Dissatisfaction with health services
Need for support
Effect on nutrition
Problems due to peoples' attitude
Effect on sex life
Role of religious faith
Miscellaneous
Number of family members (out of 50) affected by each QoL aspect
Fig 1. Main family health-related quality of
life (HRQoL) categories and their frequency
among 50 families.
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530 The Family Dermatology Life Quality Index, M.K.A. Basra et al.
individual items not adequately contributing to overall scale
score,28 while a very high a coefficient shows that items are
measuring a common latent trait with a high level of item
redundancy. Reproducibility (test–retest reliability) examines
the stability of the scale on repeat measurement. It was
determined by means of the intraclass correlation coefficient
(ICC) using a one-way random effects parallel model.35 To
ensure that only stable individuals were included for test–
retest, only those questionnaires (returned within 1–2 weeks)
were analysed in which the GQ rating about the disease
severity had not changed by more than one point on the 0–
10 scale.
Responsiveness to change
A subset of participants (family members) who confirmed that
patients’ skin condition had changed completed the FDLQI
(and GQ) 3–6 months later. To assess the sensitivity of the
FDLQI to change, the difference in the scores on the two
occasions was calculated as well as the difference in the dis-
ease severity GQ score. Based on the difference in the GQ
(disease severity) score, subjects were divided into two
groups: ‘improved’ (in whom the GQ score decreased) and
‘worsened’ (in whom the GQ score increased). The paired
sample t-test was used to assess the within-group difference
and the Mann–Whitney U-test was used to interpret the differ-
ence in FDLQI scores between improved and worsened
groups. The Spearman’s rank correlation coefficient was used
to determine the correlation between the change in FDLQI
score and the change in the disease severity score.
Validity
We assessed the construct validity of the instrument by testing
a number of a priori hypotheses.
Firstly, we hypothesized that there would be a moderate to
high correlation between the HRQoL of the patient and the
family. To assess this we compared the FDLQI scores of the
family members with the DLQI scores of the patients (only
those ‡ 16 years of age) using Spearman’s rank correlation;
here we expected to find strongly positive correlation.
Secondly, we hypothesized that family members of patients
with inflammatory skin diseases would have greater impair-
ment of their HRQoL when compared with the family mem-
bers of patients with noninflammatory diseases or isolated
lesions. To test this we divided the participants into two dis-
ease category groups: inflammatory (family members of
patients suffering from inflammatory skin diseases, e.g. acne,
eczema, psoriasis) and noninflammatory [family members
with patients having noninflammatory diseases or isolated
benign, premalignant or malignant lesions, e.g. naevi, viral
warts, basal cell carcinoma (BCC), solar keratoses]. Then we
compared the FDLQI scores between the two disease categories
using the Mann–Whitney U-test. Within-group analysis was
also carried out to examine the relationship between different
diseases and the family impact (e.g. psoriasis, eczema, acne).
Thirdly, we hypothesized that family impact would be rela-
ted to the patient’s disease severity.36 Here we tested the cor-
relation between FDLQI scores and the patient’s disease
severity score (GQ), as rated by the family members using
Spearman’s correlation coefficient.
All the statistical analysis was carried out on SPSS version
12.0 computer software (SPSS Inc., Chicago, IL, U.S.A.).
Results
Development of the questionnaire
Of the 51 family members recruited for interviews, one was
later found to have a significant heart problem and was exclu-
ded. The demographic characteristics of the remaining 50 par-
ticipants are shown in Table 1. The mean age of the family
members was 48Æ1 (range 24–82) years; 19 were male and
31 were female. Most of them were either one of the parents
(44%) or spouses/partners (44%) of the patients. The patients
(mean age ¼ 35Æ7 years; range 5 months–84 years) suffered
from one of the 21 skin diseases shown in Table 1. Interview
analysis identified 59 different aspects of family members’ life
quality which were adversely affected by the patients’ skin dis-
eases. These were grouped into main topic areas (Fig. 1). A
detailed description of these initial interviews is presented
elsewhere.37
The most frequently described aspects were psychological
distress (98%), burden of care (54%), social life (48%), holi-
days (46%) and housework (42%). The two least frequently
mentioned aspects of life were the effect on sex life and the
Table 1 Sociodemographic characteristics of subjects (n ¼ 50) in theinterview stage
Characteristics % Diagnoses %
Sex Eczemas 28Male 38 Psoriasis 14
Female 62 Discoid lupuserythematosus
8
Acne 6Marital status Ichthyosis 4
Married 66 Granuloma annulare 4Single 24 Squamous cell carcinoma 4
Divorced/widowed 10 Basal cell carcinoma 4Malignant melanoma 4
Relationship with the patient Keratoacanthoma 2Parent 44 Solar keratosis 2
Spouse/partner 44 Alopecia areata 2Daughter 4 Angioedema 2
Grandparent 4 Pemphigus 2
Grandchildren 2 Vitiligo 2Sibling 2 Haemangioma 2
Lichen sclerosis 2Ethnic origin Urticaria 2
Caucasian 88 Incontinentia pigmenti 2Asian 8 Mycosis fungoides 2
Afro-Caribbean 4 Dental sinus 2
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The Family Dermatology Life Quality Index, M.K.A. Basra et al. 531
role of religious faith in coping with the patients’ illness
(included here due to its major significance in some cultures
and mentioned by some subjects during the interviews); each
of these were described by only 8% of the participants. There
was no statistically significant difference between the male and
female participants in the frequency of any of the HRQoL
aspects.
The present 10-item version of the FDLQI questionnaire was
finalized after meticulous reviewing of the feedback from the
family members of the patients and dermatology experts dur-
ing the pilot test and initial psychometric testing of the 19
items. Items were removed if either the item–total correlation
was < 0Æ2028 or if it was endorsed by very few respondents.28
Evaluation of the questionnaire
Of the 141 family members/partners approached, five
declined to participate for personal reasons. Two more were
later dropped from analysis after finding from their medical
records that they had other significant health issues not men-
tioned at the time of recruitment. Responses of two subjects
were eliminated due to incomplete answers. The final psycho-
metric analysis was carried out on data from 132 family mem-
bers (of patients with 45 different skin conditions) who
completed the FDLQI and 109 of their related patients (only
those ‡ 16 years of age) who completed the DLQI. The socio-
demographic characteristics of study participants are shown in
Table 2 and the dermatological characteristics in Table 3.
There was no statistically significant difference in the age of
male and female participants [family members (P ¼ 0Æ10),
patients (P ¼ 0Æ24)].
Family Dermatology Life Quality Index score distribution
Scores for the overall scale (0–30) ranged from 0 to 26, med-
ian ¼ 7Æ0, mean ¼ 8Æ02 and SD ¼ 6Æ36, indicating the ability
of the instrument to detect subject variability on a continuum
of QoL impact (Table 4). There were no ceiling effects (i.e.
maximum score of 30 not reached) and a minimum of floor
effects with only 7Æ6% subjects scoring 0.
Using the mean score for individual items (range 0–3), the
most highly scoring items were the emotional impact (1Æ39),
burden of care (0Æ95), effect on the physical well-being of the
family member (0Æ86), extra household expenditure (0Æ82)
and problems due to people’s reaction to the patients’ skin
appearance (0Æ80) (Fig. 2). Using the percentage (out of 132)
responding positively (combining a little, quite a lot and very
much), the most commonly reported QoL aspects were emo-
tional aspect (79Æ5%), burden of care (60%), impact on phys-
ical well-being (53Æ1%) and extra household expenditure
(53%). Although for each of the 10 items the female mean
score was marginally higher than the male mean score, there
was neither a statistically significant difference in the total
FDLQI scores (M ¼ 7Æ24, F ¼ 8Æ43, P ¼ 0Æ31) nor in the
individual item scores (P ¼ 0Æ08–0Æ91) between male and
female subjects. However, there was a significant difference in
the FDLQI scores (P < 0Æ001) of family members of patients
12 years of age or younger (mean FDLQI score ¼ 12Æ4,
SD ¼ 6Æ6, n ¼ 16) compared with those with family members
older than 12 years (mean FDLQI score ¼ 7Æ4, SD ¼ 6Æ1,
n ¼ 116).
Caregivers can estimate the severity of patient’s disease ac-
curately.36,38 The family members’ assessment of the patients’
disease severity (GQ) score ranged from 0 to 10, with a mean
of 5Æ1 (SD ¼ 2Æ8). The patients’ own assessment of their dis-
ease severity (GQ) mean score was 5Æ3 (SD ¼ 2Æ8). There was
a high correlation between the family members’ and patients’
subjective assessment of the patients’ disease severity (r ¼0Æ69, Pearson’s rank correlation coefficient, P ¼ 0Æ01).
Factor analysis
Factor analysis of the data (from 132 participants) was per-
formed using principal component analysis followed by obli-
que rotation with Kaiser normalization.32 Based on the
guidelines suggested by Gorsuch,31 i.e. 5–10 cases for each
item, the sample size of our study (n ¼ 132 for a scale of 10
items) was considered adequate for factor analysis. Two
Table 2 Sociodemographic characteristics of participants in theevaluation stage
Total number of family members (n) 132Males 45
Females 87Mean age of family members (years) 49Æ2Relation with patient (%)
Parents 44Æ7Spouses/partners 45Æ5Son/daughter 9Æ1
Marital status (%)
Married 74Æ2Single 22Æ7Divorced/widowed/separated 3Æ1
Occupation (%)
Employed 50Retired 20Æ5Housewife 22Student 3Æ8Unemployed 3Æ8
Residence (%)
Urban 93Æ9Rural 6Æ1
Educational status (%)Primary 7Æ6Secondary 56Æ1Diploma/degree 34Æ8
Ethnic origin (%)Caucasian 90Æ1Asian 6Æ1Afro-Caribbean 3Æ8
Total number of patients (n) 132Males 58
Females 74Mean age of patients (years) 39Æ5
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp528–538
532 The Family Dermatology Life Quality Index, M.K.A. Basra et al.
main factors were identified by retaining those factors with
eigenvalues > 1; the first factor had an eigenvalue of 4Æ78 and
accounted for about 48% of the common variance, while the
second smaller factor had an eigenvalue of 1Æ18 and accounted
for about 12% of the variance (Table 5). The two factors
together accounted for 60% of the common variance, which
is higher than the minimum recommended (i.e. 50%) for a
stable factor solution.39 The two extracted factors were signifi-
cantly interrelated (r ¼ 0Æ48), confirming that our approach
of using oblique rotation31 was appropriate. Reckase40 has
suggested that unidimensionality can be assessed through the
eigenvalues of factors. A scale is considered to exhibit unidi-
mensionality if there is one dominant factor. Another method
to conclude unidimensionality is to calculate the ratio of the
first and second factor eigenvalues. If the ratio is higher than a
critical value, such as four, as suggested by some psychometri-
cians,31,41 the scale is unidimensional. In our factor analysis,
not only was there one dominant first factor but also the ratio
between the first (4Æ78) and second (1Æ18) factor eigenvalues
was greater than the minimum recommended ratio of four for
supporting unidimensionality. Figure 3 shows the scree plot
that demonstrates the eigenvalues against each factor. As can
be seen, from the first factor onwards the line is almost flat
meaning that it is only the first factor that accounts for most
Table 3 Dermatological characteristics of patients in the validationstage
Number of patients; n 132Total number of dermatoses; n 45
Group 1 (inflammatory); n (%) 24 (53Æ3)a
Group 2 (noninflammatory),
isolated lesions—benign,premalignant and malignant);
n (%)
21 (46Æ6)a
Mean duration of patients’ diseases 7Æ7 years
(range 2 weeks–55 years)
Group 1 %b Group 2 %b
Eczemas 15Æ9 Naevi 5Æ3Psoriasis 15Æ2 Solar keratosis 5Æ3Acne 15Æ2 Basal cell carcinoma 3Æ8Leg ulcer 2Æ3 Vitiligo 1Æ5Urticaria 1Æ5 Viral wart 1Æ5Pemphigoid 3Æ0 Haemangioma 1Æ5Contact dermatitis 1Æ5 Morphoea 1Æ5Fungal infection 1Æ5 Bowen disease 1Æ5Erythema nodosum 1Æ5 Squamous cell carcinoma 0Æ8Othersd 11Æ2 Otherse 9Æ6Total 68Æ8c Total 32Æ3c
aPercentage of skin diseases. bPercentage of participants
(family members of patients with these skin diseases).cBecause of rounding of figures, percentages sometimes do
not add up to 100. dNodular prurigo, folliculitis, pompho-lyx, ichthyosis, erythema multiforme, discoid lupus
erythematosus, rosacea, pityriasis rosea, lichen planus,
seborrhoeic dermatitis, lichen sclerosis, acne inversa,chronic actinic dermatitis, epidermolysis bullosa. eAlopecia
areata, milia, haematoma, hyperhidrosis, onychomycosis,epidermoid cyst, pruritus, mycosis fungoides, porokerato-
sis, Zoon’s balanitis, pityriasis lichenoides chronica.
Table 4 Family Dermatology Life Quality Index (FDLQI) scoredistribution in various family member groups and main dermatoses
n Mean score SD Range
Overall FDLQI score 132 8Æ02 6Æ4 0–26FDLQI—inflammatory 90 10Æ0 6Æ2 1–26
FDLQI—noninflammatory 42 3Æ8 4Æ3 0–17Male family members 45 7Æ2 5Æ9 0–23
Female family members 87 8Æ4 6Æ6 0–26Eczema 20 13Æ0 5Æ9 3–25
Acne 20 8Æ9 5Æ6 1–25Psoriasis 20 6Æ7 3Æ0 3–16
Naevus 7 3Æ1 3Æ1 0–8Basal cell carcinoma 5 1Æ6 1Æ7 0–4
Solar keratosis 7 1Æ1 0Æ9 0–2
0
0·2
0·4
0·6
0·8
1
1·2
1·4
1·6
1·8
Emot
ional
Physic
al well
-bein
g
Relatio
nship
s
People
reac
tion
Social
life
Leisu
re a
ctivit
ies
Burde
n of
care
House
wor
k
Job/
study
Expen
ditur
e
FDLQI Item
Mea
n It
em S
core
(m
axim
um
= 3
) Overall
Inflammatory
Noninflammatory
Fig 2. Mean Family Dermatology Life Quality Index (FDLQI) scores
for individual items; overall and for inflammatory and
noninflammatory groups (shown in order as presented in the
questionnaire).
Table 5 Factor analysis: components and their eigenvalues with % ofvariance explained by each component
Component Eigenvalues % of variance
1 4Æ780 47Æ8012 1Æ180 11Æ797
3 0Æ864 8Æ6404 0Æ768 7Æ677
5 0Æ623 6Æ2266 0Æ488 4Æ878
7 0Æ435 4Æ3468 0Æ355 3Æ554
9 0Æ290 2Æ90410 0Æ218 2Æ178
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The Family Dermatology Life Quality Index, M.K.A. Basra et al. 533
of the variance and each successive factor accounts for smaller
and smaller amounts of the total variance. It has also been
suggested40 that unidimensionality may hold if at least 20% of
the variance is accounted for by the first factor. The variance
explained by the first factor in our principal component analy-
sis, which was about 48%, again supports the assumption of a
dominant single underlying dimension. These results support
the justification for using a single total FDLQI score. Items
were then identified that loaded on each of the two compon-
ents with standard regression coefficients of more than 0Æ50
(Table 6). Factors were labelled as Psychosocial impact, which
loaded six items (emotional impact, physical well-being,
impact on relationships, peoples’ reaction, social life and leis-
ure activities) and Physical impact, which loaded four items
(burden of care, additional housework, effect on job/study
and extra household expenditure). The assignment of a par-
ticular item to a component was based on higher loading
value. The loading of ‘physical well-being’ on ‘psychosocial
impact’ may be explained by the concept of ‘burn-out’,42
which describes the fact that prolonged exposure to stress,
and to situations where demands exceed individual’s adaptive
resources, is related to physical fatigue, emotional exhaustion
and cognitive weariness. The loading of ‘additional household
expenditure’ on ‘physical impact’ may relate to the fact that
cheaper choices often require more effort—physically, emo-
tionally and cognitively.
Reliability
Internal consistency of the instrument, measured by Cron-
bach’s a coefficient (in 132 subjects) was 0Æ88, indicating
adequate internal consistency. This was not improved by
deleting individual items (0Æ86–0Æ87), demonstrating that all
the items of the scale contribute to the total score. The value
of a was more than the minimum of 0Æ70 required for a
panel of items such as the FDLQI to be considered a scale,
showing further that the individual items were contributing
adequately to the overall scale score.34 All item-to-total score
correlations were significant at P < 0Æ001.
Of the 64 (47Æ7%) respondents who returned the second
set of completed questionnaires 1–2 weeks after the first
administration, 13 were eliminated because the patients’ dis-
ease severity score self-assessed by the family member had
changed by more than one point from the initial score. One
more questionnaire was excluded due to incomplete
responses. Final reproducibility (test–retest reliability) assess-
ment was based on data from 50 respondents (37Æ8%) in
whom the clinical severity of patients’ disease had not
changed (assessed by maximum change in GQ score of one
point). Test–retest reliability of the scale assessed by ICC, was
0Æ94, reflecting the scale’s ability to show reproducible results
in stable subjects. For individual items the value of ICC ranged
between 0Æ70 and 0Æ87 indicating substantial agreement
between the two scores.43
Responsiveness to change
Responsiveness of the scale to change was assessed on 27 fam-
ily members 3–6 months after initial FDLQI administration at
the routine follow-up visit of the patient. Only those subjects
who reported that the patient’s skin condition was improved
or worsened compared with the first administration were
given the questionnaire to complete. Of these responses, only
data from subjects in whom the subjects’ self-assessed disease
severity (GQ score) was changed by two or more points were
analysed (n ¼ 27).
In those subjects who confirmed that the patients’ condition
had improved (n ¼ 17), the mean FDLQI score decreased
from 11Æ4 to 4Æ8 (mean change ¼ 6Æ6, SD ¼ 3Æ3, Fig. 4a),
which was highly significant (P < 0Æ0001); the corresponding
decrease in global rating of disease severity (GQ score) was
from 6Æ9 to 3Æ9 (mean change ¼ 3Æ0, SD ¼ 1Æ3). In subjects
who reported that the patients’ skin condition had worsened
Component number
10987654321
Eig
enva
lue
5
4
3
2
1
0
Fig 3. Scree plot showing eigenvalue against each factor; from the
first factor onwards, the line is almost flat showing each successive
factor accounting for smaller and smaller amounts of the total
variance; 48% of the common variance is explained by the first factor.
Table 6 Factor analysis: structure matrix after rotation showing the
loadings of different items on two components
FDLQI item 1 2
Emotional 0Æ797 0Æ312
Physical well-being 0Æ708 0Æ631Relationships 0Æ775 0Æ315
People’s reaction 0Æ753 0Æ322Social life 0Æ772 0Æ408
Leisure activities 0Æ773 0Æ419Burden of care 0Æ367 0Æ824
Extra housework 0Æ340 0Æ861Job/study 0Æ519 0Æ578
Extra expenditure 0Æ599 0Æ608
FDLQI, Family Dermatology Life Quality Index.
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534 The Family Dermatology Life Quality Index, M.K.A. Basra et al.
(n ¼ 10), the mean FDLQI score increased from 8Æ1 to 15Æ6(mean change ¼ 7Æ5, SD ¼ 6Æ5, P < 0Æ01, Fig. 4b) and the
corresponding change in the global disease severity rating was
an increase from 4Æ9 to 7Æ7 (mean change ¼ 2Æ8, SD ¼ 2Æ1).
The difference in the FDLQI score change between the
improved and worsened subjects grouped together was also
significant (P < 0Æ001). There was a high correlation between
the magnitude of change in the FDLQI score and the change
in GQ score (r ¼ 0Æ74, Spearman’s rank correlation coeffi-
cient, at significance level of 0Æ01).
Validity
Construct validity of the FDLQI was assessed by testing the a
priori hypotheses. There was strong correlation between the
families’ FDLQI scores and patients’ DLQI scores (r ¼ 0Æ69,
Spearman’s rank correlation, P ¼ 0Æ01) assessed in 109 paired
subjects and patients (‡ 16 years of age) (Fig. 5). The overall
mean DLQI score was 8Æ12, SD ¼ 7Æ3, range ¼ 0–29, n ¼109. This reflects that the overall level of impairment of the
HRQoL of the family member is related to that of the patient.
The mean FDLQI score for the inflammatory group
(mean ¼ 10Æ0, n ¼ 90) was much higher than for the non-
inflammatory/isolated lesion group (mean ¼ 3Æ8, n ¼ 42,
P < 0Æ0001) using the Mann–Whitney U-test. The difference
was also significant across all the individual FDLQI items
(Fig. 2) except for peoples’ reactions (P ¼ 0Æ1). This result
suggests that the magnitude and nature of the family impact
differed between the two clinical groups and most of the
items of the FDLQI were able to discriminate between the two
clinical groups. Within-group analysis for two disease categor-
ies showed that the FDLQI scores were able to discriminate
between different dermatoses (Table 4). The family impact
within the inflammatory group was seen to differ (P ¼ 0Æ001)
between psoriasis (mean ¼ 6Æ7, SD ¼ 2Æ9, n ¼ 20), eczema
(mean ¼ 13Æ1, SD ¼ 5Æ9, n ¼ 20) and acne (mean ¼ 8Æ9,
SD ¼ 5Æ6, n ¼ 20). Similarly for the noninflammatory/isol-
ated lesions group, the FDLQI scores were clearly low as
expected, e.g. benign naevi (mean ¼ 3Æ1, SD ¼ 3Æ1, n ¼ 7),
BCC (mean ¼ 1Æ6, SD ¼ 1Æ7, n ¼ 5) and solar keratosis
(mean ¼ 1Æ1, SD ¼ 0Æ9, n ¼ 7).
The FDLQI scores and disease severity of the patient (GQ
score), as assessed by the family member, were moderately
correlated (r ¼ 0Æ49, P ¼ 0Æ01, n ¼ 132, Spearman’s rank
correlation) in the postulated direction confirming the hypoth-
esis that family impact of skin disease is positively associated
with the disease severity of the patient.
Discussion
The impact of many skin diseases is not limited to the patient
but may extend to the rest of the family.44 Family members
play a central role in the care of such patients, especially those
with inflammatory skin disease, and so family impact data are
potentially important components of the measurement of the
overall burden of skin disease. In dermatology, measurement
of this ‘secondary impact’ of skin diseases on the patients’ fam-
ily members has largely been ignored except for atopic derma-
titis. Families may not get the level of psychosocial support that
they need, further affecting medical and economic outcomes.
11·35
4·76
6·88
3·88
0
2
4
6
8
10
12
1 2Time point
Sco
re
FDLQI score
Disease severity(GQ score)
8·1
15·6
4·9
7·7
0
2
4
6
8
10
12
14
16
18
1 2Time point
Sco
re
FDLQI score
Disease severity(GQ score)
(a)
(b)
Fig 4. Sensitivity of Family Dermatology Life Quality Index (FDLQI)
to change. (a) Sensitivity to improvement in patients’ clinical
condition: both FDLQI and disease severity (global question, GQ)
scores have decreased on repeat measurement (1 ¼ first
administration, 2 ¼ second administration after 3–6 months).
(b) Sensitivity to worsening in patients’ clinical condition: both
FDLQI and disease severity (GQ) scores have increased on repeat
measurement (1 ¼ first administration, 2 ¼ second administration
after 3–6 months).
FDLQI (n = 109) DLQI (n = 109)
0
5
10
15
20
25
30
Sco
re
Fig 5. Score distribution for Family Dermatology Life Quality Index
(FDLQI) (completed by the family members) and Dermatology Life
Quality Index (DLQI) (completed by the patients) with median,
quartiles, extreme values and outliers.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp528–538
The Family Dermatology Life Quality Index, M.K.A. Basra et al. 535
This paper describes the development and initial validation
of an instrument which is specific for the family members/
partners of patients with a wide range of skin diseases. The
evaluation of the FDLQI was conducted to meet the basic cri-
teria28,45 necessary for an instrument to be scientifically valid
and hence used with confidence.
The FDLQI measures family impact over the previous
1 month, a duration consistent with the episodic nature of
many skin diseases3 and a reasonable period to attain stability
after a change in therapy.6 The results of exploratory factor
analysis support the assumption of unidimensionality and
hence justify summing individual scale items to yield an over-
all scale score. The score ranged from 0 to 26, reflecting the
measure’s sensitivity to variation in the family impact. To be
sensitive to absence or presence of QoL impairment, QoL in-
strument scale scores should not have large floor or ceiling
effects.46 A scale having a ceiling effect of more than 20%
suggests that items with a higher range of impact measure-
ment are needed to differentiate moderate cases from severe.
In the same way, a floor effect of more than 20% is consid-
ered to be insensitive to important lower levels of impact on
QoL.46 In this context, the FDLQI, having a minimal floor
effect (7Æ6%) and no ceiling effect, would be expected to per-
form well in studies measuring change. The FDLQI demon-
strates content validity on the grounds that its item content
was based on the most frequently raised issues47 described by
the patients’ families, confirmed during the pilot testing as
well as by the consensus of experts in dermatology.
The reliability of the scale was clearly demonstrated; intern-
al consistency and test–retest reliability were reasonably ade-
quate with Cronbach’s a of 0Æ88 and ICC of 0Æ94. When an
instrument is required for monitoring individuals or in clinical
trials, an internal consistency reliability of Cronbach’s a in
excess of 0Æ85 is needed.48 A high ICC value indicates that the
scale was reproducible in stable subjects. Although our sample
for responsiveness was not large, we were able to show sensi-
tivity of the scale to change in subjects’ HRQoL with respect
to change in clinical severity of patients’ skin disease. The
FDLQI has demonstrated evidence of validity based on success-
fully testing the a priori hypotheses about its construct. We
were able to demonstrate that a family’s HRQoL was not only
related to the patient’s HRQoL, but it was also related to the
clinical severity of the patient’s disease in the expected direc-
tion and as shown by others.17 In fact, one study of the
family impact of atopic eczema36 demonstrated that the
caregiver’s assessment of severity of the child’s eczema was
the strongest predictor of the secondary family impact. The
known group validity of the FDLQI was also demonstrated by
its ability to differentiate between groups on the basis of the
nature of skin disease as predicted, i.e. the inflammatory skin
disease group having significantly higher score than the non-
inflammatory/isolated lesions group. One of our findings was
that eczema causes more impairment of family’s HRQoL than
psoriasis (P < 0Æ0001); this complements the previous finding
that HRQoL of eczema patients is more affected than that
of psoriasis patients.5,29 This possibly reflects the relatively
younger and more family-dependent age of onset in eczema
patients as well as the more itchy nature of eczema than
psoriasis.
There are several limitations of our study. There was a
relatively small sample size and recruitment of convenience
samples, which implies that scores and prevalence estimates
could be applied only to those who participated in the study.
Further studies in different settings and different populations
should be carried out to confirm our findings on family
impact. Although the preliminary analysis has provided some
evidence of the probable unidimensionality of the instru-
ment, we feel more work, as suggested by Mazzotti et al.,49
is required to confirm this issue. Further evidence needs to
be collected of the responsiveness of the instrument to clin-
ical change, for example before and after hospital admission
or other major intervention. There will also be a need to
define the meaning of FDLQI scores50 as well as the minimal
important difference for overall FDLQI score. The establish-
ment of validation is an ongoing process and there is no
gold standard against which QoL instruments can be valid-
ated.51 Measuring other aspects of validity, such as conver-
gent and divergent validity, by comparing FDLQI scores with
other generic family-oriented QoL scales would increase our
confidence in the use of the instrument and further streng-
then its potential usefulness in clinical practice and observa-
tional research.
One motivation for this study has been a wish to heighten
awareness of a very major consequence of skin disease, which
up to now has been largely ignored. If a concept cannot be
measured it attracts little scientific attention; by proposing for
the first time a method applicable to all skin diseases to score
the secondary impact of skin disease on life quality of family
members, it is hoped that attention will be focused on this
important aspect of clinical dermatology.
The ability to identify and score this secondary impact may
encourage the development of new strategies to alleviate this
impact and will allow the assessment of such strategies. The
FDLQI could potentially be used as an additional outcome
measure for health service research and clinical trials. A speci-
ality-specific measure, such as the FDLQI, may complement
generic health status measures in addressing issues concerning
health services funding and the distribution of resources
between specialities.52
There was very high acceptance of and interest in the
FDLQI by respondents. Many commented on the great impact
caused to them by their family members’ skin disease and
often mentioned that this burden had never before been
acknowledged by health service providers. This very strong
feedback has encouraged us to consider that these concepts do
need to be understood, measured and acted upon.
In conclusion, the FDLQI was developed to meet the need
for a comprehensive yet simple and practical instrument for
general clinical use and as an additional outcome measure in
dermatology for research and clinical trials. It is easy to com-
plete and score and seems reliable, valid and responsive to
improvement or deterioration in HRQoL.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp528–538
536 The Family Dermatology Life Quality Index, M.K.A. Basra et al.
Acknowledgments
We wish to thank the dermatology patients, their family
members and dermatology staff for their help with this study.
In particular we wish to thank Sister Anne Thomas for advice
on ethical issues and Mrs Susan Williams for her help.
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22 Braue A, Ross G, Varigos G, Kelly H. Epidemiology and impact ofchildhood Molluscum contagiosum: a case series and clinical review of
the literature. Pediatr Dermatol 2005; 22:287–94.23 Chamlin SL, Cella D, Frieden IJ et al. Development of the Childhood
Atopic Dermatitis Impact Scale: initial validation of a quality-of-life
measure for young children with atopic dermatitis and their fam-ilies. J Invest Dermatol 2005; 125:1106–11.
24 McKenna SP, Whally D, Dewar AL et al. International developmentof the Parents’ Index of Quality of Life in Atopic Dermatitis
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Development and Use, 3rd edn. Oxford: Oxford University Press, 2003.29 Lewis V, Finlay AY. 10 years experience of the Dermatology Life
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of Quantitative Methodology for the Social Sciences (Kaplan D, ed). ThousandOaks, CA: Sage Publications, 2004; 93–105.
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37 Basara MKA, Finlay AY. The family impact of skin diseases: the
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Appendix: the Family Dermatology Life QualityIndex (FDLQI)
Name: ................................................ FDLQI Score
Relationship with patient: .........................................
Patient’s diagnosis (if known): ....................................... Date:
...........................
• The questions relate to the impact of your relative/partner’s
skin disease on your quality of life over the last month.
• Please read the questions carefully and tick one box for
each.
1. Over the last month how much emotional distress have you
experienced due to your relative/partner’s skin disease (e.g.
worry, depression, embarrassment, frustration)?
Not at all/Not relevant u A little u A lot u Very much u
2. Over the last month how much has your relative/partner’s
skin disease affected your physical well-being (e.g. tiredness,
exhaustion, contribution to poor health, sleep/rest disturb-
ance)?
Not at all/Not relevant u A little u Quite a lot u Very much u
3. Over the last month how much has your relative/partner’s
skin disease affected your personal relationships with him/her
or with other people?
Not at all/Not relevant u A little u Quite a lot u Very much u
4. Over the last month how much have you been having
problems with other peoples’ reactions due to your relative/
partner’s skin disease (e.g. bullying, staring, need to explain to
others about his/her skin problem)?
Not at all/Not relevant u A little u Quite a lot u Very much u
5. Over the last month how much has your relative/partner’s
skin disease affected your social life (e.g. going out, visiting or
inviting people, attending social gatherings)?
Not at all/Not relevant u A little u Quite a lot u Very much u
6. Over the last month how much has your relative/partner’s
skin disease affected your recreation/leisure activities (e.g. holi-
days, personal hobbies, gym, sports, swimming, watching TV)?
Not at all/Not relevant u A little u Quite a lot u Very much u
7. Over the last month how much time have you spent on look-
ing after your relative/partner (e.g. putting on creams, giving
medicines or looking after their skin)?
Not at all/Not relevant u A little u Quite a lot u Very much u
8. Over the last month how much extra housework have you
had to do because of your relative/partner’s skin disease (e.g.
cleaning, vacuuming, washing, cooking)?
Not at all/Not relevant u A little u Quite a lot u Very much u
9. Over the last month how much has your relative/partner’s
skin disease affected your job/study (e.g. need to take time off,
not able to work, decrease in the number of hours worked, hav-
ing problems with people at work)?
Not at all/Not relevant u A little u Quite a lot u Very much u
10. Over the last month how much has your relative/partner’s
skin disease increased your routine household expenditure (e.g.
travel costs, buying special products, creams, cosmetics)?
Not at all/Not relevant u A little u Quite a lot u Very much u
Thank you for completing the questionnaire.
� MKA Basra, AY Finlay. Cardiff University 2005.
The FDLQI is copyright. For more information please contact
the authors.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp528–538
538 The Family Dermatology Life Quality Index, M.K.A. Basra et al.
PHOTOBIOLOGY DOI 10.1111/j .1365-2133.2006.07670.x
Repeated low-dose ultraviolet (UV) B exposures of humansinduce limited photoprotection against the immune effectsof erythemal UVB radiationJ. Narbutt, A. Lesiak, A. Sysa-Jedrzejowska, A. Wozniacka, A. Cierniewska-Cieslak,* J. Boncela,� C. Jochymski,�W. Kozlowski,� A. Zalewska, M. Skibinska§ and M. Norval–
Department of Dermatology and *Centre for Molecular and Medical Biophysics, Medical University of Lodz, Krzemieniecka 5, 94-017 Lodz, Poland
�Center of Medical Biology, Polish Academy of Sciences, Lodz, Poland
�Department of Pathology, Military Medical Institute, Warsaw, Poland
§Basildon Hospital, Basildon, Essex, U.K.
–Medical Microbiology, University of Edinburgh Medical School, Edinburgh, U.K.
CorrespondenceJoanna Narbutt.
E-mail: joanna.narbutt@onet.pl
Accepted for publication30 August 2006
Key wordscyclooxygenase-1, cyclooxygenase-2, cytokines,
photoprotection, thymine dimers, ultraviolet-
induced immunosuppression
Conflicts of interestNone declared.
Summary
Background Exposure of human subjects to ultraviolet (UV) B radiation causes im-munosuppression. Most experiments to date have not tested the effects of lowdaily doses of UVB radiation.Objectives To ascertain whether photoprotection against several UV-inducedimmune effects might develop following repeated exposure.Methods Groups of approximately 30 healthy individuals were given whole-bodyUVB irradiation on each of 10 consecutive days with 0.7 minimal erythema dose,or whole-body irradiation as before followed by a single erythemal UVB dose ona small body area, or irradiated only with a single erythemal UVB dose on asmall body area, or were not irradiated. They were sensitized with diphenyl-cyclopropenone (DPCP) 24 h after the final dose, and skin biopsies collected toassess cytokine mRNA expression and the number of cells with thymine dimersand expression cyclooxygenase (COX)-1 and COX-2.Results The contact hypersensitivity (CHS) response to DPCP was significantlylower in the three irradiated groups compared with the unirradiated controls,while cutaneous interleukin (IL)-1b, IL-6, IL-10 and tumour necrosis factor-amRNAs, COX-1 and COX-2 and thymine dimers were all significantly higher.When the single erythemal UVB dose was given following the repeated lowexposures, a slight downregulation in cytokine expression and thymine dimerformation was indicated.Conclusions The repeated low doses of UVB protected to a limited extent againstthe effects of an erythemal UVB dose on cytokine expression and thymine dimerformation, but not on CHS or COX enzymes.
Solar ultraviolet (UV) radiation reaching the surface of the
earth contains predominantly UVA wavelengths (315–
400 nm) with a smaller component of UVB wavelengths
(280–315 nm). As UVB is more effective than UVA at indu-
cing biological damage, UVB is thought to contribute towards
about 80% of the harmful effects associated with sun expos-
ure.1 One of these effects is suppression of cell-mediated
immune responses. The process involved is complex.2–4 It is
initiated by chromophores in the upper layers of the skin that
are capable of absorbing the radiation. One such chromophore
is DNA5 with the commonest photoproduct being cyclobutane
pyrimidine dimers (thymine dimers, dTT). These dimers are
repaired, but this process is slow with an estimated half-life of
33 h.6 The DNA damage leads to the increased synthesis of
several cytokines.7–11 In addition, an upregulation in the
production of platelet activating factor,12 histamine and
prostaglandin E2 (PGE2)13,14 occurs. Cyclooxygenase (COX)
enzymes are the first in a series that converts arachidonic acid
into prostaglandins and thromboxane. Two isoforms of COX
are found:15 COX-1 is constitutively expressed in almost all
cell types including keratinocytes, while COX-2 is induced in
many tissues in response to stimuli such as injury or inflam-
mation. UV radiation can stimulate COX-2 expression in basal
keratinocytes.16
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547 539
Modulation of the contact hypersensitivity (CHS) response is
frequently used to evaluate immunity in human subjects follow-
ing UV exposure.17 In most cases the sensitizer, a hapten such as
diphenylcyclopropenone (DPCP), is applied to the skin within a
few days of the UV radiation, with the elicitation phase taking
place several weeks later. To measure local immunosuppression,
the hapten is placed directly on an irradiated skin site. A primary
allergic response (PAR) can develop within 1–2 weeks of sensi-
tization in many individuals who have not previously come in
contact with the particular sensitizer. This response is thought
to indicate the onset of hapten-specific sensitization.18
To date, most information regarding UV-induced immuno-
suppression in human subjects has been gathered from study-
ing the induction phase of CHS following exposure of a small
area of the body to a single UV dose, often > 1 minimal ery-
thema dose (MED).19–21 However, such a protocol does not
mimic natural conditions as most people are exposed to the
sun for short periods of time on a daily basis over the sum-
mer months with many experiencing occasional burning UV
exposures on limited areas of the body. There is the possibility
that, as the skin adapts to the chronic UV radiation by epider-
mal thickening and tanning, photoprotection against the
effects of UV on the immune system may develop. In the pre-
sent study, we aimed to test this possibility by repeatedly
irradiating volunteers with suberythemal UVB, followed by a
single erythemal UVB dose on a small body area. CHS, cytokine
and COX expression, and DNA damage were then assessed in
comparison with unirradiated subjects or others irradiated
with the single erythemal dose only.
Materials and methods
Subjects
The study included 140 healthy subjects of either phototype II
or III, as assessed by Fitzpatrick score.22 These are the most
commonly occurring skin types in Poland and include subjects
who do not burn easily and develop a tan on sun exposure.
The volunteers were recruited following advertising in the
local newspapers and within the University of Lodz. They
were without any skin or other disease and were not receiving
any medication. Subjects exposed to high doses of sunlight or
sunlamps within 2 months prior to the study, or previously
sensitized with DPCP, were excluded. The experimental pro-
cedures were conducted in the winter months to decrease any
influence of natural sunlight. Each volunteer gave written
informed consent before entry into the study and underwent a
thorough physical examination and full blood count. The
experimental plan was approved by the Local Ethics Commit-
tee of the Medical University of Lodz and was conducted
according to the Declaration of Helsinki principles.
Phototesting and ultraviolet B irradiation
Phototesting of each volunteer was undertaken approximately
1 week before the study began using a Waldmann Medizin-
technik UV 109 device (Waldmann Medizintechnik, Villingen-
Schwenningen, Germany) containing TL-12 tubes (Philips,
Eindhoven, the Netherlands) emitting 58.6% UVB and 41.4%
UVA. The test was performed with an incremental dose series
on six squares (1 · 1 cm) on the back. The MED was defined
as a just perceptible erythema 24 h later. From this value, 0.7
MED was calculated for each individual although it is recog-
nized that, due to undulations on the body surface and varia-
tions in MED with site, some areas may receive slightly higher
and others slightly lower doses.
The volunteers were divided into five groups as shown in
Table 1: 40 nonirradiated individuals served as the control
group (group A), 30 subjects were given whole-body UV
irradiation for 10 consecutive days with a dose of 0.7 MED
(group B), 30 subjects were given whole-body UV irradiation
for 10 consecutive days with a UV dose of 0.7 MED followed
24 h later by a single UV dose of 3 MED (left buttock,
10 · 10 cm) (group C), 30 subjects were irradiated with a
single UV dose of 3 MED (left buttock, 10 · 10 cm) (group
D) and 10 individuals were irradiated on the left buttock
(10 · 10 cm) with a single UV dose of 4 MED (group E).
The TL-12 lamps, with an erythema effectiveness irradiance
for UVB (280–315 nm, biologically weighted with the CIE
erythema action spectrum) of 0.64 mW cm)2, were used for
the whole-body exposures, and the phototesting device was
used to irradiate the buttock. Measurement of the intensity of
the lamps was performed using a type 1 UV meter calibrated
against a spectrophotometer (Waldmann Medizintechnik). For
erythema, pigmentation, PAR and CHS measurements, all 140
subjects were included. For the cytokine mRNAs (n ¼ 40),
Table 1 Characteristics of subjects in groups A–E
Group
Number of
volunteers
Mean age,
years (range) Sex, F/M Phototype, II/III
MED, J cm)2,
mean (range)
A: unirradiated 40 25.0 (18–36) 17/23 19/21 0.18 (0.09–0.21)B: 10 · 0.7 MED, whole body 30 30.0 (19–38) 19/11 14/16 0.16 (0.09–0.19)
C: 10 · 0.7 MED, whole body(C1) + 3 MED, 10 · 10 cm (C2)
30 27.8 (19–32) 16/14 18/12 0.15 (0.07–0.17)
D: 3 MED, 10 · 10 cm 30 29.4 (18–37) 16/14 11/19 0.14 (0.09–0.19)E: 4 MED, 10 · 10 cm 10 30.6 (19–34) 4/6 5/5 0.14 (0.07–0.19)
MED, minimal erythema dose.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547
540 Photoprotection following repeated UVB exposures, J. Narbutt et al.
COX proteins (n ¼ 21) and dTT (n ¼ 21), not all the subjects
in each group were used on ethical grounds, as these assess-
ments required the collection of skin biopsies, up to three per
person in some instances.
Erythema and pigmentation
Erythema and pigmentation were quantified using the UV
Optimise 555 device (Chromo-Light, Espergaerde, Denmark)
before and after irradiation, but before sensitization, on the
buttock skin in the UV-exposed groups (groups B–E). The
mean values of three readings were calculated.
Sensitization and elicitation of contact hypersensitivity
Sensitization and elicitation of CHS were performed using
DPCP (Fluka Chemie GmbH, Buchs, Switzerland) as described
by Narbutt et al.23 In brief, the subjects were sensitized on the
left irradiated (groups B–E, 24 h after the final UVB exposure)
or nonirradiated (group A) buttock skin. The sensitization site
was assessed for the development of a PAR, including its
length of time, severity and size. Three weeks later, all the
volunteers received an antigenic challenge on the unirradiated
upper inner left arm skin using a series of 20 lL acetone con-
taining 0.4, 0.8, 1.6, 3.2 and 6.4 lg DPCP. The highest con-
centration of DPCP was applied only if no PAR was observed.
The CHS response was evaluated after 48 h by a subjective vis-
ual scoring system: 0, no reaction; 1, macular erythema; 2,
erythema with infiltration; 3, erythema with infiltration and
papules or vesicles; 4, bullous reaction. This score was deter-
mined by one dermatologist who did not know the group
assignations.
Assessment of interleukin (IL)-1b, IL-6, IL-10 and tumour
necrosis factor-a mRNA expression by relative
quantitative reverse transcription–polymerase chain
reaction
The control samples consisted of 3-mm punch biopsies from
10 subjects in group A, 15 in group C (before irradiation
began) and 15 in group D (before irradiation began), making
40 in total. The irradiated samples consisted of two more
biopsies from the same 15 individuals in group C (C1: 24 h
after 10 days of 0.7 MED UVB, and C2: 24 h after 3 MED
UVB) and one more biopsy from the same 15 individuals in
group D (24 h after 3 MED UVB). The samples (50 mg) were
dissolved in Fenozol reagent and total mRNA prepared using a
total RNA Prep Plus Kit (A&A Biotechnology, Gdynia, Poland).
The first strand of cDNA was synthesised according to the
manufacturer’s instructions (Promega, Madison, WI, U.S.A.)
using (dT)20 primer with 2 lg of total RNA. Then mRNAs
for tumour necrosis factor (TNF)-a, interleukin (IL)-10, IL-6
and IL-1b were determined by relative quantitative reverse
transcription–polymerase chain reaction (RT–PCR) using
b-actin mRNA as an internal standard,24 using the following
primers: 5¢-GTAGCCCATGTTGTAGCAAACC-3¢ and 5¢-GAG-
GACCTGGGAGTAGATGAGG-3¢ for TNF-a, 5¢-GAGAACAGCT
GCACCCACTTCC-3¢ and 5¢-CTGGGTCTTGGTTCTCAGCTTGG-
3¢ for IL-10, 5¢-ACCTGAACCTTCCAAAGATGG-3¢ and 5¢-GACT
GCAGGAACTCCTTAAAGC-3¢ for IL-6, 5¢-CTCGCCAGTGAAAT-
GATGG-3¢ and 5¢-GCATCTTCCTCAGCTTGTCC-3¢ for IL-1band 5¢-CAGCAGATTCAAGCAGCTATGG-3¢ and 5¢-GTCTGTGG-
TGCTGATCTCATCC-3¢ for b-actin. The RT–PCR assay con-
tained 5 lL cDNA sample, 10 · Taq polymerase buffer
(Epicentre Biotechnologies, Madison, WI, U.S.A.), 2 mmol
L)1 MgCl2, 1 · PCR enhancer, deoxyribonucleoside triphos-
phate mix, and 25 pmol of each primer in 50 lL reaction vol-
ume. The samples were denatured at 95 �C for 5 min, then
cooled on ice before 1.25 U Taq DNA polymerase (Epicentre
Biotechnologies) was added. The reaction was performed in
25–30 extension cycles consisting of a 30-s denaturation step
at 94 �C, a 30-s annealing step at 60 �C and a 40-s polymer-
ase extension step at 72 �C. Finally, each reaction was termin-
ated with a 10-min elongation step at 72 �C. The final
products were electrophoresed in 7% polyacrylamide gels
using the genetic size marker 100 bp DNA Ladder (Promega).
Bands were visualized by UV radiation, and the results were
recorded photographically and analysed densitometrically
using LKB Ultrascan XL Enhanced Laser Densitometer (Phar-
macia LKB, Uppsala, Sweden). Concentrations of mRNAs were
normalized in each sample relative to b-actin mRNA. Usually,
four to seven bands were analysed for each sample and the
mean value was calculated. The mean ± SD was then calcu-
lated for each group.
Expression of cyclooxygenase (COX)-1 and COX-2, and
assessment of DNA damage (thymine dimers)
Three-millimetre biopsies were collected from buttock skin
of seven unirradiated individuals (group A), seven individu-
als in group C (C1: 24 h after 10 days of 0.7 MED UVB,
and C2: 24 h after 3 MED UVB), and seven individuals in
group D (24 h after 3 MED UVB). The samples were put in
liquid nitrogen immediately, and stored at )80 �C until
analysis.
For COX-1 and COX-2 assessment, 4-lm frozen sections
were collected on to Superfrost Plus slides (Menzel, Braun-
schweig, Germany), fixed in acetone for 10 min and air
dried. Endogenous peroxidase was blocked by incubating for
5 min with 3% hydrogen peroxide before washing in phos-
phate-buffered saline (PBS) and incubation for 30 min in 10%
normal horse serum in Tris–HCl–PBS and 1% bovine serum
albumin (BSA). After rinsing, the sections were incubated
overnight at 4 �C with the COX-1 monoclonal antibody
(mAb) (dilution 1 : 40, NCL-COX-1, clone 12E12; Novocas-
tra, Newcastle upon Tyne, U.K.) or COX-2 mAb (dilution
1 : 50, NCL-COX-2, clone 4H12; Novocastra). Detection
was performed with LSAB+ System-HRP (Dako Cytomation,
Glostrup, Denmark). High-sensitivity diaminobenzidine (Dako
Cytomation) chromogenic substrate system was used. The
slides were counterstained with haematoxylin and dehydrated.
Negative controls were performed by omitting the primary
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547
Photoprotection following repeated UVB exposures, J. Narbutt et al. 541
antibody. The presence of brown colour at the sites of the tar-
get antigens (in cellular membrane, cytoplasm) indicated posi-
tive immunoreactivity. The number of COX-1+ or COX-2+
cells in the epidermis was counted by a computer-image pro-
gram (SIS Analysis; Olympus, Tokyo, Japan). At least two
sections were examined from each subject at · 400 magnifica-
tion, and the mean ± SD number of positive cells mm)2 for
each group calculated.
For the assessment of dTT, 4-lm cryostat sections were col-
lected on to Superfrost Plus slides (Menzel), fixed for 1 h in
4% buffered formalin at room temperature and air dried. The
sections were treated for 15 min of microwave oven heating
in Target Retrieval Solution (citrate buffer, pH 6.0; Dako Cyto-
mation) at 800 W and then transferred to distilled water.
Endogenous peroxidase was blocked by incubation for 30 min
with 0.3% hydrogen peroxide in methanol. Slides were
washed in PBS and incubated for 30 min with 10% normal
rat serum in Tris–HCl–PBS and 1% BSA. After rinsing, the sec-
tions were incubated overnight at 4 �C with the dTT mAb
(dilution 1 : 4000, clone KTM53; Kamiya Biomedical Com-
pany, Seattle, WA, U.S.A.). Detection, colour development and
counterstaining were performed as outlined above for COX.
Negative controls were performed by omitting the primary
antibody. Stained sections were examined at · 400 magnifica-
tion throughout the whole of the epidermis. Presence of
brown colour at the target antigen in cellular nucleus indicat-
ed positive immunoreactivity. The number of dTT+ nuclei
was counted by a computer-image program (SIS Analysis). At
least two sections were examined from each subject and the
mean ± SD number of positive cells mm)2 for each group
calculated.
Statistical analysis
The Mann–Whitney test, Wilcoxon pair test and v2 test were
used to analyse the results, with P < 0.05 being considered
statistically significant.
Results
Repeated suberythemal ultraviolet (UV) B exposure
caused erythema and pigmentation development, and
was slightly protective against the erythemal effects of a
subsequent high UVB dose
Clinical examination revealed no visible erythema after
10 days of suberythemal whole-body UVB but a slight
pigmentation was evident. Irradiation with 3 or 4 MED UVB
on the small body area caused well-defined oedematous ery-
thema. Table 2 shows the detailed results for the measurement
of erythema and pigmentation in groups B–E. In all four
groups, the erythema values were significantly higher
(P < 0.005) following the UV exposure than before irradia-
tion. In group C, after 10 days of irradiation with suberythe-
mal UVB followed by irradiation with 3 MED on the small
body area, the erythema value was significantly lower than in
group D (P < 0.0001) and group E (P < 0.0001). Suberythe-
mal UVB on 10 consecutive days led to a significant increase
in pigmentation (P ¼ 0.001 for group B and 0.0019 for
group C), while the additional erythemal UVB dose decreased
the pigmentation value in group C (P ¼ 0.000004). Follow-
ing erythemal UVB, pigmentation was significantly lower in
group D compared with group C (P ¼ 0.0004). The decrease
in pigmentation observed in groups D and E may have resul-
ted from the intense erythema and oedema which cause
changes in the optical properties of the skin.
Repeated suberythemal ultraviolet (UV) B exposures
suppressed the occurrence of primary allergic response
and contact hypersensitivity, and did not protect against
the immunosuppressive effects of a subsequent
erythemal UVB dose
PAR was detectable a mean of 8.8 days after sensitization.
The response ranged from erythema with definite borders
Table 2 Erythema and pigmentation following ultraviolet (UV) exposure in groups B–E (mean ± SD, n ¼ 30 in groups B–D and n ¼ 10 ingroup E)
Group B:10 · 0.7 MED,
whole body
Group C: 10 · 0.7 MED,whole body + 3 MED,
10 · 10 cm
Group D: 3 MED,
10 · 10 cm
Group E: 4 MED,
10 · 10 cm
Erythema% value light reflectance before UV 28.1 ± 5.8 26.0 ± 5.3 29.2 ± 5.8 25.2 ± 4.6
% value light reflectance after 10 · 0.7 MED 36.6 ± 7.0 37.8 ± 6.7 – –% value light reflectance after
3 or 4 MED (10 · 10 cm)
– 48.9 ± 7.4 62.6 ± 9.9 60.4 ± 7.1
Pigmentation
% value light reflectance before UV 20.6 ± 5.7 21.6 ± 7.1 21.1 ± 8.0 23.2 ± 6.2% value light reflectance after 10 · 0.7 MED 24.0 ± 5.8 24.9 ± 6.7 – –
% value light reflectance after 3 or4 MED (10 · 10 cm)
– 22.1 ± 8.3 13.9 ± 9.0 17.4 ± 8.1
MED, minimal erythema dose.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547
542 Photoprotection following repeated UVB exposures, J. Narbutt et al.
to strong erythema with oedema, and vesicles in some
cases, but without blistering. The diameter of the PAR var-
ied between 9 and 19 mm. The percentage of subjects in
the unirradiated group with PAR (60%) was significantly
higher than in all the irradiated groups (26.7%, 33.3%,
26.7% and 10% in groups B, C, D and E, respectively;
P < 0.05 for all comparisons). There were no statistical dif-
ferences between the irradiated groups B–E with respect
to the occurrence of PAR, its length of time, severity or
diameter.
The CHS response at the highest concentration of DPCP was
not analysed statistically as the eliciting dose of DPCP was
applied only in the volunteers in whom PAR did not develop.
UV radiation in all the groups suppressed the CHS response,
and it was observed at all the DPCP concentrations (P < 0.05
for all comparisons, data not shown). As analysis of the visual
assessment of the CHS revealed statistically significant differ-
ences between the unirradiated group and any of the irra-
diated groups at any of the DPCP concentrations and because,
in the majority of the irradiated volunteers, the CHS was
assessed as 0 (absent), we decided to simplify the analysis by
treating the CHS response as a binominal trait where 0 repre-
sented no reaction and 1 represented a response. These results
are shown in Figure 1. Statistical analysis of the CHS response
showed no significant differences between subjects in group B
and group C (P > 0.05), or between subjects in group C and
group D (P > 0.05).
Repeated suberythemal ultraviolet (UV) B exposures
increased the expression of interleukin (IL)-1b, IL-6,
IL-10 and tumour necrosis factor-a mRNAs in skin
biopsies, and slightly protected against the increased
expression of these cytokines induced by a subsequent
erythemal UVB dose
Following the suberythemal UVB exposures for 10 consecu-
tive days (group C1), the expression of the mRNAs of
IL-1b, IL-6, IL-10 and TNF-a increased significantly
(Fig. 2a–d). After the additional exposure to erythemal UVB
(group C2), there was a further, although smaller, increase
in IL-1b, IL-6 and IL-10 mRNA expression and a decrease in
TNF-a mRNA expression. The single erythemal UVB dose
induced a large rise in the expression of all four cytokine
mRNAs (group D). The increase was greater than that fol-
lowing the repeated suberythemal exposures and also follow-
ing the repeated suberythemal exposures plus the additional
erythemal exposure.
0102030405060708090
100%
of
sub
ject
s w
ith
no
resp
on
se
0·4 μgDPCP
0·8 μgDPCP
1·6 μgDPCP
3·2 μgDPCP
EEEEA A A AB B B B CCCC DDDD
Fig 1. Percentage of subjects with no contact hypersensitivity
response to the challenge dose of 0.4, 0.8, 1.6 and 3.2 lg
diphenylcyclopropenone (DPCP) in groups A–E.
0
10000
20000
30000
40000
50000
60000
(b)
(a)
(c)
(d)
Exp
ress
ion
of
IL-1
bet
a m
RN
A(a
rbit
rary
un
its)
P<0·000001
P=0·0000063 P<0·01
P=0·04 P=0·00048
P=0·000013
DC2C1Unirradiated
0
10000
20000
30000
40000
50000
60000
Exp
ress
ion
of
IL-6
mR
NA
(arb
itra
ry u
nit
s)
P<0·000001
P=0·0045P<0·01
P=0·017
P<0·001
P=0·000037
DC2C1Unirradiated
0
10000
20000
30000
40000
50000
60000
Exp
ress
ion
of
TN
F-a
lph
a m
RN
A(a
rbit
rary
un
its)
P<0·0001
P<0·001P=0·0045
P>0·05
P=0·017
P=0·046
DC2C1Unirradiated
0
10000
20000
30000
40000
50000
60000
Exp
ress
ion
of
IL-1
0 m
RN
A(a
rbit
rary
un
its)
P<0·000001
P=0·004P<0·01
P>0·05
P=0·0011
DC2C1Unirradiated
Fig 2. Expression of (a) interleukin (IL)-1b, (b) IL-6, (c) tumour
necrosis factor (TNF)-a and (d) IL-10 mRNAs in skin biopsies from
unirradiated subjects (n ¼ 40), subjects irradiated with 0.7 minimal
erythema dose (MED) ultraviolet (UV) B for 10 consecutive days
(C1, n ¼ 15) followed by 3 MED UVB (C2, n ¼ 15), and subjects
irradiated with 3 MED UVB (D, n ¼ 15). The cytokine expression was
normalized to the b-actin mRNA intensity and is presented in
arbitrary units. The mean ± SD for each group is shown.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547
Photoprotection following repeated UVB exposures, J. Narbutt et al. 543
Repeated suberythemal ultraviolet (UV) B exposures
increased the number of cyclooxygenase (COX)-1+ and
COX-2+ cells in skin biopsies, and did not protect
against the increased number induced by a subsequent
erythemal UVB dose
In unirradiated skin biopsies, COX-1+ cells were observed
only occasionally, mainly in the basal layer of the epidermis,
and no COX-2+ cells were seen. The number of both COX-
1+ and COX-2+ cells increased significantly following UVB
exposure (P < 0.05 for all comparisons, Fig. 3). In irradiated
skin, the COX-1+ cells were located in both the basal layers
and the upper part of the epidermis, while the COX-2+
cells were present mainly in the suprabasal layer of the epi-
dermis (Fig. 4). The additional erythemal UVB dose follow-
ing the repeated suberythemal doses (group C2) induced
almost the same number of COX-1+ and COX-2+ cells as
the single erythemal UVB dose on its own (group D)
(Fig. 3), indicating that photoprotection for this factor had
not developed.
Repeated suberythemal ultraviolet (UV) B exposures
increased the number of thymine dimer cells in skin
biopsies, and slightly protected against the increased
number induced by a subsequent erythemal UVB dose
In unirradiated skin, only a few dTT cells were observed
(15 mm)2), scattered within the epidermis, and the staining
was considered weak. The number increased significantly fol-
lowing UV exposure (P < 0.05 for all comparisons, Fig. 5).
The highest was seen after irradiation with the single erythe-
mal dose (group D) and this number was significantly greater
than that following the repeated suberythemal doses followed
by the single erythemal dose (group C2) (P ¼ 0.00001). The
dTT+ cells were found mainly in the upper parts of the epi-
dermis following the repeated suberythemal doses of UVB,
while they were present throughout the epidermis following
the single erythemal dose.
Discussion
In a previous study we attempted to simulate the natural ex-
posure of subjects with skin phototypes II/III to summer sun-
light by whole-body irradiation with 0.3 MED solar-simulated
0
100
200
300
400
500
600
Nu
mb
er o
f C
OX
-1+
an
dC
OX
-2+
cells
mm
–2
COX-2
D C2 C1 A
COX-2 COX-2 COX-2 COX-1 COX-1 COX-1 COX-1
Fig 3. Number of cyclooxygenase (COX)-1+ and COX-2+
cells mm)2 in the epidermis of unirradiated subjects (A, n ¼ 7),
subjects irradiated daily with 0.7 minimal erythema dose (MED)
ultraviolet (UV) B for 10 consecutive days (C1, n ¼ 7) followed by 3
MED UVB (C2, n ¼ 7), and subjects irradiated with 3 MED UVB (D,
n ¼ 7). The mean ± SD for each group is shown.
(a)
(b)
Fig 4. Expression of cyclooxygenase (COX)-1 (a) and COX-2 (b) in
human epidermis after 0.7 minimal erythema dose (MED) ultraviolet
(UV) B for 10 consecutive days followed by 3 MED UVB as shown by
immunohistochemistry (original magnification · 400).
0 100 200 300 400 500 600 700 800 900
1000
Nu
mb
er o
f ep
ider
mal
dT
T+
cells
mm
–2
P=0·000001
P<0·000001
D C2 C1 A
Fig 5. Number of thymine dimers (dTT+) cells mm)2 in the
epidermis of unirradiated subjects (A, n ¼ 7), subjects irradiated daily
with 0.7 minimal erythema dose (MED) ultraviolet (UV) B for 10
consecutive days (C1, n ¼ 7) followed by 3 MED UVB (C2, n ¼ 7),
and subjects irradiated with 3 MED UVB (D, n ¼ 7). The mean ± SD
for each group is shown.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547
544 Photoprotection following repeated UVB exposures, J. Narbutt et al.
radiation (SSR) daily for up to 30 days.23 Their immune
responses to DPCP were then assessed. We found that both
the PAR and the CHS were reduced. These effects were de-
pendent on the cumulative UV dose as the downregulation
became more apparent as the number of days of exposure in-
creased. Therefore photoadaptation of this immune response
had not occurred, despite the development of some pigmenta-
tion in the skin.
In the present study, broadband UVB lamps were used
rather than SSR but, as the UVB waveband is the most biolo-
gically active and has similar immunological effects as SSR, we
assume that the changes induced by such lamps mimic closely
those following exposure to natural sunlight. We wished to
establish whether photoprotection against several effects of an
erythemal UVB dose was generated by pre-exposure of sub-
jects to suberythemal whole-body UVB radiation for 10 days.
We estimate that the dose of UVB given daily was equivalent
to being in the sun for about 35 min around midday on a
clear sky summer day in mid-Europe. The repeated irradia-
tions resulted in a small but significant degree of protection
against the erythemal effects of the subsequent high UVB
dose. This might be expected as the subjects were all of photo-
type II/III (the commonest skin type in Poland) with the
ability to tan to some extent in response to solar UV radiation.
The repeated low UVB doses resulted in the suppression of
both the PAR and CHS, indicating a lack of photoadaptation
and corroborating our previous study.23 In addition, these ex-
posures did not offer any protection against the downregulat-
ing effects of a subsequent erythemal UVB dose on PAR and
CHS. In one of the few previous reports to monitor CHS after
several exposures to low doses of UVB, Cooper et al.19 showed
that irradiation of a small body area with 0.7 MED UVB on
four consecutive days caused suppression of the CHS response
in 68% of subjects. Damian et al.25 demonstrated that the recall
response to nickel in nickel-allergic volunteers was suppressed
by exposure of a small area of the skin to suberythemal SSR
and that this effect was maintained even after 4 weeks of
repeated UV exposures. Long-term UVB phototherapy given to
patients with psoriasis was reported to impair subsequent sen-
sitization to DPCP.26
Following UV radiation of human skin, there is an upregu-
lation in the expression of various cytokines,10, 27–33 induced
by various mediators such as calcitonin gene-related peptide,
a-melanocyte stimulating hormone, platelet activating factor,
histamine and PGE2.3 The vast majority of the studies monit-
oring cytokine changes has been performed either in vivo
following a single erythemal dose of UVB, or in vitro in
keratinocyte cultures. As far as we are aware, the situation
in vivo following repeated suberythemal exposures has not been
examined previously. We found that the mRNAs of the four
selected cytokines, IL-1b, IL-6, IL-10 and TNF-a, were
increased as a result of the 10 daily suberythemal UVB expos-
ures. However, the rise for each cytokine was significantly less
than that following the single erythemal UVB exposure, indi-
cating that the extent of the change was not dependent on the
cumulative dose of UVB. It was also noted that a small but
significant photoprotection against the mRNA induction of all
four cytokines occurred in the group irradiated daily for
10 days before receiving the single erythemal UVB exposure
(group C2, Fig. 2a–d) compared with the group irradiated
with the single erythemal UVB dose (group D, Fig. 2a–d).
PGE2 is the predominant prostaglandin formed in the
human epidermis, and its production is increased after UV
exposure.34 Of the two major forms of COX, COX-2 is
induced by both UVB and UVA-II radiation.35 The COX
enzymes have important effects on apoptosis and proliferation
in the skin,36–38 and PGE2 has been shown to play a critical
role in UV-induced systemic immunosuppression in mice.13,16
It was demonstrated by Athar et al.39 that the extent of COX-2
protein expression in the epidermis of UVB-irradiated mice
strongly correlated with dose; after several weeks of exposure,
COX-2 was present in all the layers of the epidermis but most
strongly in the basal layer. We found a similar pattern of
expression of COX-2 in human skin following the 10 daily
suberythemal exposures to UVB. The UVB radiation increased
COX-1 and COX-2 protein expression even when no clinical
signs of erythema were present. When the numbers of
COX-1+ and COX-2+ cells in the epidermis of subjects irradi-
ated with the single erythemal UVB dose were compared with
the numbers in subjects preirradiated with 10 daily subery-
themal doses followed by the erythemal dose, no evidence for
the development of photoprotection was obtained.
One of the major absorbers of UV radiation in the skin is
DNA, and DNA photoproducts, such as dTT, are recognized as
critical molecular triggers for local and systemic suppression
of CHS.7,40 IL-12, given either before or after the UV expos-
ure, can prevent this effect.41–44 DNA damage is associated
with many biological changes in the skin other than immuno-
modulation, including erythema and sunburn cell forma-
tion,45,46 and it also enhances melanogenesis.47 In the present
study, all the UV protocols increased the number of dTT+
epidermal cells, most after the single erythemal UVB dose.
The number in the skin of subjects irradiated with the 10
daily doses of suberythemal UVB before administration of the
single erythemal dose was about 34% less than this figure.
Thus some photoprotection against DNA damage had been
generated by the repeated pre-exposures. As erythema corre-
lates with DNA damage, the small degree of photoprotection
observed against erythema may explain this result. However,
the protection was by no means absolute as, in the repeatedly
exposed subjects, the subsequent single erythemal UV dose in-
creased the number of dTT+ cells substantially, from about
363 to about 573 mm)2. In confirmation of this result,
Sheehan et al.48 showed that dTT steadily accumulated during
12 daily suberythemal SSR exposures of volunteers with skin
types II and IV, and that a tan may not be the major factor in
photoprotection against erythema.
In conclusion, we have shown that repeated exposures of
individuals to suberythemal UVB resulted in the generation of
slight erythema and pigmentation, the suppression of PAR and
CHS, and an increase in the mRNA expression of four cyto-
kines, in COX-1 and COX-2 proteins, and in dTT. The repeated
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp539–547
Photoprotection following repeated UVB exposures, J. Narbutt et al. 545
irradiations protected to a limited extent against the subse-
quent effects of an erythemal UVB dose on erythema, cytokine
expression and dTT formation, but not on the suppression of
PAR and CHS or the expression of the COX enzymes. Thus
sun protection measures are recommended, even in subjects
who have developed a tan.
Acknowledgments
This study was funded by the European Union project QTL-
CT-2001-0022IHA-UV and the Medical University of Lodz
projects 503-1019-1 and 502-11-352. The dTT monoclonal
antibody was kindly provided by Professor H. van Loveren.
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Photoprotection following repeated UVB exposures, J. Narbutt et al. 547
THERAPEUTICS DOI 10.1111/j .1365-2133.2006.07621.x
A pilot study of the safety and efficacy of picolinic acid gelin the treatment of acne vulgarisM.P. Heffernan, M.M. Nelson and M.J. Anadkat
Division of Dermatology, Washington University School of Medicine, St Louis, MO, U.S.A.
CorrespondenceMichael P. Heffernan, Division of Dermatology,
Wright State University School of Medicine,
Dayton, OH 45408, U.S.A.
E-mail: michael.heffernan@wright.edu
Accepted for publication25 July 2006
Key wordsacne vulgaris, picolinic acid
Conflicts of interestM.P.H. has been active in industry-sponsored
research with picolinic acid. He has conducted
reimbursed investigations for Novactyl, Inc. and
has received travel grants from Novactyl, Inc.
M.M.N. and M.J.A. have no financial conflicts of
interest to declare.
Summary
Background Cost limitations, adverse effects or lack of efficacy limit the use of cur-rent topical therapies in mild to moderate acne vulgaris.Objectives To determine the safety and efficacy of picolinic acid, a novel zinc fingertherapy, in the treatment of mild to moderate acne vulgaris.Methods Twenty subjects with mild to moderate acne vulgaris were treated at ourcentre during an open-label study with 10% picolinic acid gel (PCL-016) twicedaily to the face over 12 weeks.Results Fifteen patients completed the 12-week open-label study. A reduction of58Æ2% (P < 0Æ001) in mean total lesion count, 55Æ5% (P < 0Æ001) in meaninflammatory lesion count and 59Æ7% (P < 0Æ005) in noninflammatory lesioncount was seen in this population. No serious adverse events or clinically signifi-cant changes in laboratory values were noted.Conclusions Results from this study suggest that 10% picolinic acid gel appliedtwice daily may be safe and effective in the treatment of mild to moderate acnevulgaris.
Acne vulgaris is a widely prevalent condition that affects
nearly 80% of the U.S. population aged 11–30 years, and
most of the population at some point in their life.1–3
Patients with this condition are most bothered by their
appearance.4,5 Affected patients are prone to embarrassment,
social withdrawal, depression, anxiety, anger, scorn and stig-
matization.6–10 Higher unemployment rates have also been
demonstrated in adults with acne vulgaris compared with
those without acne.11 Patients with acne vulgaris have sim-
ilar levels of social, emotional and psychological impair-
ments as patients with chronic illnesses such as asthma,
arthritis and diabetes.12
The pilosebaceous unit is believed to be the primary site of
pathology in acne vulgaris. The distribution of acne favours
sites with the highest concentration of pilosebaceous units,
such as the face, chest or back.2,6,13,14 Four main processes
have been suggested as factors in the development of acne
vulgaris: sebaceous gland hyperplasia with increased sebum
production, altered follicular growth and differentiation, fol-
licular colonization with Propionibacterium acnes, and inflamma-
tion.13,15–18 The primary lesion of acne vulgaris, the
microcomedo, is formed as a result of sebaceous gland hyper-
plasia and alteration in follicular growth and differentiation.
Resultant noninflammatory (open and closed comedones) or
inflammatory (papule, pustule, nodule) lesions may later
evolve. Scarring often results in severe cases, but may occur in
instances of mild to moderate disease.19,20
Increasingly effective therapies have evolved in recent years
as the pathogenesis of acne vulgaris has become better under-
stood. Topical retinoids, antibiotics, benzoyl peroxide, azelaic
acid and salicylic acid are currently the mainstays of treatment
for mild to moderate acne vulgaris. Many patients fail to
improve with these agents due to cost, adverse effects leading
to noncompliance (i.e. irritation), or lack of therapeutic bene-
fit. The use of oral antibiotics or systemic retinoids increases
both the cost and the risks for adverse effects.
Picolinic acid is an intermediate metabolite of the amino
acid tryptophan. It appears to play a key role in zinc transport.
As a therapeutic agent, the molecule seems to work by per-
turbing zinc binding in zinc finger proteins (ZFPs). Picolinic
acid has antiviral and antibacterial properties in vitro and in vivo,
and also modifies the immune response alone and in conjunc-
tion with other cytokines. Picolinic acid was first evaluated in
the treatment of herpes labialis.
Here we report the results in 20 subjects with mild to mod-
erate acne vulgaris treated at our centre during an open-label,
phase I study of 10% picolinic acid gel (PCL-016).
Patients and methods
Patients
Subjects were eligible for study participation if they were at
least 18 years of age and had a clinical diagnosis of mild to
� 2006 The Authors
548 Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp548–552
moderate acne vulgaris of the face at baseline. Subjects were
required to have between 10 and 100 inflammatory lesions
(papules and pustules) and no more than two nodules. Sub-
jects were judged to be in generally good health as deter-
mined by the principal investigator. Subjects were excluded if
they had other active skin diseases that may interfere with
evaluation; had a beard or other facial hair that may interfere
with evaluation; had a history of an allergic reaction or signifi-
cant sensitivity to constituents of the study drug; had a poorly
controlled medical condition; were female and pregnant or
breastfeeding or considering becoming pregnant during the
study; had a history of clinically significant drug or alcohol
abuse in the last year; had participated in a clinical research
study within 30 days of enrolment; or were considered unre-
liable or unable to understand protocol directions in the esti-
mation of the investigator. Subjects with other forms of acne
such as acne rosacea, acne excoriee, chloracne, acne conglo-
bata, acne fulminans or drug-induced acne were excluded.
Subjects with moderate to severe nodulocystic acne deemed
by the investigator to require systemic treatment were also
excluded.
All subjects provided written informed consent before
inclusion into the study. The study protocol was approved by
the Institutional Review Board of the Washington University
School of Medicine.
Methods
All subjects received open-label 10% picolinic acid (PCL-016)
gel (NV-02; Novactyl, Inc., St Louis, MO, U.S.A.) to be
applied in a thin layer to affected areas of the face twice daily
for 12 weeks. Picolinic acid was stored at room temperature
at the study site. Subjects stored study medication at home at
room temperature after it was dispensed to them.
Subjects were not permitted to use any treatments for acne
vulgaris during the study. Subjects discontinued topical astrin-
gents for 1 day and other topical medications, such as antibi-
otics, antiseptics, corticosteroids and retinoids, for at least
2 weeks. Subjects discontinued systemic corticosteroids and
oral antibiotics for at least 4 weeks. Systemic retinoids were
discontinued for at least 6 months. All other systemic therapy
for acne vulgaris required discontinuation for at least
3 months. Women using oral contraception were required to
be taking the same contraceptive for at least 3 months prior to
study entry and to agree to continue this therapy until after
completion of the study.
Clinical and laboratory assessments were performed at
weeks 0, 4, 8 and 12 after the start of therapy. Clinical assess-
ments included physical examination and an acne lesion
count. A Physician Global Improvement score was also deter-
mined at the final visit. Laboratory assessments included a full
blood count, serum chemistry, urine pregnancy test (if applic-
able) and plasma picolinic acid level. Concomitant medications
and drug compliance were reviewed at each visit. All adverse
events were assessed and recorded including date and time of
onset, description, severity, time course, duration, outcome
and relationship to study drug. An additional visit was con-
ducted at week 1 following the initiation of therapy for
review of concomitant medications, adverse event query and
laboratory assessments.
Photographs of subjects who gave written informed consent
were taken at weeks 0, 4, 8 and 12. Photographs were taken
with a Nikon N80 camera. Photographs were performed to
document efficacy and were not used for statistical analysis.
The primary efficacy endpoints included improvement in
the total inflammatory lesion count, total lesion count and
global severity score at week 12 compared with baseline. Sec-
ondary efficacy endpoints included improvement in the total
noninflammatory lesion count at week 12 compared with
baseline. Safety variables evaluated included the incidence of
adverse events, abnormalities in routing laboratory monitor-
ing, and comparisons of pre- and post-treatment plasma picol-
inic acid levels. Efficacy and safety outcomes were designed to
direct planning for larger, blinded phase II–III investigations.
Statistical analyses
P-values were obtained for both per protocol and intent-to-
treat analyses using a paired t-test. Analyses performed using a
single-sample t-test yielded identical values.
Results
Baseline characteristics
Twenty subjects who met all of the inclusion criteria and none
of the exclusion criteria were enrolled in this study. Fifteen
patients completed the 12-week open-label study per protocol.
Four patients were lost to follow-up and one patient withdrew
due to a localized adverse event (discussed later). Table 1
summarizes the baseline characteristics for the participants.
Efficacy
All 15 patients who completed the study demonstrated reduc-
tion in total lesion count and noninflammatory lesion count.
Fourteen of these patients (93Æ3%) had a reduction in inflam-
matory lesion count. A reduction of 58Æ2% (from 85Æ33 to
Table 1 Baseline characteristics
Male 5Female 15
Age range (years) 20–48 (mean 29Æ6)White 12
African-American 6Hispanic 1
Asian 1Total baseline lesions 14–172 (mean 78Æ15)
Total baseline inflammatory lesions 12–58 (mean 27Æ45)Total baseline noninflammatory lesions 2–148 (mean 50Æ7)
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp548–552
Picolinic acid gel for acne vulgaris, M.P. Heffernan et al. 549
35Æ67; P ¼ 0Æ0001) in mean total lesion count, 55Æ5% (from
30Æ40 to 13Æ33; P ¼ 0Æ0002) in mean inflammatory lesion
count and 59Æ7% (from 54Æ93 to 22Æ33; P ¼ 0Æ0007) in non-
inflammatory lesion count was seen using per protocol analy-
sis (Fig. 1).
Similar findings were demonstrated using intent-to-treat an-
alysis for all 20 subjects enrolled into the study. A reduction
of 50Æ6% (from 78Æ15 to 38Æ60; P ¼ 0Æ0001) in mean total
lesion count, 47Æ2% (from 27Æ45 to 14Æ30; P ¼ 0Æ0001) in
mean inflammatory lesion count and 52Æ4% (from 50Æ70 to
24Æ30; P ¼ 0Æ0012) in mean noninflammatory count was seen
using intent-to-treat analysis.
A Physician Global Improvement score was determined for
each of the 15 patients completing the study at the final visit.
Seven (47%) of these patients were judged to be almost clear
(90–99% clearance) or to have marked improvement (75–
89% clearance). Three patients (20%) had moderate (50–74%
clearance) or minimal (25–49% clearance) improvement. Five
patients (33%) were judged to have no change (0–24% clear-
ance) from baseline.
Safety
Nineteen of the 20 patients enrolled were exposed to study
drug for at least 1 week, 17 for at least 4 weeks, and 15 for
all 12 weeks of the study. There were no deaths or serious
adverse events noted during this study. There were no clinic-
ally significant changes in any laboratory values (full blood
count and serum chemistry) during the study. No patients
became pregnant during the study.
One of the 20 subjects enrolled (patient 2) experienced a
drug-related adverse event leading to withdrawal from the
study. This patient described ‘burning’ after application of
study drug lasting 4 h that was ‘too much’. This was classified
by the investigator to be mild and probably related to study
drug. There were no sequelae from this event. Two other
patients described adverse events determined as probably rela-
ted to study drug. Patient 15 noted burning after application
lasting up to 1 h that resolved after the first week of therapy.
Patient 6 described a sensation of ‘facial tingling’ on the first
day of study drug application only. Table 2 summarizes each
of the documented adverse events from this study.
Plasma measurements of picolinic acid were done using a
validated assay developed by ABC Laboratories (now known as
ABC Pharma Services, Columbia, MO, U.S.A.) for Novactyl,
Inc. There were no stipulations regarding timing of blood
draws relative to last dosing. Samples were collected in vacu-
tainers containing powdered heparin. After gentle mixing, the
samples were kept on wet ice and centrifuged within 15 min
of collection. Resulting plasma was aliquoted and kept at
)20 �C until analysis. Plasma levels of picolinic acid varied
widely between patients and within patients during the study
(Table 3). The maximum level detected was 113 ng mL)1. No
correlation between plasma picolinic acid levels and the occur-
rence of adverse events was evident.
Discussion
This study was designed to assess the clinical efficacy and safety
of 10% picolinic acid gel, a novel immunomodulatory therapy
targeting ZFPs, in the treatment of mild to moderate acne
vulgaris. Results from this open-label study show picolinic acid
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Week
Les
ion
Co
un
t
Total Lesions 85.33 83.73 59.40 47.40 35.67Inflammatory Lesions 30.40 30.00 20.27 15.20 13.33Noninflammatory Lesions 54.93 53.73 39.13 32.20 22.33
0 1 4 8 12
Fig 1. Mean lesion counts using per protocol
analysis.
Table 2 Adverse events
Patient Adverse event Week
Relation
to studydrug
2 Burning at application site 1 Probable
6 Tingling at application site 1 Probable15 Burning at application site 1 Probable
12 Sinusitis 5 Possible15 Allergic rhinitis 12 Possible
9 Vaginal yeast infection 8 Unlikely11 Upper respiratory viral infection 9 Unlikely
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp548–552
550 Picolinic acid gel for acne vulgaris, M.P. Heffernan et al.
to be highly efficacious with 100% of the subjects who com-
pleted the study per protocol achieving a reduction in total and
noninflammatory lesion count and 93Æ3% of subjects achieving
a reduction in inflammatory lesion count. A clinically signifi-
cant reduction in mean total lesion count, mean inflammatory
lesion count and mean noninflammatory lesion count was seen
using both intent-to-treat and per protocol analysis. In add-
ition, nearly half of the patients (47%) who completed the
study were almost clear or had marked improvement at the
final visit using the Physician Global Improvement score.
Picolinic acid exists in vivo as a metabolite of the essential
amino acid tryptophan. It is produced in approximately 25–
50-mg quantities daily, assuming normal dietary intake. Picol-
inic acid chelates transition metal ions (i.e. Zn2+) and is
involved in the absorption and transport of transition metal
ions.
Picolinic acid has been demonstrated to possess immune-
modulating properties. Zinc binding within ZFPs is perturbed
in the presence of picolinic acid. This leads to an alteration in
chemokine expression independently and in the presence of
other chemokines such as interferon-c.21,22 Picolinic acid has
also been shown to have antiviral properties against herpes
simplex virus type 1 and type 2. ZFPs are involved in viral
replication, viral packaging and normal cell homeostatic func-
tions. An Investigational New Drug application has recently
been submitted by the manufacturer for picolinic acid cream
in the treatment of herpes labialis.
A phase I cumulative irritant study was recently completed by
Novactyl, Inc. assessing 10% picolinic acid cream in healthy vol-
unteers. Patients applied 200-mg quantities daily under a patch
for 21 consecutive days. The result of this irritant study was that
10% PCL-016 cream was clinically comparable with BlistexTM
(Blistex Inc., Oak Brook, IL, U.S.A.).
There was only one adverse effect, burning at the applica-
tion site, which led to patient discontinuation from this study.
This was judged to be mild in intensity and probably related
to study drug by the investigator. Another patient experienced
burning after application of study drug that resolved within
1 week of the study. One patient described facial tingling pre-
sent only on day 1 of study drug application. The remaining
adverse effects discussed earlier were mild and judged as poss-
ibly related or unlikely to be related to study drug. There were
no deaths or serious adverse events noted in this study. There
were no significant laboratory abnormalities during the study.
It is estimated by the manufacturer that 10–20 mg of PCL-
016 is delivered to the skin with each application of 10%
picolinic acid gel. There was both intrapatient and interpatient
variability in plasma levels of picolinic acid during the study.
The maximum plasma level of picolinic acid detected was
113 ng mL)1. No correlation between plasma picolinic acid
levels and the occurrence of adverse events was evident. The
minimum plasma picolinic acid level achievable before adverse
effects are expected to occur, if a relation exists, remains to be
elucidated.
In conclusion, the results of this study suggest that 10% pi-
colinic acid gel applied twice daily may be safe and effective
in the treatment of mild to moderate acne vulgaris. This is the
first reported use of NV-02 in humans, and the first reported
use of a zinc finger drug in the treatment of acne vulgaris.
Future randomized, double-blind, multicentre trials will be
necessary to confirm and better define these findings.
References
1 Kraning KK, Odland GF, eds. Prevalence, morbidity, and cost ofdermatological diseases. J Invest Dermatol 1979; 73:395–513.
2 Leyden JJ. New understandings of the pathogenesis of acne. J AmAcad Dermatol 1995; 32:S15–25.
3 Cunliffe WJ, Gould DJ. Prevalence of facial acne vulgaris in lateadolescence and in adults. BMJ 1979; i:1109–10.
4 Jowett S, Ryan T. Skin disease and handicap: analysis of the impactof skin conditions. Soc Sci Med 1985; 20:425–9.
5 Shuster S, Fisher GH, Harris E, Binnell D. The effect of skin diseaseon self-image. Br J Dermatol 1978; 99 (Suppl. 16):18–19.
6 Plewig G, Kligman AM. Acne and Rosacea, 3rd edn. New York: Sprin-ger-Verlag, 2000.
7 Kellet SC, Gawkrodger DJ. The psychological and emotional impactof acne and the effect of treatment with isotretinoin. Br J Dermatol
1999; 140:273–82.8 Koo J. The psychosocial impact of acne: patients’ perceptions. J Am
Acad Dermatol 1995; 32:S25–30.9 Wu SF, Kinder BN, Trunnell N, Fulton JE. Role of anxiety and
anger in acne patients: a relationship with the severity of the dis-order. J Am Acad Dermatol 1988; 18:325–33.
10 Aktan S, Ozmen E, Sanli B. Anxiety, depression, and nature of acnevulgaris in adolescents. Int J Dermatol 2000; 39:354–7.
11 Cunliffe WJ. Acne and unemployment. Br J Dermatol 1986; 115:386(Letter).
Table 3 Picolinic acid drug levels (ng mL)1)
Patient Week 0 Week 1 Week 4 Week 8 Week 12
1 ND 13Æ6 17Æ6 11Æ3 BQL
2 BQL BQL3 ND 17Æ9 54Æ5 BQL 47Æ04 BQL 52Æ3 90Æ5 BQL BQL5 ND BQL BQL BQL BQL
6 BQL 16Æ6 24Æ3 BQL BQL7 ND 72Æ6 113Æ0 44Æ9 BQL
8 BQL BQL BQL BQL BQL9 BQL 15Æ5 BQL BQL BQL
10 BQL 96Æ0 91Æ211 BQL 21Æ2 24Æ3 BQL BQL
12 BQL 17Æ3 31Æ1 56Æ9 BQL13 BQL
14 BQL BQL BQL BQL BQL15 BQL BQL BQL BQL BQL
16 BQL 31Æ917 BQL 22Æ3 BQL BQL 7Æ9618 BQL BQL BQL BQL BQL
19 BQL BQL BQL20 BQL 39Æ8 57Æ4 BQL 29Æ3
ND, none detected on assay; BQL, below quantitative limit(< 10 ng mL)1).
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Picolinic acid gel for acne vulgaris, M.P. Heffernan et al. 551
12 Mallon E, Newton JN, Klassen A et al. The quality of life in acne: acomparison with general medical conditions using generic ques-
tionnaires. Br J Dermatol 1999; 140:672–6.13 Gollnick HPM, Zouboulis CC, Akamatsu H et al. Pathogenesis and
pathogenesis-related treatment of acne. J Dermatol 1991; 18:489–99.14 Cunliffe WJ, Gollnick H. Acne: Diagnosis and Management. London: Mar-
tin Dunitz, 2001.15 Cunliffe WJ, Holland DB, Clark SM, Stables GI. Comedogenesis:
some new aetiological, clinical and therapeutic strategies. Br JDermatol 2000; 142:1084–91.
16 Cunliffe WJ, Simpson NB. Disorders of the sebaceous gland. In:
Textbook of Dermatology (Champion RH, Burton JL, Burns DA,Breathnach SM, eds), 6th edn. Oxford: Blackwell Science, 1998;
1927–84.17 Burton JL, Shuster S. The relationship between seborrhoea and acne
vulgaris. Br J Dermatol 1971; 84:600–1.
18 Leyden JJ, McGinley KJ, Mills OH, Kligman AM. Propionibacteriumlevels in patients with and without acne. J Invest Dermatol 1975;
65:382–4.19 Webster GF. Inflammation in acne vulgaris. J Am Acad Dermatol
1995; 33:247–53.20 Layton AM, Henderson CA, Cunliffe WJ. A clinical evaluation of
acne scarring and its incidence. Clin Exp Dermatol 1994; 19:303–8.21 Bosco MC, Rapisarda A, Massazza S et al. The tryptophan catabolite
picolinic acid selectively induces the chemokines macrophageinflammatory protein-1 alpha and -1 beta in macrophages. J Immunol
2000; 164:3283–91.
22 Melillo G, Cox GW, Biragyn A et al. Regulation of nitric-oxidesynthase mRNA expression by interferon-gamma and picolinic
acid. J Biol Chem 1994; 269:8128–33.
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552 Picolinic acid gel for acne vulgaris, M.P. Heffernan et al.
THERAPEUTICS DOI 10.1111/j .1365-2133.2006.07647.x
Treatment of refractory erosive oral lichen planus withextracorporeal photochemotherapy: 12 casesA.D. Guyot, D. Farhi,* S. Ingen-Housz-Oro,* A. Bussel,� N. Parquet,� C. Rabian,� H. Bachelez* and C. Frances
Service de Dermatologie-Allergologie, AP-HP, Hopital Tenon, Universite Paris VI, 4 rue de la Chine, 75020 Paris, France
*Service de Dermatologie 1, �Service d’Hemapherese Therapeutique and �Service d’Immunologie Biologique, AP-HP, Hopital Saint Louis, Universite Paris VII,
Paris, France
CorrespondenceCamille Frances.
E-mail: camille.frances@tnn.aphp.fr
Accepted for publication27 August 2006
Key wordsextracorporeal photochemotherapy, oral erosive
lichen planus
Conflicts of interestNone declared.
Summary
Background Case reports have suggested that extracorporeal photochemotherapy(ECP) might be beneficial for the treatment of erosive oral lichen planus (OLP)recalcitrant to conventional immunosuppressive therapies.Objectives To evaluate over a long-term period the clinical efficacy and toxicity ofECP in a series of patients with refractory OLP, and to monitor peripheral bloodlymphocyte subset counts under treatment.Methods Twelve patients with refractory OLP underwent a standardized protocol ofECP. Sessions were performed twice weekly for 3 weeks, and then the treatmentschedule was adapted according to clinical benefit. The disease severity was eval-uated monthly on a clinical basis. Complete remission was defined as the absenceof any erosion and partial remission as a decrease of at least 50% of erosion sur-face. Blood cell counts with CD4+ and CD8+ lymphocyte subsets were evaluatedevery 3 months.Results All patients showed a decrease of the erosive surface; nine (75%) achieveda complete remission and three (25%) a partial remission. Seven of the eightpatients followed for more than 3 years had recurrences of erosions when ECPsessions became less frequent or were stopped. After resumption of an initiallyaccelerated regimen of ECP, all again showed partial or complete remission.Blood lymphocyte counts decreased during treatment, without statistically signifi-cant changes in CD4+/CD8+ ratio, and increased during relapse.Conclusions ECP is an effective alternative therapy in erosive OLP showing resistanceto classical treatments. The decrease in blood lymphocyte counts appears to paral-lel the clinical improvement under treatment.
Erosive oral lichen planus (OLP) is a chronic, inflammatory
disease which may be very painful with a great impact on
quality of life.1 Spontaneous remissions are rare, observed in
2Æ8–6Æ5% of cases.2–4 The first-line therapy in OLP is high-
potency topical corticosteroids.4 For patients refractory to this
treatment, topical ciclosporin or tacrolimus may represent a
valuable second-line therapy.4,5 Long-term systemic steroids
(> 0Æ5 mg kg)1 daily) are usually effective but are associated
with a well-known spectrum of side-effects and dramatic
relapses following steroid withdrawal. Local or systemic retin-
oids are usually poorly tolerated.6,7 Systemic immunosuppres-
sive agents such as azathioprine or methotrexate are frequently
unhelpful.6 Given the dramatic improvement of OLP lesions
observed in a patient treated with extracorporeal photochemo-
therapy (ECP) for an associated cutaneous T-cell lymphoma,
and preliminary short-term results obtained in a limited series
of patients,8 we initiated a long-term follow-up study to
investigate the clinical efficacy and toxicity of ECP in 12
patients with refractory erosive OLP. We also attempted to
investigate the correlations between clinical variables and the
course of blood lymphocyte counts.
Patients and methods
Twelve patients entered the study. All suffered from histo-
logically proven, erosive OLP with an erosive surface
> 1 cm2. They were 10 women and two men (Table 1).
Their mean age at the onset of ECP was 60Æ6 years (range 40–
74). Mean duration of the disease was 7Æ5 years (range 1–
15). Three had a history of autoimmune disorders (pernicious
anaemia, type I diabetes mellitus, and primary biliary cirrhosis
with Gougerot–Sjogren syndrome, respectively). None had
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp553–556 553
chronic hepatitis C. All were refractory to local corticosteroids.
Other ineffective treatments used prior to ECP are listed in
Table 1. Erosive lesions mostly involved the tongue, cheeks
and lips. Amalgam fillings were not replaced, as the four
patients with only oral involvement had diffuse erosive OLP,
and only one or two amalgam fillings. No topographic rela-
tionship could be established between erosive OLP and amal-
gam fillings. Others had widespread disease with genital
involvement. Indeed, vulvar erosions were observed in eight
cases, and cutaneous lesions in four.
ECP was performed twice a week for the initial 3 weeks,
and then the frequency of sessions was progressively reduced
according to clinical evolution, to once every 4 months.
Finally, treatment was stopped when complete, stable remis-
sion was reached for at least 6 months. All systemic treatments
were withdrawn or were stable at least 6 months before start-
ing ECP.
At each session, mononuclear cells were extracted from two
blood masses (80 mL kg)1) using a blood cell separator
(Spectra; Gambro BCT, Denver, CO, U.S.A.). Soluble 8-meth-
oxypsoralen was added ex vivo to the cytapheresis product
(200 ng mL)1). Cells were then irradiated with ultraviolet
(UV) A at 3 J cm)2 with an UV irradiator (Vilbert-Lourmat,
Marne-la-Vallee, France), and reinfused to the patients.
Clinical follow up was performed monthly for 6 months,
then every 3 months, by the same investigator. The erosive
surface areas were estimated by measurement of the diameter
of the largest erosions. Clinical efficacy was evaluated on the
basis of the reduction in erosive surface: partial remission was
defined as a 50% or more decrease of the whole erosive sur-
face, and complete remission as a complete healing of all
lesions. Other clinical disease factors, such as erythema or
white asymptomatic lesions, were not taken into consideration.
Blood cell counts and immunophenotyping analysis of lym-
phocyte subsets were performed at each ECP session. Absolute
CD4+ and CD8+ cells were quantified in fresh blood samples
by quadruple fluorescence with beads, using a FACSCalibur
analyser (Becton Dickinson, Franklin Lakes, NJ, U.S.A.). Varia-
tions observed in absolute lymphocyte count, CD4+ and
CD8+ populations and CD4+/CD8+ ratio were statistically
analysed using Student’s t-test.
Results
Complete remission (Fig. 1) was achieved in nine patients
(75%), after a mean period from ECP onset of 11Æ5 months
(range 1–36) and a mean number of 21 sessions (range 6–
47). The other three patients achieved partial remission after a
mean period of 2Æ6 months (range 1–5) and a mean number
of 11 sessions (range 6–16).
Table 1 Description of patients with erosive oral lichen planus
Patient Sex Age (years) Duration of LP (years) Genital/skin LP Previous systemic treatments Previous topical treatments
1 F 58 6 +/) CT –
2 F 60 14 +/+ CT, acitretin, AZA –3 F 47 3 +/+ – –
4 F 54 1 +/) – Ciclosporin, tretinoin5 F 74 12 +/+ CT, AZA Ciclosporin
6 F 62 15 )/) CT –7 F 40 2 )/) – Ciclosporin
8 F 60 5 +/+ – Ciclosporin9 F 68 7 +/) Tretinoin
10 F 70 5 +/) Tretinoin11 M 60 ? )/) CT Ciclosporin
12 M 74 5 )/) CT Tretinoin
LP, lichen planus; CT, corticosteroid; AZA, azathioprine.
(b)
(a)
Fig 1. (a) Erosive lichen planus of the lip before extracorporeal
photochemotherapy. (b) Complete remission with disappearance of
erosions.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp553–556
554 Treatment of refractory erosive OLP with ECP, A.D. Guyot et al.
One patient did not have recurrence of oral erosions 4 years
after ECP withdrawal. The 11 other patients relapsed: three
relapsed during a session schedule of one per month or fewer,
and eight after ECP was stopped, at a mean of 8Æ3 months
(range 5–10) after the last session.
For eight patients who relapsed, an initial accelerated regi-
men of ECP was used again, with two sessions a week, and
treatment was then progressively reduced. Clinical improve-
ment was observed in all patients (Table 2). Five had com-
plete remission after 3–7 months of treatment (mean number
of sessions: 12Æ2). Three had only partial remission after 3, 8
and 12 months of treatment (mean number of sessions: 18).
Evolution of vulvar erosions ran in parallel with that of oral
ulcerations with earlier healing. The tolerance to the treatment
was excellent. No patient presented transient hypotension,
low-grade pyrexia or an infectious complication of induced
lymphopenia. ECP had to be discontinued in one patient
because of difficult venous access.
The mean blood lymphocyte count was 1Æ713 · 109 L)1
before the onset of ECP. It progressively decreased during
treatment to reach 0Æ982 · 109 L)1 before the last session of
ECP, when clinical response was obtained. This decrease was
statistically significant (P < 0Æ01). At the time of relapse, the
mean lymphocyte count had increased to 1Æ593 · 109 L)1
(P < 0Æ05). The decrease of lymphocyte subsets at the time of
remission was predominant for CD4+ T lymphocytes (51%,
from 0Æ750 to 0Æ368 · 109 L)1, P < 0Æ01) as well as CD8+
T lymphocytes (42%, from 0Æ499 to 0Æ292 · 109 L)1,
P < 0Æ01). The variations affecting the CD4+/CD8+ ratio
were not statistically significant.
Discussion
Therapeutic management of chronic erosive lichen planus may
be problematic when current treatments are ineffective. Sup-
pression of all irritant factors is required. Removal of amalgam
dental fillings is usually recommended when oral lesions are
close to amalgam, especially in cases of a positive patch test
reaction to its constituents.9,10 In this series, no triggering or
causative factor was suggested by clinical data.
ECP has been proposed as an immunomodulatory strategy,
allowing clinical benefit in cutaneous T-cell lymphoma, mostly
Sezary syndrome, and in nonmalignant disorders such as car-
diac transplant rejection and graft-versus-host disease (GvHD).
Indeed, the interest in ECP for severe recalcitrant forms of
lichen planus has been raised in view of clinical benefits in
patients with GvHD, and of the clinical and physiopathological
similarities between lichen planus and lichenoid GvHD. Fur-
thermore, ECP appears relatively safe, without evidence for an
increased risk of opportunistic infections.11 However, ECP is
expensive and time consuming. Patients are immobilized dur-
ing half a day for each session. Adequate venous access is
required.
In the present, long-term follow-up study we confirm the
safety of ECP, as none of our patients showed any infectious
complication during study. We also show evidence for striking
efficacy, as all patients experienced a complete or partial
remission under treatment. However, the effect of ECP was
only palliative, and the remission was dependent on a persist-
ent treatment with spaced sessions. Only one patient showed
stable remission for several years after ECP discontinuation.
Others, who experienced recurrence of erosions after ECP
withdrawal, requested further treatment with ECP as they con-
sidered this to be the most effective and the most well-toler-
ated treatment. ECP was effective again on relapses. Similar
results were obtained in a recently reported case study per-
formed in four patients with erosive OLP.12
The clinical response was associated with a contraction of
peripheral blood T-lymphocyte subsets, while B-lymphocyte
and natural killer cell counts remained relatively stable. Even
more interestingly, a peripheral blood T-cell expansion was
observed prior to the onset of relapse, suggesting that homeo-
static proliferation in the T-cell compartment might be a key
event in the occurrence of relapse. However, these results
should be interpreted cautiously, as the number of patients
was insufficient to provide an accurate comparative analysis of
lymphocyte counts in patients with partial remission vs. those
with complete remission. Further studies are currently ongo-
ing, in order to determine more accurately the lymphocyte
phenotype affected by ECP in OLP.
The mechanisms underlying the efficacy of ECP in vivo are
not fully elucidated. In lichen planus, immunological reactions
mediated by T cells are probably directed against one or sev-
eral as yet unknown foreign or self-modified antigens.13 Both
activated CD4+ and CD8+ T lymphocytes are present within
lichen lesions. CD4+ cells may activate cytotoxic CD8+ cells
that cause epidermal cell damage. The predominant secretion
of interferon-c and tumour necrosis factor-a suggested a type
1 cytokine pattern.14,15
As in GvHD, apoptosis of circulating lymphocytes is not the
sole mechanism of action of ECP. In animal models of
contact hypersensitivity, these apoptotic cells may induce
Table 2 Response of oral erosions to extracorporealphotochemotherapy (ECP)
Patient
Response
to ECP
Durationof remission
(months) Relapse
Responseto ECP
resumption
1 CR 5 Yes CR2 CR 33 Yes CR
3 PR 10 Yes PR4 CR 30 Yes –
5 CR 38 Yes PR6 PR 6 Yes –
7 CR 53 No –8 CR 34 Yes PR
9 PR 18 Yes –10 CR 19 Yes CR
11 CR 15 Yes CR12 CR 2 Yes CR
CR, complete remission; PR, partial remission.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp553–556
Treatment of refractory erosive OLP with ECP, A.D. Guyot et al. 555
antigen-specific regulatory T cells.16 In GvHD treated with
ECP, monocytes, which appear to be resistant to apoptotic
effects induced by ECP, have an enhanced synthesis of immuno-
suppressive cytokines such as interleukin (IL)-10 and IL-1
receptor antagonist; they differentiated into immature CD83+
dendritic cells actively phagocytosing apoptotic cells.17 ECP
induces an increase in the plasmacytoid DC2 population with
subsequent increased production of Th2 cytokine factors.17,18
In conclusion, ECP may be effective as a salvage therapeutic
option for erosive lichen planus refractory to current treat-
ments. Further prospective studies are needed in order to
determine more accurately the risk/benefit ratio, and the
mechanisms underlying tolerance induction in this context.
References
1 Lundqvist EN, Wahlin YB, Bergdahl M, Bergdahl J. Psychologicalhealth in patients with genital and oral erosive lichen planus. J Eur
Acad Dermatol Venereol 2006; 20:661–6.2 Silverman S, Gorsky M, Lozada-Nur F. A prospective follow-up
study of 570 patients with oral lichen planus: persistence, remis-sion, and malignant association. Oral Surg Oral Med Oral Pathol 1985;
60:30–4.3 Silverman S, Gorsky M, Lozada-Nur F et al. A prospective study of
findings and management in 214 patients with oral lichen planus.
Oral Surg Oral Med Oral Pathol 1991; 72:665–70.4 Conrotto D, Carbone M, Carrozzo M et al. Ciclosporin vs. clobetasol
in the topical management of atrophic and erosive oral lichen pla-nus: a double blind, randomized controlled trial. Br J Dermatol
2006; 154:139–45.5 Byrd JA, Davis MDP, Bruce AJ et al. Response of oral lichen planus to
topical tacrolimus in 37 patients. Arch Dermatol 2004; 140:1508–12.6 Cribier B, Frances C, Chosidow O. Treatment of lichen planus: an
evidence-based medicine analysis of efficacy. Arch Dermatol 1998;134:1521–30.
7 Hersle K, Mobacken H, Sloberg K, Thilander H. Severe oral lichenplanus: treatment with an aromatic retinoid (etretinate). Br J Derma-
tol 1982; 106:77–80.8 Becherel PA, Bussel A, Chosidow O et al. Extracorporeal photo-
chemotherapy for chronic erosive lichen planus. Lancet 1998;351:805.
9 Laeijendecker R, Dekker SK, Burger PM et al. Oral lichen planusand allergy to dental amalgam restorations. Arch Dermatol 2004;
140:1434–8.10 Rogers RS III, Bruce AJ. Lichenoid contact dermatitis: is inorganic
mercury the culprit? Arch Dermatol 2004; 140:1524–5.
11 McKenna KE, Whittaker S, Rhodes LE et al. Evidence-based practiceof photopheresis 1987–2001: a report of a workshop of the British
Photodermatology Group and the U.K. Skin Lymphoma Group. Br JDermatol 2006; 154:7–20.
12 Kunte C, Erlenkeuser-Uebelhoer I, Michelsen S et al. Behandlungdes therapieresistenten erosiven, oralen Lichen ruber mit extra-
korporaler Photopherese (ECP). J Dtsch Dermatol Ges 2005; 3:889–94.
13 Kastelan M, Prpic Massari L, Gruber F et al. The role of perforin-mediated apoptosis in lichen planus lesions. Arch Dermatol Res 2004;
296:226–30.14 Sklavounou A, Chrysomali E, Scorilas A et al. TNF-alpha expression
and apoptosis regulating proteins in oral lichen planus: a compara-tive immunohistochemical evaluation. J Oral Pathol Med 2000;
29:370–5.15 Khan A, Farah CS, Savage NW et al. Th1 cytokines in oral lichen
planus. J Oral Pathol Med 2003; 32:77–83.16 Maeda A, Schwarz A, Kernebeck K et al. Intravenous infusion of
syngeneic apoptotic cells by photopheresis induces antigen-specificregulatory T cells. J Immunol 2005; 174:5968–76.
17 Fimiani M, Di Renzo M, Rubegni P. Mechanism of action of extra-corporeal photochemotherapy in chronic graft-versus-host disease.
Br J Dermatol 2004; 150:1055–60.18 Heshmati F. Mechanisms of action of extracorporeal photochemo-
therapy. Transfus Apheresis Sci 2003; 29:61–70.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp553–556
556 Treatment of refractory erosive OLP with ECP, A.D. Guyot et al.
CONCISE COMMUNICATION DOI 10.1111/j .1365-2133.2006.07705.x
Reporting of study design in titles and abstracts of articlespublished in clinically oriented dermatology journalsR. Ubriani, N. Smith* and K.A. Katz
Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, PA, U.S.A.
*Department of Medicine, Christiana Hospital, Newark, DE, U.S.A.
CorrespondenceKenneth Katz, 1439B Corccrun St NW,
Washington, DC 20009, U.S.A.
E-mail: Kenneth.Katz@post.harvard.edu
Accepted for publication21 September 2006
Key wordscritical appraisal, epidemiology, evidence-based
medicine, medical literature, study design
Conflicts of interestNone declared.
Summary
Background Dermatologists may have difficulty in identifying the types of study designused in published articles, hindering their ability to appraise the literature critically.Objectives To assess the frequency with which titles or abstracts of articles pub-lished in clinically oriented dermatology journals reported the type of studydesign using standard key words, including ‘randomized control trial’, ‘nonrand-omized control trial’, ‘double-blind’, ‘placebo control’, ‘crossover trial’, ‘before–after trial’, ‘gold standard’, ‘blinded or masked comparison’, ‘cohort’, ‘inceptioncohort’, ‘validation cohort’, ‘validation sample’, ‘survey’, ‘case series’, ‘cost-effectiveness analysis’, ‘cost-benefit analysis’, ‘cost-utility analysis’, ‘cross-sectional study’ and ‘case–control’.Methods A cross-sectional study analysed articles published between December2004 and November 2005 in the ‘Epidemiology and Health Services Research’and ‘Therapeutics’ sections of the British Journal of Dermatology (BJD), in the ‘Studies’section of the Archives of Dermatology (Arch Dermatol) and in the ‘Reports’ section ofthe Journal of the American Academy of Dermatology (JAAD).Results In the BJD, 15 of 37 articles (40Æ5%, 95% confidence interval, CI 24Æ8–57Æ9%) included at least one standard key word in the title or abstract, comparedwith 43 of 87 articles (49Æ4%, 95% CI 38Æ5–60Æ4%) in the Arch Dermatol and 19of 93 articles (20Æ4%, 95% CI 12Æ8–30Æ1%) in the JAAD (P < 0Æ001).Conclusions Most articles in the three journals did not report the study design usedin the title or abstract. A consistent and clear indication of the design used instudies may better enable editors, reviewers and readers to assess critically articlespublished in clinically oriented dermatology journals.
Evidence-based medicine has increasingly attracted the interest
of dermatologists.1–8 One critical step in appraising evidence
is identifying the type of study presented in a report.2,9–11
Doing so allows the reader to determine where the report falls
in a hierarchy of evidence that gives more weight to random-
ized controlled trials, for example, than to single case
reports.3,12 It also allows the reader to assess how inherent
strengths and weaknesses of the study’s design might affect
the results and, ultimately, the conclusions presented in the
report.13,14
In our experience, identifying the type of study reported in
a paper is a major challenge for dermatologists in practice and
in training. We have also noticed that some journal
abstracts—the most frequently read and accessed parts of jour-
nal articles15—either do not report the type of study design
used, or report a design using nonstandard terminology. The
purpose of this study was to investigate the frequency with
which articles published in three clinically oriented dermatol-
ogy journals report study design using standard terminology.
Materials and methods
We performed a cross-sectional study of articles published
between December 2004 and November 2005 in the ‘Epidemi-
ology and Health Services Research’ and ‘Therapeutics’ sections
of the British Journal of Dermatology (BJD), the ‘Studies’ section of
the Archives of Dermatology (Arch Dermatol) and the ‘Reports’ section
of the Journal of the American Academy of Dermatology (JAAD). K.A.K.
(a board-certified dermatologist with training in epidemiology
and statistics) and R.U. (a resident in dermatology) independ-
ently reviewed the abstract of each article and determined whe-
ther it contained at least one of the 19 study design-related key
words (Table 1) that were proposed in a 1987 report in the
Annals of Internal Medicine16 and are currently recommended for use
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp557–559 557
in the ‘Design’ section of a structured abstract by the Arch
Dermatol.17 The BJD and JAAD do not publish recommended
design-related key words in their author instructions. A key
word present anywhere in the title or abstract of an article was
considered acceptable. Disagreements were resolved by consen-
sus. We also performed an electronic search of all of the titles
and abstracts of the articles for one term—‘systematic review’—
not included in the list of recommended key words. A v2 test
comparing proportions of articles that included a key word in
the three journals (not including ‘systematic review’) was per-
formed using Stata 9.0 (Stata Corporation, College Station, TX,
U.S.A.).
Results
In the BJD, 15 of 37 articles (40Æ5%, 95% confidence interval,
CI 24Æ8–57Æ9%) included at least one standard key word in
the title or abstract, compared with 43 of 87 articles (49Æ4%,
95% CI 38Æ5–60Æ4%) in the Arch Dermatol and 19 of 93
abstracts (20Æ4%, 95% CI 12Æ8–30Æ1%) in the JAAD
(P < 0Æ001). Table 1 shows the frequency of use of each
standard key word in each journal. Of the 19 key words, 12
(63%), 10 (53%) and 13 (68%) were not used in any articles
in the BJD, the Arch Dermatol and the JAAD, respectively. The
term ‘systematic review’ appeared in two of 37 articles (5%)
from the BJD. This term did not appear in any of the articles
from the Arch Dermatol or the JAAD.
Discussion
Among articles from the BJD, the Arch Dermatol and the JAAD
included in this study, fewer than half included a standard
study design-related key word in the title or abstract. Articles
published in the Arch Dermatol had the highest percentage
(49%) of key word inclusion, perhaps reflecting the fact that
it is the only journal among the three to include a list of key
Table 1 Frequency of use of study design-related key words in titles or abstracts of
articles published between December 2004and November 2005 in the British Journal of
Dermatology, the Archives of Dermatology and theJournal of the American Academy of Dermatology,
classified by type of study design17Key word
Frequency of use in article titles and abstracts
British Journal
of Dermatology(37 articles)
Archives of
Dermatology(87 articles)
Journal of theAmerican Academy
of Dermatology(93 articles)
Intervention studies
Randomized control trial 8 8 4Nonrandomized control trial 0 0 0
Double-blind 7 6 4Placebo control 5 4 1
Crossover trial 0 0 1Before–after trial 0 1 0
Studies of screening and diagnostic testsGold standard 0 0 0
Blinded or masked comparison 0 0 0Studies of prognosis
Cohort 0 9 5
Inception cohort 0 0 0Validation cohort 0 0 0
Validation sample 0 0 0Descriptions of clinical features of medical disorders
Survey 3 9 7Case series 1 6 0
Studies with a formal economic evaluationCost-effectiveness analysis 0 0 0
Cost-benefit analysis 0 0 0Cost-utility analysis 0 0 0
Other studiesCross-sectional study 1 3 0
Case–control 1 1 0
Totala 26 47 22
aThe total number of key words is greater than the number of articles containing a key
word because some articles contained more than one key word in the title and abstract.The table does not include articles with the term ‘systematic review’ in the title or
abstract. ‘Systematic review’ was not in the list of recommended key words.17 Two of 37
articles (5%) of articles from the British Journal of Dermatology included this term; none fromthe Archives of Dermatology or the Journal of the American Academy of Dermatology did.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp557–559
558 Study design reporting, R. Ubriani et al.
words16 in its ‘Instructions to Authors’.17 This finding also
parallels the results of a study of the quality of dermatology
abstracts that were published in 2000.18 In that study,
abstracts from the Arch Dermatol received higher quality scores
than those from the JAAD on all characteristics assessed,
including reporting of study design, and higher quality scores
than those from the BJD on seven of eight characteristics
assessed, including study design.18 The study also assessed the
use of study design-related key words in article abstracts (but
not in titles), although it was one of three factors included in
an overall assessment of study design; frequency of key word
use in each journal was not reported.18
There are several ways in which the frequency of reporting
of type of study design in journal abstracts could be increased.
Firstly, journals could raise awareness by emphasizing the
importance of reporting study design in published ‘Instruc-
tions to Authors,’ as the Arch Dermatol already does. Secondly,
journals could review and edit abstracts more strictly to ensure
that study design type is reported. JAMA adopted such an
approach in 1999,15 after preliminary results of a study dem-
onstrated deficiencies in JAMA abstracts.15,19 Within 1 year,
the quality of abstracts in JAMA had significantly improved.20
Thirdly, journals could require authors to select a type of
study design from a pull-down menu of standard key
words16,17 on an electronic submission website, just as article
type might be selected. This selection could then either be
automatically entered into an abstract or compared with the
study design reported in the submitted abstract. Finally, jour-
nals could introduce a separate element of the structured
abstract, called ‘Type of study design used’, with text limited
to standard key words.16,17 With any of these approaches,
providing definitions of key words (or links to websites that
include this information)16 would also be useful. These
approaches might also help eliminate the use of ambiguous
and confusing nonstandard words and phrases that sometimes
appear in descriptions of study design.
This study has limitations. Firstly, we examined only 12
issues from each of three major dermatology journals. Sec-
ondly, the ‘Methods’ sections of a manuscript might include
information about type of study design not found in the
abstract and, therefore, not be captured in this study. Thirdly,
we did not assess the accuracy of study design reporting.
However, given the importance of the abstract to readers and
browsers of the medical literature,15 lack of information about
study design in the title or abstract itself is still concerning.
Finally, although we did search for one design type—system-
atic review—that was not included in the list of recommen-
ded key words, the list of search terms is not exhaustive and
might not include all relevant study designs.16,17 Future lists
of recommended key words might be more effective if they
are more inclusive of research typically conducted by derma-
tologists.
Evidence-based medicine is increasingly important to der-
matologists. Improving the reporting of study design types in
dermatology journals might facilitate dermatologists’ ability to
make a critical appraisal of articles in the literature that are
relevant to care of their patients.
Acknowledgments
Dr. Katz’s fellowship was funded by a Kirschstein National
Research Service Award from the National Institutes of
Health.
References
1 Abeni D, Girardelli CR, Masini C et al. What proportion of derma-tological patients receive evidence-based treatment? Arch Dermatol
2001; 137:771–6.2 Barzilai DA, Freiman A, Dellavalle RP et al. Dermatoepidemiology.
J Am Acad Dermatol 2005; 52:559–73.3 Bigby M. Evidence-based medicine in a nutshell. A guide to find-
ing and using the best evidence in caring for patients. Arch Dermatol1998; 134:1609–18.
4 Bigby M, Williams H. Appraising systematic reviews and meta-analyses. Arch Dermatol 2003; 139:795–8.
5 Dellavalle RP, Stegner DL, Deas AM et al. Assessing evidence-baseddermatology and evidence-based internal medicine curricula in US
residency training programs: a national survey. Arch Dermatol 2003;139:369–72.
6 Straus SE, Sackett DL. Bringing evidence to the clinic. Arch Dermatol1998; 134:1519–20.
7 Williams HC. Applying trial evidence back to the patient. ArchDermatol 2003; 139:1195–200.
8 Williams HC, Strachan DP. The Challenge of Dermato-epidemiology. BocaRaton, FL: CRC Press, 1997.
9 Guyatt G, Rennie D (eds). American Medical Association. Users’ Guides tothe Medical Literature: a Manual for Evidence-Based Clinical Practice. Chicago,
IL: AMA Press, 2002.10 Sackett DL. Evidence-Based Medicine: How to Practice and Teach EBM, 2nd
edn. Edinburgh: Churchill Livingstone, 2000.11 Sackett DL, Rosenberg WM, Gray JA et al. Evidence based medicine:
what it is and what it isn’t. BMJ 1996; 312:71–2.12 Guyatt GH, Haynes RB, Jaeschke RZ et al. Users’ Guides to the
Medical Literature: XXV. Evidence-based medicine: principles forapplying the Users’ Guides to patient care. Evidence-Based Medi-
cine Working Group. JAMA 2000; 284:1290–6.13 Greenhalgh T. How to read a paper. Getting your bearings (deci-
ding what the paper is about). BMJ 1997; 315:243–6.
14 Hennekens CH, Buring JE, Mayrent SL. Epidemiology in Medicine, 1stedn. Boston, MA: Little, Brown, 1987.
15 Winker MA. The need for concrete improvement in abstractquality. JAMA 1999; 281:1129–30.
16 Ad Hoc Working Group for Critical Appraisal of the MedicalLiterature. A proposal for more informative abstracts of clinical
articles. Ann Intern Med 1987; 106:598–604.17 Archives of Dermatology (homepage on the Internet). Chicago, IL:
AMA, c2006. Instructions for Authors (about 20 screens). Availablefrom: http://archderm.ama-assn.org/misc/ifora.dtl (last accessed
22 February 2006).18 Dupuy A, Khosrotehrani K, Lebbe C et al. Quality of abstracts in 3
clinical dermatology journals. Arch Dermatol 2003; 139:589–93.19 Pitkin RM, Branagan MA, Burmeister LF. Accuracy of data in
abstracts of published research articles. JAMA 1999; 281:1110–11.20 Pitkin RM, Branagan MA, Burmeister LF. Effectiveness of a journal
intervention to improve abstract quality. JAMA 2000; 283:481.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp557–559
Study design reporting, R. Ubriani et al. 559
CASE REPORT DOI 10.1111/j .1365-2133.2006.07599.x
Solitary plaque mycosis fungoides on the penis respondingto topical imiquimod therapyL.Y.T Chiam and Y.C. Chan
National Skin Centre, 1 Mandalay Road, Singapore 308205
CorrespondenceYuin-Chew Chan.
E-mail: ycchan@nsc.gov.sg
Accepted for publication11 July 2006
Key wordsmycosis fungoides, penis, topical imiquimod
Conflicts of interestNone declared.
Summary
Mycosis fungoides (MF) presenting in the genitalia is rare. We report a case oflong-standing penile MF in a young man. Commonly used treatments for limitedplaque MF include topical corticosteroids and ultraviolet light therapy. There area few anecdotal reports on the use of topical imiquimod in MF. Our patientresponded well and remained in complete remission after treatment with topicalimiquimod.
Cutaneous T-cell lymphoma (CTCL) is a rare disease with an
incidence of approximately 0Æ36 per 100 000 person-years in
the United States.1 Mycosis fungoides (MF) is the most com-
mon variant of CTCL. The lesions of MF have a predilection
for nonexposed areas. There have been reports of unilesional
MF affecting the trunk, upper extremities, inguinal region,
foot and back.2
Case report
A healthy 32-year-old Bangladeshi man was referred to our
centre by his primary health care physician for a chronic pink
plaque on his penis. It had been present for 15 years, but in-
creased growth and pain over the past year had prompted him
to seek medical attention.
Physical examination revealed a 19 · 11 mm well-demarca-
ted psoriasiform scaly pink plaque on the left glans penis
(Fig. 1). There were no other skin lesions. His testes were
normal in size. He had no enlarged lymph nodes or hepato-
splenomegaly.
Histological examination revealed laminated hyperkeratosis
in the stratum corneum, with irregular epidermal hyperplasia
(Fig. 2). The basal keratinocytes had an infiltrate of atypical
lymphocytes. The nuclei were hyperchromatic with halos.
A Pautrier’s microabscess was seen. This was consistent with
MF.
His full blood count, liver and renal function tests and chest
radiograph were normal. Peripheral blood film for Sezary cells
was negative.
He was treated with clobetasol propionate ointment for
6 weeks with no improvement. Treatment was changed to Fig. 1 Psoriasiform plaque on the glans penis.
� 2006 The Authors
560 Journal compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp560–562
imiquimod 5% cream and he was instructed to apply this on
the plaque once every 2 days. Our patient experienced local
pain during the first week of treatment. He also developed
local skin erosion in the third month of therapy, necessitating
temporary withdrawal of the topical application for a few
days. After 4Æ5 months of topical imiquimod, he was clinically
in complete remission and remained so on follow-up
6 months later.
Discussion
The differential diagnoses of a chronic nonhealing plaque on
the glans penis include psoriasis, lichen sclerosus, Bowen dis-
ease, squamous cell carcinoma, extramammary Paget disease
and MF. Differentiating between MF and lichen sclerosus can
be difficult clinically and histologically, especially if no Pautri-
er’s microabscess is seen. In these cases, T-cell rearrangement
studies may be necessary to support the diagnosis of MF.
MF involving only the genitalia is rare. A single report of
MF involving the vulva has been described.3
Long-term prognosis and survival outcome of early MF is
excellent. MF patients with clinical stage IA disease have a
favourable long-term outcome in which life expectancy is
similar to matched control populations.4
Treatment of MF is indicated to reduce symptoms, improve
clinical appearance, prevent complications and prevent pro-
gression of disease. Treatment for MF includes topical and
systemic agents, which can be administered alone or in
combination. Various topical therapies for limited plaque MF
include steroids,5 psolaren plus ultraviolet (UV) A,6 narrow-
band UVB and nitrogen mustard. Systemic therapies include
interferon, retinoids, denileukin diftitox (licensed in the
U.S.A.) and chemotherapy.
Early MF can be treated with excellent outcomes with non-
aggressive topical treatments. Systemic chemotherapy such as
CHOP has not been shown to improve survival in these
patients.
Imiquimod is a topical immunomodulator. It is a potent sti-
mulator of T-helper 1 cytokines, including interferon (IFN)-a,
IFN-c, and interleukin-12. It increases natural killer-cell activity
and also enhances antigen presentation by Langerhans cells.7
It has been approved by the Food and Drug Administration for
the treatment of genital warts and actinic keratosis. There is
growing evidence pointing to many other potential therapeutic
benefits of this drug in cutaneous neoplastic lesions.8
There have been anecdotal reports in the literature demon-
strating clinical use of imiquimod for the treatment of MF.9 In
a recent preliminary open-label study, topical imiquimod 5%
cream was associated with a 50% clinical response rate in
stage IA–IIB MF.10
Application site reactions are commonly seen in patients
undergoing treatment with topical imiquimod. The localized
skin reactions are mild to moderate, and the most frequently
reported application site reactions include pruritis, erythema,
oedema, discharge, papular rash, pain and occasionally hypo-
pigmentation or hyperpigmentation. Adverse effects were well
tolerated and largely limited to lesions that cleared with treat-
ment.
Conclusion
This is a rare case of penile MF in a young man, which was
steroid-resistant but responded well to topical imiquimod.
References
1 Weinstock MA, Gardstein B. Twenty-year trends in the reportedincidence of mycosis fungoides and associated mortality. Am J Public
Health 1999; 89:1240–4.2 Vang R, Medeiros LJ, Malpica A et al. Non-Hodgkin’s lymphoma
involving the vulva. Int J Gynecol Pathol 2000; 19:236–42.3 Kim YH, Jensen RA, Watanabe GL et al. Clinical stage 1A (limited
patch and plaque) mycosis fungoides. A long-term outcome analy-sis. Arch Dermatol 1996; 132:1309–13.
4 Zackheim HS, Kashani-Sabet M, Amin S. Topical corticosteroids formycosis fungoides: experience in 79 patients. Arch Dermatol 1998;
134:949–54.5 Querfeld C, Rosen ST, Kuzel TM et al. Long term follow-up of
patients with early-stage cutaneous T-cell lymphoma who achievedcomplete remission with psoralen plus UV-A monotherapy. Arch
Dermatol 2005; 141:305–11.
Fig 2. Pautrier’s microabscess and hyperchromatic nuclei with halos
(haematoxylin and eosin; original magnification · 40).
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp560–562
MF on the penis and topical imiquimod therapy, L.Y.T Chiam and Y.C. Chan 561
6 Kim YH, Martinez G, Varghese A, Hoppe RT. Topical nitrogenmustard in the management of mycosis fungoides: update of the
Stanford experience. Arch Dermatol 2003; 139:165–73.7 Sauder DN. Immunomodulatory and pharmacological properties of
imiquimod and S-27609. J Am Acad Dermatol 2000; 43 (1 Pt 2):S6–11.8 Navi D, Huntley A. Imiquimod 5 percent cream and the treatment
of cutaneous malignancy. Dermatol Online J 2004; 10:4.
9 Suchin KR, Junkins-Hopkins JM, Rook AH. Treatment of stage 1Acutaneous T-cell lymphoma with topical application of the
immune response modifier imiquimod. Arch Dermatol 2002;138:1137–9.
10 Deeths MJ, Chapman JT, Dellavalle RP et al. Treatment of patch andplaque stage mycosis fungoides with imiquimod 5 percent cream.
J Am Acad Dermatol 2005; 52:275–80.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp560–562
562 MF on the penis and topical imiquimod therapy, L.Y.T Chiam and Y.C. Chan
CASE REPORT DOI 10.1111/j .1365-2133.2006.07605.x
Paraneoplastic pemphigus without an underlying neoplasmG.T. Park, J.H. Lee,* S.J. Yun,* S.C. Lee* and J.B. Lee*
Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710, Korea
*Department of Dermatology, Chonnam National University Medical School, 8-Hak-dong, Gwangju 501-757, Korea
CorrespondenceJee-Bum Lee.
E-mail: jbmlee@chonnam.ac.kr
Accepted for publication25 July 2006
Key wordsparaneoplastic pemphigus, underlying neoplasm
Conflicts of interestNone declared.
Summary
We describe a 52-year-old man with paraneoplastic pemphigus (PNP) withoutany evidence of an underlying neoplasm over an 8-year follow-up period. Hehad a chronic relapsing vesiculobullous eruption for approximately 7 years (fromApril 1998 to May 2005). Initially, scattered flaccid vesicles with crusts devel-oped on the face and trunk, which waxed and waned several times. Our patientwas diagnosed as having PNP based on immunopathological criteria for PNP, i.e.histopathological, immunoblotting and immunoprecipitation analyses. However,physical and laboratory examinations including serial blood tests with peripheralblood smear, whole-body positron emission tomography/computed tomographyand abdominal ultrasound were unable to detect any underlying neoplasm overan 8-year follow-up period.
Paraneoplastic pemphigus (PNP), which was first described by
Anhalt et al.1 in 1990, is an autoimmune blistering disease that
is characterized by the production of autoantibodies (aAbs)
mainly directed against the proteins of the plakin family.
Overlapping aAb specificity distributions have been reported
for PNP, pemphigus vulgaris (PV) and pemphigus foliaceus
(PF), suggesting a close relationship between the different
types of pemphigus.2–9 In addition, the diverse clinical mani-
festations of PNP and the lack of specificity of its histological
criteria imply that it is a pleomorphic disease entity.10,11 Inter-
estingly, despite the implication of various neoplasms in
almost all cases of PNP,1,10–16 only one case of PNP without
an underlying tumour has been reported.17 PNP was recently
reported to be caused by fludarabine, a synthetic nucleoside
analogue.18
We report a patient with PNP without any obvious underly-
ing tumour or illness as determined by physical and laboratory
examinations conducted over approximately 8 years of follow
up.
Case report
A 52-year-old man, who had been healthy prior to his first
visit to our hospital in April 1998, experienced scattered pru-
ritic vesicular eruptions and erosions on the face and trunk
(Fig. la). Six months before his first visit, he had noticed
newly developed skin eruptions of pruritic erythematous ma-
cules and tiny vesicles with crusts confined to the face, which
did not respond to conventional therapy for eczematoid
dermatitis at a private clinic. On his first visit to our hospital
he appeared healthy except for the skin lesions, which showed
scattered multiple vesiculobullae with crusts surrounded by
erythema on the face, upper chest and upper back, and ero-
sions on the lips. Mucosa of both conjuctiva and mouth (not
genital mucosa) were involved. He denied taking any medica-
tions and did not have any other disease, especially a neo-
plasm. Physical examination showed no organomegaly or
lymphadenopathy, and with the exception of an elevated IgE
level (407 IU mL)1, normal 0–100) all other laboratory tests
were negative or normal. These tests included full blood cell
count, peripheral blood smear, chest X-ray, electrolyte levels,
lactate dehydrogenase, urine analysis, prothrombin and partial
thromboplastin times, complement C3 and C4, liver enzymes,
blood urea nitrogen, creatinine, rheumatoid factor, lupus
erythematosus cell phenomenon and antinuclear antibody test.
Skin biopsy from a vesicle on the face revealed dyskeratotic
cells and basal vacuolization in the epidermis, an infiltrate
containing lymphohistiocytic cells and melanophages, and red
blood cell extravasation in the dermis (Fig. 2). Direct immu-
nofluorescence (IF) showed IgG deposits in epidermal inter-
cellular spaces (data not shown). Indirect IF using the
patient’s serum was performed with normal human foreskin
and rat bladder as substrates. IgG aAbs bound both the epithe-
lial cellular surface (ECS) and the basement membrane zone
(BMZ) of rat bladder (Fig. 3a). Similar findings were obtained
on human foreskin (data not shown). However, rat bladder
did not react with the serum of a patient with PV as a nega-
tive control (Fig. 3b). The titres of circulating aAbs were
examined by indirect IF using normal human foreskin as a
substrate. These titres were increased or decreased according
to disease activity from 1 : 1280 to 1 : 20. We also per-
formed immunoblotting using normal human foreskin epider-
mal extract. Regardless of disease severity, both 210- and
190-kDa bands corresponding to envoplakin and periplakin
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp563–566 563
were always detected on the immunoblot at a titre of 1 : 20.
Moreover, these were consistent with those demonstrated by
PNP patient serum (Fig. 4a, lanes 1 and 2). In contrast, bul-
lous pemphigoid (BP) and PV patient sera did not react with
these two bands, as was expected (Fig. 4a, lanes 3 and 4). To
confirm these immunoblotting analysis results, we performed
immunoprecipitation analysis. As shown in Fig. 4b, our
patient’s serum and PNP patient serum immunoprecipitated
with periplakin and envoplakin protein. However, PV patient
serum used as a negative control showed no reaction with
either protein (Fig. 4b). We also performed enzyme-linked
immunosorbent assay (ELISA) using recombinant purified de-
smoglein (Dsg) 1 antigen (MBL Co., Nagoya, Japan) with our
patient’s serum, and four PNP sera associated with underlying
neoplasms, four PF and three PV sera as positive controls, and
normal serum and two BP sera as negative controls. According
to the manufacturer’s protocol, the results of ELISA revealed
that our patient’s serum (anti-Dsg 1, 78 U mL)1), two of four
PNP sera, four of four PF sera and two of three PV sera
showed positive (anti-Dsg 1, ‡ 20 U mL)1) values, but that
all negative control sera showed negative (anti-Dsg 1,
< 14 U mL)1) values.
Despite thorough laboratory examinations including whole-
body positron emission tomography/computed tomography
(General Electric Medical Systems, Milwaukee, WI, U.S.A.),
abdominal ultrasound, peripheral blood smear, Venereal Dis-
ease Research Laboratory test, carcinoembryonic antigen, a-
fetoprotein, prostate-specific antigen and serum/urine immu-
(b)(a)
Fig 1. Vesicles and erosions with crusting on
the entire face, involving the mucosa of both
eyes and lips and upper chest in the active
disease state (a), and following recovery (b).
Fig 2. Histological examination revealed dyskeratotic cells and basal
vacuolization in the epidermis, a dermal infiltrate of lymphohistiocytic
cells and melanophages, and red blood cell extravasation in the dermis
(haematoxylin and eosin; original magnification · 200).
(b)(a)
Fig 3. Indirect immunofluorescence of the
patient’s serum shows IgG deposits in the
transitional epithelium of rat bladder as a
substrate. (a) Our patient’s serum; (b) serum
of a patient with pemphigus vulgaris (PV) as
negative control. Original magnification
· 200.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp563–566
564 PNP without an underlying neoplasm, G.T. Park et al.
noelectrophoresis, we were unable to detect any tumour over
a period of approximately 8 years. During the follow-up per-
iod, our patient’s skin lesions were treated with prednisolone,
immunosuppressant (azathioprine) and dapsone. However, his
symptoms gradually worsened and spread to both extremities,
and the treatment was stopped in November 2004. Fortu-
nately, the chronic vesiculobullous eruption improved sponta-
neously after a duration of 7 years, and since May 2005 our
patient has been healthy and off all medication (Fig. 1b). Indi-
rect IF of our patient’s serum in a state of complete remission
was examined on normal human foreskin and rat bladder. IgG
aAbs of our patient’s serum did not bind to the ECS or BMZ
of human foreskin and rat bladder at a titre of 1 : 10, and
only negligible 190- and 210-kDa bands were seen in immu-
noblotting at a titre of 1 : 20 (data not shown).
Discussion
A vesiculobullous disease termed PNP with distinct aAbs was
first described in 1990.1 A diagnosis of PNP is established in
patients who have at least four of the following five criteria
proposed by Anhalt et al.: a polymorphic eruption that affects
the skin and mucous membranes; histopathological features
that include intraepidermal acantholysis and dyskeratosis with
vacuolar changes; an intraepidermal and/or BMZ deposition of
IgG and C3 on direct IF; serum aAbs to multiple epithelia;
and the recognition of the following antigenic bands in im-
munoblotting and/or immunoprecipitation assays: 250, 230,
210 and 190 kDa.1,9,17 Amagai et al.3 have demonstrated both
by immunoprecipitation and by ELISA that PNP sera have
pathogenic antibodies against Dsg 3 and Dsg 1. Thus, the PNP
autoantibody complex is composed of anti-Dsg (Dsg 3 and
Dsg 1), antiplakin (desmoplakins I and II, BP antigen 1, en-
voplakin, periplakin, plectin) and anti-170-kDa (unidentified)
antibodies. Of the various target antigens recognized by indi-
vidual patients with PNP, the most specific serological findings
are aAbs against periplakin and envoplakin.19 These serological
findings are important because these aAbs differ from those
found in PV, BP and other autoimmune bullous diseases.
These aAbs were always detected by immunoblotting analysis
in our patient’s sera obtained during the active disease state.
This suggests that aAbs against periplakin and envoplakin are
still detectable serological markers, unless PNP is treated com-
pletely. Most PNP cases have been reported in patients with a
history of neoplasia, such as lymphoma, chronic lymphocytic
leukaemia, poorly differentiated sarcoma and Castleman’s dis-
ease.1,10–16 However, PNP is not always accompanied by neo-
plasia.17 The present case suggests that not all patients with
PNP have a definite neoplasm or even a history of one. How-
ever, we cannot rule out the possibility that other factors such
as drugs and inflammatory diseases can trigger aAbs related to
PNP in the absence of a neoplasm even though we followed
this patient to find underlying tumours during a long-term 8-
year follow-up period. In addition, patients with PF have been
shown to acquire aAbs against both 190- and 210-kDa anti-
gens of desmosomal plaques by an epitope-spreading phe-
nomenon even though aAbs against Dsg 1 were detected by
ELISA using recombinant purified Dsg 1.5
PNP can mimic a variety of dermatological diseases includ-
ing PV, BP, erythema multiforme and lichen planus.1,2,10–12,16
Owing to this clinical and histological variability, other speci-
fic examinations such as immunoblotting, immunoprecipita-
tion and indirect IF with rat bladder are required for a
definitive diagnosis of PNP.9,20 Thus far, its association with
lymphoproliferative disorders, positive indirect IF staining
with rat bladder, and positivity for envoplakin and/or peripla-
kin by immunoblotting and immunoprecipitation are gener-
ally regarded as sensitive and specific evidence for a diagnosis
of PNP.9,21
Despite no association with a neoplasm, the present case
was diagnosed by a positive indirect IF result using rat
bladder, and by immunoblotting and immunoprecipitation
analyses. Interestingly, our patient recovered from the disease
after 7 years, as is often observed for other autoimmune
bullous diseases such as PV, and has lived well without
medication, i.e. with a favourable outcome. However, the
question remains as to the source of these aAbs against the
plakin family in the absence of a neoplasm. This case indicates
that more studies are required to identify the factors that elicit
an immune reaction and induce PNP in the absence of an
underlying neoplasm.
(a) (b)
Fig 4. The serum of our patient reacted with periplakin and
envoplakin. (a) Immunoblotting (IB) analysis of the sera of various
patients. Lane 1, control paraneoplastic pemphigus (PNP) serum
reacted with 210- and 190-kDa antigens; lane 2, our patient’s serum;
lane 3, control bullous pemphigoid (BP) serum reacted with 230- and
180-kDa antigens; lane 4, control pemphigus vulgaris (PV) serum
reacted with 160- and 130-kDa antigens. (b) Immunoprecipitation
(IP) analysis of the sera of several patients. Control PNP serum, our
patient’s serum and PV serum were incubated with normal human
epidermal extract. Each precipitate was immunoblotted using
antiperiplakin and anti-envoplakin antibodies. The arrows indicate
envoplakin and periplakin, respectively. Control PNP serum and our
patient’s serum reacted with both envoplakin and periplakin, but PV
serum did not. Lower panels show endogenous periplakin and
envoplakin, respectively.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp563–566
PNP without an underlying neoplasm, G.T. Park et al. 565
References
1 Anhalt GJ, Kim SC, Stanley JR et al. Paraneoplastic pemphigus. An
autoimmune mucocutaneous disease associated with neoplasia. NEngl J Med 1990; 323:1729–35.
2 Joly P, Thomine E, Gilbert D et al. Overlapping distribution ofautoantibody specificities between pemphigus vulgaris and para-
neoplastic pemphigus. J Invest Dermatol 1994; 103:65–72.3 Amagai M, Nishikawa T, Noussari HC et al. Antibodies against de-
smoglein 3 (pemphigus vulgaris antigen) are present in sera frompatients with paraneoplastic pemphigus and cause acantholysis in
vivo in neonatal mice. J Clin Invest 1998; 102:775–82.4 Joly P, Gilbert D, Thomine E et al. Immunofluorescence and immu-
noelectron microscopy analyses of a human monoclonal anti-epi-thelial cell surface antibody that recognizes a 185-kD polypeptide:
a component of the paraneoplastic pemphigus antigen complex? JInvest Dermatol 1993; 101:339–45.
5 Ghohestani R, Joly P, Gilbert D et al. Autoantibody formationagainst a 190-kDa antigen of the desmosomal plaque in two
patients with pemphigus foliaceus. Br J Dermatol 1997; 137:774–9.6 Joly P, Mokhtar I, Gilbert D et al. Immunoblot and immunoelectron
microscopic analysis of endemic Tunisian pemphigus. Br J Dermatol1999; 140:44–9.
7 Chorzelski TP, Maciejowski E, Jablonska S et al. Coexistence of pem-phigus and bullous pemphigoid. Arch Dermatol 1974; 109:849–53.
8 Korman NJ, Stanley JR, Woodley DT. Coexistence of pemphigusfoliaceus and bullous pemphigoid: demonstration of autoantibodies
that bind to both the pemphigus foliaceus antigen complex andthe bullous pemphigoid antigen. Arch Dermatol 1991; 127:387–90.
9 Joly P, Richard C, Gilbert D et al. Sensitivity and specificity of clin-
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10 Tankel M, Tannenbaum S, Parekh S. Paraneoplastic pemphigus pre-senting as an unusual bullous eruption. J Am Acad Dermatol 1993;
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11 Mehregan DR, Oursler JR, Leiferman KM et al. Paraneoplastic pem-phigus: a subset of patients with pemphigus and neoplasia. J Cutan
Pathol 1993; 20:203–10.12 Lam S, Stone MS, Goeken JA et al. Paraneoplastic pemphigus, cic-
atricial conjunctivitis, and acanthosis nigricans with pachydermato-glyphy in a patient with bronchogenic squamous cell carcinoma.
Ophthalmology 1992; 99:108–13.13 Fullerton SH, Woodley DT, Smoller BR, Anhalt GJ. Paraneoplastic
pemphigus with autoantibody deposition in bronchial epitheliumafter autologous bone marrow transplantation. JAMA 1992;
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14 Camisa C, Helm TN, Liu YC et al. Paraneoplastic pemphigus: areport of three cases including one long-term survivor. J Am Acad
Dermatol 1992; 27:547–53.15 Berg WA, Fishman EK, Anhalt GJ. Retroperitoneal reticulum cell
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16 Stevens SR, Griffiths CEM, Anhalt GJ et al. Paraneoplastic pemphiguspresenting as a lichen planus pemphigoides like eruption. Arch Der-
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18 Gooptu C, Littlewood TJ, Frith P et al. Paraneoplastic pemphigus:an association with fludarabine? Br J Dermatol 2001; 144:1255–61.
19 Hashimoto T, Amagai M, Watanabe K et al. Characterization ofparaneoplastic pemphigus autoantigens by immunoblot analysis.
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Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp563–566
566 PNP without an underlying neoplasm, G.T. Park et al.
CASE REPORT DOI 10.1111/j .1365-2133.2006.07699.x
Allogeneic bone marrow transplantation in a 7-year-old girlwith congenital erythropoietic porphyria: a treatmentdilemmaS.M. Taibjee, O.E. Stevenson,* A. Abdullah,� C.Y. Tan,� P. Darbyshire, C. Moss, H. Goodyear,* A. Heagerty,*S. Whatley� and M.N. Badminton�
Departments of Haematology and Dermatology, Birmingham Children’s Hospital, Birmingham B4 6NL, U.K.
*Departments of Paediatrics and Dermatology, Heart of England NHS Trust, Birmingham, U.K.
�Department of Dermatology, City Hospital, Birmingham, U.K.
�Department of Medical Biochemistry, University Hospital Wales, Cardiff, U.K.
CorrespondenceSaleem Taibjee.
E-mail: saleemtaibjee@blueyonder.co.uk
Accepted for publication25 September 2006
Key wordsallogeneic, bone marrow transplantation, congenital
erythropoietic porphyria, graft-versus-host disease,
Gunther’s disease, postinflammatory
hypopigmentation
Conflicts of interestNone declared.
Summary
Congenital erythropoietic porphyria (CEP, Gunther’s disease) has a very variablephenotype. In the more severely affected, bone marrow transplantation (BMT) ispotentially curative, but is not without risks. We describe a 7-year-old girl withCEP characterized by severe photosensitivity but only mild anaemia, in whomthe difficult decision to proceed with allogeneic BMT was made after discussionin a multidisciplinary team. She has shown successful engraftment, accompaniedby biochemical and clinical resolution of her metabolic disease. She remains well3 years later, the oldest patient with CEP receiving BMT to survive beyond12 months. However, she has experienced significant morbidity including floridcutaneous graft-versus-host disease with postinflammatory hypopigmentation.Her case is important in highlighting the delay in diagnosis not uncommon inthis condition and the complex decision-making process involved in proceedingwith BMT.
Congenital erythropoietic porphyria (CEP, Gunther’s disease)
is a rare autosomal recessive porphyria resulting from marked
deficiency of uroporphyrinogen III synthase (UROS) enzyme
activity (10q25.2-q26.3). It has a variable clinical phenotype,
ranging from hydrops fetalis and intrauterine death, severe
lifelong photosensitivity, skin fragility, transfusion-dependent
haemolytic anaemia and hypersplenism with curtailed life
expectancy, to only mild cutaneous features in some cases.1,2
At least 36 different UROS gene mutations have been identi-
fied in CEP3 and the estimation of functional activity of
mutant enzyme in prokaryotic expression systems has enabled
genotype–phenotype correlation.1,2,4 The biochemical disturb-
ance reflected by endogenous porphyrin profiles also predicts
phenotypic severity in patients.5
Treatment options are based on clinical severity, and
include photoprotection, b-carotene, activated charcoal, hyper-
transfusion, hydroxycarbamide and splenectomy.2,6–10 In vitro
studies indicate that retroviral-mediated gene therapy of hae-
matopoietic stem cells may be a potential treatment option in
the future.11,12 In severe cases the prognosis tends to be poor
and bone marrow transplantation (BMT) may be justified as
it is potentially curative. Successful engraftment leads to a
replacement of bone marrow with erythroblasts with normal
UROS activity. Porphyrin levels normalize and photosensitivity
subsides. It was first used in 1991 in a 10-year-old girl with
CEP who demonstrated biochemical and clinical resolution,
but died of overwhelming cytomegalovirus infection, high-
lighting the considerable risks involved.13 Since then at least
a further eight cases have been reported where either
BMT1,14–19 or cord-blood stem cell transplantation20 has been
successful in this condition (this may be an underestimate,
after identification of at least one further successful case fol-
lowing personal communication with Prof. Alain Fischer,
Hopital Necker-Enfants Malades, Paris, 2005). Most patients
were transfusion dependent prior to BMT, but with notable
exceptions.18,19 One patient subsequently required retrans-
plantation.16,19 The management dilemmas in CEP, particularly
concerning BMT, have previously been alluded to by Dawe
et al.21 where they described a 35-year-old man with severe
CEP where allogeneic BMT was considered but discounted
based on the perceived risks.
We report a 7-year-old girl with CEP where the decision to
proceed with BMT was a difficult one, involving a multidisci-
plinary team and the family.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp567–571 567
Case report
Our patient was born to consanguineous Pakistani parents. She
had an unremarkable perinatal history. Her parents had
noticed pink-stained nappies during her infancy. Aged
8 months she had been assessed for failure to thrive and diar-
rhoea, without a clear cause identified. Aged 2 years, she was
seen by a paediatric dental surgeon regarding her discoloured
brown teeth, and at 3 years endocrine causes for her excessive
hair were excluded. She had skin changes, which had initially
been attributed to atopic eczema. The unifying diagnosis of
CEP was eventually made at age 5 years by both a senior
dermatologist and a paediatrician. In retrospect her parents
described a blistering eruption affecting the face and hands
since infancy, present all year round but worse after exposure
to sunlight. Examination showed blistering, prominent scar-
ring and hypertrichosis on the face and dorsum of hands
(Figs 1, 2), erythrodontia (Fig. 3) with pink fluorescence
under Wood’s light (Fig. 4) and discoloured toenails.
Blood and urine porphyrin analysis was characteristic for
CEP, with total urinary porphyrin 20 141 nmol L)1 (reference
range 20–320), comprising 87% uroporphyrin isomer I, and
plasma total porphyrin 444Æ6 nmol L)1 (reference ran-
ge < 11Æ2) with plasma porphyrin fluorescence emission spec-
troscopy confirming a peak at 618 nm. Faecal porphyrin
analysis showed a total porphyrin of 2143 nmol g)1 dry
weight (reference range 0–200), comprising 91% copro-
porphyrin isomer I. UROS enzyme assay confirmed a markedly
reduced red cell uroporphyrin III synthesis activity of
41 nmol mL)1 erythrocytes h)1 (mean activity in three con-
temporaneous control samples 183 nmol mL)1 h)1). Full
blood count showed a mild normocytic anaemia with haemo-
globin (Hb) 10Æ8 g dL)1 (normal 11Æ5–16Æ5) and normal
leucocyte and platelet count. Bone marrow aspiration showed
a normocellular marrow with mild dyserythropoiesis only.
No mutation could be identified after sequencing exon and
intron-exon flanking regions of the UROS gene.
The initial management included sun avoidance and sun
protection using high factor sunblock, with minimal success.
A particular factor was the family’s trips to Pakistan, which
included the Pakistani summer months. Our patient continued
to display ongoing severe photosensitivity and scarring.Fig 1. Dorsum of hands at age 5 years: scarring, milia and active
blisters.
Fig 2. Face at age 5 years: scarring, dyspigmentation and
hypertrichosis.
Fig 3. Erythrodontia.
Fig 4. Dentition showing pink fluorescence under Wood’s light.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp567–571
568 Allogeneic bone marrow transplantation in a girl with CEP, S.M. Taibjee et al.
A multidisciplinary team was established, involving the derma-
tologist, paediatric haematologist, bone marrow transplant
team and general practitioner. The school and family were
both involved. Based on previous reports of successful BMT in
CEP, the long process of discussion culminated in the decision
to proceed with BMT 2 years after her initial diagnosis. Rele-
vant factors included her severe dermatological manifestations,
failure to thrive and short stature, ongoing anaemia (Hb
8Æ1 g dL)1 at 1 month prior to BMT), significant lifestyle
restrictions, poor school attendance, the views expressed by
the family itself, as well as the availability of a sibling with a
full HLA match (HLA typing: A2, A11; B37, B60; Cw6, Cw3;
DR10, DR11, DR52; DQ7, DQ1).
In January 2003, aged 7 years, she underwent allogeneic
BMT from her 4-year-old brother at Birmingham Children’s
Hospital. Conditioning consisted of fludarabine (days )14
to )10; 25 mg m)2 daily), busulphan (days )9 to )6;
3Æ5 mg kg)1 daily) and cyclophosphamide (with mesna, days
)5 to )2; 50 mg kg)1 daily). She received ciclosporin (from
day )1; 2Æ5 mg kg)1 twice daily) for graft-versus-host disease
(GVHD) prophylaxis, granulocyte colony-stimulating factor
(from day +8; 5 lg kg)1 daily) and weekly intravenous im-
munoglobulin (Vigam�; BPL, Elstree, U.K.; from day +2;
0Æ4 g kg)1 weekly). Following bone marrow infusion (defined
as day 0), her neutrophil count was first noted to be greater
than 0Æ5 · 109 L)1 on day +13, suggesting successful engraft-
ment, and this was subsequently confirmed by repeated inter-
phase fluorescent in situ hybridization analyses demonstrating
100% of cells with donor (XY) chromosome complement,
with no cytogenetic evidence of a host (XX) cell line, i.e. full
donor chimerism. She remained photosensitive for only
4 weeks post-BMT, with only occasional new blisters in this
initial period. This was reflected in the steady biochemical
improvement in her porphyrin profile (Table 1). Her anaemia
gradually resolved (Hb 12Æ8 g dL)1 at 5 months post-BMT).
Unfortunately her recovery from BMT was punctuated by a
series of problems, most notably GVHD, anorexia and psycho-
social issues. Acute GVHD commenced at week 3 post-BMT
with a typical maculopapular rash of approximately 30% sur-
face area associated with palmar-plantar erythema. It was most
florid at weeks 4 and 5 post-BMT, and then gradually resolved
with desquamation and hypopigmentation. This was accom-
panied by moderate (grade 2) gut GVHD leading to diarrhoea
and vomiting, confirmed on upper gastrointestinal endoscopy
and colonoscopy. Topical treatments comprised emollients and
antiseptics only. The initial GVHD systemic treatment included
ciclosporin (maximum 3 mg kg)1 twice daily) and predniso-
lone (maximum 1 mg kg)1 daily, or intravenous methyl-
prednisolone when noncompliant with oral medications;
maximum 2 mg kg)1 daily). Two months post-BMT, due to
ongoing gut GVHD and poor compliance with ciclosporin
(which was stopped), she was commenced on mycophenolate
mofetil (continued until 4 months post-BMT; maximum
600 mg m)2 twice daily) and FK506 (gradually weaned off
by February 2004; maximum 0Æ1 mg kg)1 twice daily). Her
diarrhoea persisted intermittently until almost 4 months post-
BMT. Nutritional problems necessitated a period of total
parenteral nutrition. Three months post-BMT, as a result of
persistent anorexia and poor compliance with medication, she
went on to require gastrostomy insertion for enteral feeding,
which was continued for several weeks.
Although there was no evidence of skin sclerosis or other
features of chronic GVHD, she developed widespread post-
inflammatory hypopigmentation and alopecia (Figs 5, 6).
Despite referral to the cosmetic camouflage service and the
input of a clinical psychologist, she remains distressed by her
Table 1 Urine, erythrocyte and plasma porphyrin results before and after bone marrow transplantation (BMT)
At diagnosis
(pre-BMT)
1 month
post-BMT
3 months
post-BMT
9 months
post-BMT
Reference
range
TUP (nmol L)1) 20 141 944 355 604 20–320TUP : creatinine ratio (nmol mmol)1) 2722 175 222 123 < 34
Erythrocyte porphyrin (lmol L)1) 7Æ8 4Æ7 Not done Not increased 0Æ4–1Æ7Plasma porphyrin (nmol L)1) 444Æ6 37Æ4 Not done 15Æ1 < 11Æ2
TUP, total urinary porphyrin.
Fig 5. Postinflammatory hypopigmentation following graft-versus-
host disease.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp567–571
Allogeneic bone marrow transplantation in a girl with CEP, S.M. Taibjee et al. 569
appearance. She has been difficult to rehabilitate due to var-
ious behavioural problems. Her reluctance to mobilize resulted
in fixed contractures at the elbows and shoulders despite phy-
siotherapy. She was finally discharged from hospital 6 months
post-BMT. At the latest review, 3 years post-BMT, she is hap-
pier and integrating well into school. She still has marked
hypopigmentation, with slow but steady repigmentation, and
wears a wig. The disruption to the family unit caused by her
disease and prolonged hospital admission may have contribu-
ted to the separation of her parents and behavioural problems
in one of her brothers. However, members of her family
express no regrets with their decision to proceed with BMT.
Discussion
The decision to proceed with BMT in CEP should be based on
clinical assessment of the disease’s impact, as well as estimat-
ing the long-term prognosis wherever possible. Our patient
was mildly anaemic and not transfusion dependent. Moore
et al.22 considered the onset of dermatological changes includ-
ing scarring in the first decade of life as a poor prognostic fac-
tor, although there is little evidence for this assertion.
Freesemann et al.5 have previously demonstrated the correl-
ation between degree of porphyrin excess and disease severity
in nine patients with CEP. Total urinary porphyrins ranged
between 2291 and 101 788 nmol per 24 h, but this is hard
to correlate with the levels in our patient, expressed as
nmol mmol)1 creatinine (Table 1). In the same study, whole
blood erythrocyte porphyrins ranged from 0Æ6 to 12Æ0 lmol L)1
(median 2Æ0; reference range 0Æ09–0Æ67) which compares
with 7Æ8 lmol L)1 erythrocytes (i.e. packed red blood cells)
in our patient. Although the different assay methodologies
make a direct comparison difficult, the median baseline eryth-
rocyte porphyrin reported by Freesemann et al. was three times
the assay upper reference limit (URL), compared with 4Æ5times the URL in our patient, confirming that she had a rela-
tively severe baseline biochemical phenotype.
Genotype–phenotype correlation, using in vitro prokaryote
expression of UROS constructs to estimate functional activity
of the mutant enzyme, is extremely useful in CEP. Patients
with severe disease demonstrate mutations leading to near-
absent enzymatic activity, whereas milder cases without trans-
fusion dependence have at least one allelic mutation resulting
in significant residual UROS activity.1,2,5 In view of the paren-
tal consanguinity, a homozygous gene defect was expected in
our patient. However, despite the markedly reduced UROS
enzyme activity, a mutation was not identified using the cur-
rent gene sequencing approach which includes sequencing of
the erythroid-specific region. Using this approach our current
sensitivity of mutation detection is 75% (S.W., unpublished
data). Two other specialist centres have reported allele detec-
tion frequencies of 89–92%,2,23 although neither centre has
reported a patient in whom no mutation was identified on
either allele. This suggests other sites for mutations within this
34-kb gene, including regulatory elements which may be
located thousands of base pairs upstream or downstream from
the coding region. The role of possible modifying genes on
UROS expression is also yet to be established.
Despite immunosuppressive prophylaxis, GVHD remains a
common complication of allogeneic BMT. It appears that our
patient was the only one showing significant GVHD, but there
are no clear reasons for this. Her sibling donor was HLA iden-
tical, and the prophylactic regimen she received was similar to
that in previous reports in CEP, where all patients received
prophylaxis consisting of ciclosporin, although in some cases
combined with methotrexate or corticosteroids. Although she
is the oldest of the 10 patients with CEP now reported to have
undergone BMT, increasing age only emerges as a risk factor
for GVHD in data including mixed adult and paediatric popu-
lations. Age does not appear to be a risk factor in paediatric
cohorts alone.24 The degree of postinflammatory hypopigmen-
tation observed is likely to relate to her Fitzpatrick skin
type V.
Although our patient presented to a variety of specialties
with classical features of CEP, the unifying diagnosis was not
made until 5 years of age. This highlights the diagnostic delay
not uncommon in this condition, as the dermatological signs
may be subtle in early years and many clinicians are unfamil-
iar with this rare disease.
To our knowledge, our patient is the oldest child with CEP
undergoing BMT to survive beyond 12 months. Her case
highlights the complex decision to proceed with BMT, involv-
ing a multidisciplinary team and the family. To date, all 10 of
the patients with CEP receiving BMT reported in the literature
(and the additional unreported patient identified after personal
Fig 6. Postinflammatory hypopigmentation following graft-versus-
host disease, with gastrostomy.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp567–571
570 Allogeneic bone marrow transplantation in a girl with CEP, S.M. Taibjee et al.
communication with Alain Fischer, Paris) have shown
successful resolution of their disease, although one patient
subsequently died of transplant-associated complications.
An assessment of clinical and psychosocial impact of the dis-
ease, including prognostic factors such as porphyrin levels,
enzyme assays and genotyping, should be balanced against the
potential morbidity and mortality of this intervention.
Acknowledgments
We thank the laboratory staff, Department of Medical Bio-
chemistry, University Hospital Wales, for performing bio-
chemical analyses on our patient, the dedicated staff on the
BMT unit, Birmingham Children’s Hospital, who helped our
patient through her long period of rehabilitation, and our
patient and family for their positive outlook throughout.
References
1 Tezcan I, Xu W, Gurgey M et al. Congenital erythropoietic porphy-ria successfully treated by allogenic bone marrow transplantation.
Blood 1998; 92:4053–8.2 Desnick RJ, Astrin KH. Congenital erythropoietic porphyria: advan-
ces in pathogenesis and treatment. Br J Haematol 2002; 117:779–95.3 Human Gene Mutation Database. http://www.hgmd.cf.ac.uk/ac/
gene.php?gene¼UROS (last accessed 26 October 2006).4 Desnick RJ, Glass IA, Xu W et al. Molecular genetics of congenital
erythropoietic porphyria. Semin Liver Dis 1998; 18:77–84.5 Freesemann AG, Bhutani LK, Jacob K, Doss MO. Interdependence
between degree of porphyrin excess and disease severity in con-genital erythropoietic porphyria (Gunther’s disease). Arch Dermatol
Res 1997; 289:272–6.6 Badminton MN, Elder GH. Management of acute and cutaneous
porphyrias. Int J Clin Pract 2002; 56:272–8.7 Guarini L, Piomelli S, Poh-Fitzpatrick MB. Hydroxyurea in congen-
ital erythropoietic porphyria. N Engl J Med 1994; 330:1091–2.8 Hift RJ, Meissner PN, Kirsch RE. The effect of oral activated char-
coal on the course of congenital erythropoietic porphyria. Br JDermatol 1993; 129:14–17.
9 Pimstone NR, Gandhi SN, Mukerji SK. Therapeutic efficacy of oralactivated charcoal in congenital erythropoietic porphyria. N Engl J
Med 1987; 316:390–3.10 Piomelli S, Poh-Fitzpatrick MB, Seaman C et al. Complete suppres-
sion of the symptoms of congenital erythropoietic porphyria bylong-term treatment with high level transfusions. N Engl J Med
1986; 314:1029–31.
11 Kauppinen R, Glass IA, Aizencang G et al. Congenital erythropoieticporphyria: prolonged high-level expression and correction of
the heme biosynthetic defect by retroviral-mediated gene transferinto porphyric and erythroid cells. Mol Genet Metab 1998; 65:10–
17.12 Mazurier F, Geronomi F, Lamrissi-Garcia I et al. Correction of defi-
cient CD34(+) cells from peripheral blood after mobilisation in apatient with congenital erythropoietic porphyria. Mol Ther 2001;
3:411–17.13 Kauffmann L, Evans DIK, Stevens RF, Weinkove C. Bone marrow
transplantation for congenital erythropoietic porphyria. Lancet 1991;
337:1510–11.14 Dupuis-Girod S, Akkari V, Ged C et al. Successful match-unrelated
donor bone marrow transplantation for congenital erythropoieticporphyria (Gunther disease). Eur J Pediatr 2005; 164:104–7.
15 Harada FA, Shwayder TA, Desnick RJ, Lim HW. Treatment ofsevere CEP with bone marrow transplant. J Am Acad Dermatol 2001;
5:279–82.16 Lagarde C, Hamel-Teillac D, De Prost Y et al. Allogenic bone
marrow transplantation in congenital erythropoietic porphyria.Gunther’s disease. Ann Dermatol Venereol 1998; 125:114–17.
17 Lanino E, Morreale G, Gabarino L et al. Severe congenitalerythropoietic porphyria. Complete correction after BMT from
an unrelated donor. European Group for Blood and MarrowTransplantation 2001; http://congress.akm.ch/abstract/abstract/
abt.ausg_pkt_ses?xssprache¼ENGOPT&xnkon_nr¼45&xnSESSION_NR¼1612&xnCO_PROFILE¼356788# (last accessed 26 October
2006).18 Shaw PH, Mancini AJ, McConnell JP et al. Treatment of CEP in
children by allogenic stem cell transplantation: a case reportand review of the literature. Bone Marrow Transplant 2001; 27:101–5.
19 Thomas C, Ged C, Nordmann Y et al. Correction of congenital ery-thropoietic porphyria by bone marrow transplantation. J Pediatr
1996; 129:453–6.20 Zix-Kieffer I, Langer B, Eyer D et al. Successful cord blood stem
cell transplant for congenital erythropoietic porphyria (Gunther’sdisease). Bone Marrow Transplant 1996; 18:217–20.
21 Dawe SA, Stephens AD, Peters TJ et al. Congenital eythropoieticporphyria: dilemmas in present day management. Clin Exp Dermatol
2002; 27:680–3.22 Moore MR, McColl KEL, Fitzsimmons EJ et al. The porphyrias. Blood
Rev 1990; 4:88–96.23 De Verneuil H, Ged C, Moreau-Gaudry F. Congenital erythropoietic
porphyria. In: The Porphyrin Handbook: Medical Aspects of Porphyrins
(Kadish KM, Smith KM, Guilard R, eds), Vol. 14. San Diego:Academic Press, 2003; 43–63.
24 Bolwell BJ. Are predictive factors clinically useful in bone marrowtransplantation? Bone Marrow Transplant 2003; 32:853–61.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp567–571
Allogeneic bone marrow transplantation in a girl with CEP, S.M. Taibjee et al. 571
Gene Corner
Galli–Galli disease is an acantholytic variant of Dowling–Degos disease
DOI: 10.1111/j.1365-2133.2006.07703.x
Dowling–Degos disease (DDD; MIM 179850) is a rare auto-
somal dominant disorder characterized by reticulate flexural
hyperpigmentation associated with hyperkeratotic papules,
pitted perioral scars and comedo-like lesions or cysts.1 Charac-
teristic histopathological features include filiform epithelial
downgrowths of the rete ridges, typically involving the folli-
cular infundibulum, basilar hyperpigmentation and dermal
melanosis. Follicular retention cysts and perivascular mono-
nuclear infiltrates can also be present.1 Galli–Galli disease is a
term that has been used to describe patients displaying promin-
ent acantholytic changes on histology, in addition to clinical
and pathological features resembling those of DDD.2 Two muta-
tions in KRT5, encoding one of the two major basal epidermal
keratin intermediate filaments, were recently reported to under-
lie DDD in a number of European cases.3 In the present study,
we report a novel mutation in KRT5 in a patient with clinical and
histopathological features typical of Galli–Galli disease.
Case and methods
A 43-year-old woman of Arab Moslem origin was referred
because of an exceedingly pruritic papular rash involving axil-
lae, inframammary areas and groins (Fig. 1a), and suggestive
of Hailey–Haily disease (MIM 169900), Darier disease (MIM
124200) or pemphigus vegetans. She reported that her
deceased father and eldest sister were similarly affected. Clin-
ical examination revealed brownish reticulate pigmentation
and densely distributed erosive papules in all skin flexural
areas (Fig. 1a). A number of skin biopsies revealed findings
typical of DDD including acanthosis, epidermal rete ridges
with bud-like and filiform projections, a mixed dermal inflam-
matory infiltrate of variable severity, and dermal melano-
phages (Fig. 1b). Numerous foci of suprabasal acantholysis
were also observed (Fig. 1c). To address the possibility that
the patient had an autoimmune blistering disease, possibly
underlying the acantholytic changes observed on histology,
we performed indirect and direct immunofluorescence studies
(aimed at detecting the presence of anti-epidermal antibodies
in patient skin and serum). Both tests were negative. Taken
together, clinical and pathological data suggested a diagnosis
of DDD – Galli–Galli variant.
The patient provided her written and informed consent to
participate in the study according to a protocol previously
approved by the Committee for Genetic Studies of the Israeli
Ministry of Health. Genomic DNA was extracted from whole
blood samples using the salt/chloroform extraction method
and polymerase chain reaction (PCR) amplified using primer
pairs spanning the entire coding sequence as well as intron–
exon boundaries of the KRT5 gene as described elsewhere.4
Mutation p.M1? was verified using PCR–restriction fragment
length polymorphism (RFLP) analysis, exploiting the fact that
it abolishes a recognition site for endonuclease NlaIII. Briefly,
a 795-bp long DNA fragment was amplified using forward
primer 5¢-AGCTCTGTTCTCTCCAGCAC-3¢ and reverse primer
5¢-CAGTCTAATTCAGAACGTGTCC-3¢ and digested with NlaIII.
Results and discussion
We established the entire KRT5 coding sequence in our
patient, as previously described.4 DNA samples from other
family members were not available for analysis. We identified
a single deleterious change consisting of a heterozygous
T fi C transition at KRT5 cDNA position 2 (Fig. 1d). A PCR–
RFLP assay was established based upon the fact the mutation
c.T2C destroys a recognition site for endonuclease NlaIII.
Using this PCR–RFLP assay, we excluded the mutation from a
panel of 100 population-matched chromosomes (not shown).
The c.T2C mutation is predicted to disrupt the KRT5 initi-
ation codon (p.M1?) and therefore to lead to either haplo-
insufficiency by causing lack of protein translation or to the
usage of a downstream alternative initiation codon, resulting
in the synthesis of a N-deleted KRT5 mutant protein as previ-
ously reported in other monogenic disorders.5,6
Most pathogenic sequence alterations described to date in
keratin genes in general, and in KRT5 in particular are mis-
sense mutations located within or at the boundaries of the
central a-helical rod domain.7,8 Clinical and histopathological
consequences of mutations affecting these conserved regions
of epidermal keratin genes classically consist of skin blister-
ing/hyperkeratosis and keratinocyte vacuolar degeneration,
respectively, as seen in epidermolysis bullosa simplex caused
by mutations in KRT5/KRT14.8,9 In contrast, mutations affect-
ing the keratin variable head and tail domains are often non-
sense/frameshift mutations, associated with markedly atypical
phenotypes. Notable examples include mutations resulting in
KRT1 or KRT5 tail domain elongation and causing ichthyosis
hystrix of Curth Macklin10 (MIM 146590) or epidermolysis
bullosa simplex with migratory circinate erythema11 (MIM
� 2007 The Authors
572 Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp572–574
609352), respectively; and nonsense/frameshift mutations
affecting the early N-terminal domain of KRT5 and KRT14 and
resulting in DDD3 or Naegeli–Franceschetti–Jadassohn syn-
drome12 (MIM 161000), respectively.
Acantholysis is a typical feature of a number of diseases
such as Hailey–Hailey disease and pemphigus vulgaris.13,14
However, it has also been described as a rare incidental find-
ing in numerous skin diseases such as squamous cell carci-
noma, melanocytic lesions and irritant dermatitis.15–17 This
histopathological finding always reflects abnormal function of
the desmosomal plaque, although the cause for desmosomal
dysfunction varies from one disease to another. The function
of the keratin head domain is not yet known in detail
although a direct interaction between desmoplakin, an import-
ant desmosomal protein, and the KRT5 head domain has been
demonstrated.18 It could therefore be envisaged that haplo-
insufficiency or N-terminal deletion of KRT5, which can be
predicted by all mutations described so far in DDD (Betz et al.3
and this study), may prevent the proper maturation of the
desmosomal plaques, causing abnormal cell–cell adhesion. Of
interest, one of the patients found by Betz et al.3 to carry the
founder KRT5 Ile140fs mutation had previously been reported
as affected with Galli–Galli disease,2 again suggesting a (direct
or indirect) causal relationship between KRT5 mutations and
acantholysis. In addition, abnormal location of desmocollin 3,
another component of the desmosomal plaque, has been
reported in DDD.19 Why have acantholytic changes been
reported in only a minority of DDD cases remains unex-
plained. The location and/or type of KRT5 causative mutation
is unlikely to account for the variable occurrence of this histo-
logical feature as the same mutation (Ile140fs) has been
reported both in Galli–Galli disease and in DDD.3 Independent
modifying genetic traits, including polymorphic sequence
alterations in KRT5, may possibly modulate the phenotypic
expression of mutations in DDD. In this regard, our patient
was found to carry, in addition to the causative mutation, a
number of known polymorphic nucleotide heterozygous
changes in KRT5 (IVS1+9C fi T, c.C594A, c.C594A, c.G732A,
(a) (b)
(c) (d)
Fig 1. Clinical, pathological and genetic assessment of a patient with Galli–Galli disease. (a) On examination, brownish reticulate pigmentation
and numerous erosive reddish papules are observed in the right axilla. Histopathological examination reveals (b) typical filiform elongation of the
rete ridges, melanophages in the dermis and perivascular mononuclear infiltrates (haematoxylin and eosin; original magnification ·100) as well as
(c) numerous foci of suprabasal acantholysis (haematoxylin and eosin; original magnification ·400). (d) Sequence analysis reveals a heterozygous
T fi C transition at cDNA position 2 resulting in loss of KRT5 initiating codon (upper panel). The wild-type (WT) sequence is given for
comparison (lower panel).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp572–574
Galli–Galli disease is an acantholytic variant of DDD, E. Sprecher et al. 573
cA1582G, c.G1627A). Finally, acantholysis may not be invari-
ably present at all times and anatomical sites in DDD, and
therefore could occasionally be overlooked.
Regardless of the pathomechanism involved, the present
observation and a previous study3 indicate that Galli–Galli dis-
ease is merely a variant of DDD. Whether acantholysis is truly
rare in DDD, or represents a common histological feature of
this disease, which has so far been under-reported, remains to
be determined.
Acknowledgments
We are grateful to our patient for her participation in our
study. We thank Vered Friedman for her help with nucleic
acid analysis. This study was supported in part by a grant pro-
vided by the Bureau for Economic Growth, Agriculture, and
Trade, Office of Economic Growth and Agricultural Develop-
ment, U.S. Agency for International Development, under the
terms of Award No. TA-MOU-01-M21-023.
E . S PR ECH ER*��M. INDE LMAN*
Z. KHAMAY S I*
J . LUGA S SY*�D. PETRON IU S*
R . BERGMAN*�
*Laboratory of Molecular Dermatology and
Department of Dermatology, Rambam Health
Care Campus, POB 9602, Haifa 31096, Israel
�Bruce Rappaport Faculty of Medicine and
�Rappaport Institute for Research in the Medical
Sciences, Technion, Israel Institute of Technology,
Haifa, Israel
E-mail: e_sprecher@rambam.health.gov.il
References
1 Kim YC, Davis MD, Schanbacher CF, Su WP. Dowling–Degos disease
(reticulate pigmented anomaly of the flexures): a clinical and histo-pathologic study of 6 cases. J Am Acad Dermatol 1999; 40:462–7.
2 Braun-Falco M, Volgger W, Borelli S et al. Galli–Galli disease: anunrecognized entity or an acantholytic variant of Dowling–Degos
disease? J Am Acad Dermatol 2001; 45:760–3.3 Betz RC, Planko L, Eigelshoven S et al. Loss-of-function mutations
in the keratin 5 gene lead to Dowling–Degos disease. Am J HumGenet 2006; 78:510–19.
4 Ciubotaru D, Bergman R, Baty D et al. Epidermolysis bullosa sim-plex in Israel: clinical and genetic features. Arch Dermatol 2003;
139:498–505.5 Cruts M, Gijselinck I, van der Zee J et al. Null mutations in pro-
granulin cause ubiquitin-positive frontotemporal dementia linkedto chromosome 17q21. Nature 2006; 442:920–4.
6 Paulsen M, Lund C, Akram Z et al. Evidence that translation reinitia-tion leads to a partially functional Menkes protein containing two
copper-binding sites. Am J Hum Genet 2006; 79:214–29.7 Omary MB, Coulombe PA, McLean WH. Intermediate filament pro-
teins and their associated diseases. N Engl J Med 2004; 351:2087–100.
8 Irvine AD. Inherited defects in keratins. Clin Dermatol 2005; 23:6–14.
9 Rugg EL, Leigh IM. The keratins and their disorders. Am J Med GenetC Semin Med Genet 2004; 131C:4–11.
10 Sprecher E, Ishida-Yamamoto A, Becker OM et al. Evidence for
novel functions of the keratin tail emerging from a mutation caus-ing ichthyosis hystrix. J Invest Dermatol 2001; 116:511–19.
11 Gu LH, Kim SC, Ichiki Y et al. A usual frameshift and delayed ter-mination codon mutation in keratin 5 causes a novel type of epi-
dermolysis bullosa simplex with migratory circinate erythema.J Invest Dermatol 2003; 121:482–5.
12 Lugassy J, Itin P, Ishida-Yamamoto A et al. Naegeli–Franceschetti–Jadassohn syndrome and dermatopathia pigmentosa reticularis: two
allelic ectodermal dysplasias caused by dominant mutations inKRT14. Am J Hum Genet 2006; 79:724–30.
13 Lanza A, Cirillo N, Femiano F, Gombos F. How does acantholysisoccur in pemphigus vulgaris: a critical review. J Cutan Pathol 2006;
33:401–12.14 Szigeti R, Kellermayer R. Autosomal-dominant calcium ATPase
disorders. J Invest Dermatol 2006; 126:2370–6.15 Kane CL, Keehn CA, Smithberger E, Glass LF. Histopathology of
cutaneous squamous cell carcinoma and its variants. Semin Cutan MedSurg 2004; 23:54–61.
16 Hutcheson AC, Nietert PJ, Maize JC. Incidental epidermolytichyperkeratosis and focal acantholytic dyskeratosis in common
acquired melanocytic nevi and atypical melanocytic lesions. J AmAcad Dermatol 2004; 50:388–90.
17 Naraghi ZS, Mansouri P, Mortazavi M. A clinicopathological studyon acute cutaneous lesions induced by sulfur mustard gas (yper-
ite). Eur J Dermatol 2005; 15:140–5.18 Kouklis PD, Hutton E, Fuchs E. Making a connection: direct
binding between keratin intermediate filaments and desmosomalproteins. J Cell Biol 1994; 127:1049–60.
19 Braun-Falco M, Ring J. Enhanced cytoplasmic expression of desmo-collin 3 in epidermal rete ridges of Dowling–Degos syndrome. Br J
Dermatol 2003; 149:1293–6.
Accepted for publication: 26 September 2006
Key words: Dowling–Degos disease, keratin, mutation, pigmentation
Conflicts of interest: none declared.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp572–574
574 Galli–Galli disease is an acantholytic variant of DDD, E. Sprecher et al.
Correspondence
A survey of approach to wound healing bysecondary intention among British Society forDermatological Surgery members
DOI: 10.1111/j.1365-2133.2006.07657.x
SIR, Secondary intention healing is a simple and swift ap-
proach to wound healing that, when used appropriately,
results in a satisfactory cosmetic and cost-effective outcome.1
There are, however, no guidelines on how best to manage
wounds healing by secondary intention postoperatively. A
study comparing hydrocolloid occlusive dressings and conven-
tional wound management (twice daily wound cleansing and
topical application of bacitracin ointment and an adhesive
dressing) found that wounds treated with occlusive dressings
healed quicker and were less painful.2 However, this study
looked only at shave biopsy and 3-mm punch biopsy sites. A
recent Cochrane review of dressings and topical agents for
general surgical wound healing by secondary intention3 was
unable to make any firm conclusions as only small, poor-qual-
ity trials exist. We decided to investigate what choices individ-
ual dermatologists with an interest in surgery make regarding
dressings and topical agents for wounds allowed to heal by
secondary intention.
British Society for Dermatological Surgery (BSDS) members
were sent a single-page questionnaire via e-mail and were
invited to participate in this study in September 2005. Two
months later, a reminder was e-mailed to members.
One hundred and eighty-two BSDS members with an e-mail
address were invited to participate. Fifty-one (28%) replied.
Twenty-nine of 51 (57%) had one dedicated surgical session
per week with the remainder having two to four surgical ses-
sions per week. Five (10%) carried out an average of fewer
than five operations per week, while 22 (43%) undertook five
to 10 per week and 24 (47%) performed > 10 per week. Sec-
ondary intention wound healing was used by 42 (82%) BSDS
members on fewer than three wounds per week, by eight
(16%) members on three to six wounds per week, and by a
single member (2%) on more than six wounds per week. The
choices made with regard to dressing type and topical agents
for wounds healing by secondary intention are illustrated in
Fig. 1a,b. The majority (n ¼ 40; 78%) said their patients had
fewer than three follow-up visits at the dermatology depart-
ment for wound care. Thirty-six (71%) members arranged for
follow-up wound care at the patient’s general practitioner and
most (71%) gave their patients a postoperative wound care
information sheet.
The questionnaire was kept brief to encourage uptake
among BSDS members. A response rate of less than a third was
therefore disappointing as published data on response rates
to physician surveys suggest that response rates are routinely
40–50%.4 The brevity of the questionnaire we used limited
the breadth of information obtained by this survey and issues
such as the influence of patient comorbidities or wound site
and size on selection of wounds for secondary intention heal-
ing were necessarily overlooked. None the less, we can make
some conclusions from the responses received. We surveyed a
group of dermatologists with a special interest in surgery. Their
specialist interest is confirmed by their workload recorded –
almost half of those surveyed performed > 10 operations per
week. Wounds were left to heal by secondary intention by the
respondents in a minority of cases. Perhaps this is a facet of
the population of dermatologists surveyed – those with a
greater interest and experience in surgical techniques may
prefer to close wounds with grafts, flaps etc. rather than allow
healing by secondary intention. There was no consensus on
either dressing type or topical agent used for wound care,
although alginate-based dressings and topica Polyfax� (poly-
myxin B+ bacitracin) ointment (PLIVA, Petersfield, U.K.) and
Bactroban� (mupirocin) cream (GlaxoSmithKline, Uxbridge,
02468
101214161820
Num
ber
of r
eplie
s
Dressing types
02468
1012141618
Num
ber
of r
eplie
s
Topical agents
Polyfax Bactroban Other agent Nothing No reply
Alginate
(a)
(b)
Gauze Hydrocolloid Foam Combination No reply
Fig 1. (a) Types of dressing used on wounds healing by secondary
intention. (b) Types of topical agent used by dermatologists on
wounds healing by secondary intention.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612 575
U.K.) were the most popular choices. Clearly there is intercen-
tre variation and choices regarding secondary intention wound
healing are most likely to be influenced by personal preference
and experience. The fact that almost one-fifth of those
surveyed use a combination of dressing types confirms the
variation in approach to postoperative care with secondary
intention healing. A study looking at larger dermatological
wounds would be required to establish whether one dressing
type or topical preparation carries benefits over another with
regard to satisfactory outcome of surgical wounds when
factors such as scarring and postoperative infection are
assessed. Furthermore, information on indication for surgery,
site and size of wound, comorbidities and facilities for self-
wound care postoperatively are all important factors that
should be taken into consideration in any future studies.
Acknowledgments
We thank Sean Semple of the BAD office for his assistance in
e-mailing the questionnaire to BSDS members. We also thank
all BSDS members who participated in the survey.
A . LA L LY
R . TURNER
Department of Dermatology,
Churchill Hospital, Oxford OX3 7LJ, U.K.
E-mail: Aoife.Lally@orh.nhs.uk
References
1 Christenson LJ, Phillips PK, Weaver AL, Otley CC. Primary closurevs second-intention treatment of skin punch biopsy sites: a random-
ized trial. Arch Dermatol 2005; 141:1093–9.2 Nemeth AJ, Eaglstein WH, Taylor JR et al. Faster healing and less
pain in skin biopsy sites treated with an occlusive dressing. ArchDermatol 1991; 127:1679–83.
3 Vermeulen H, Ubbink D, Goossens A et al. Dressings and topicalagents for surgical wounds healing by secondary intention. Cochrane
Database Syst Rev 2004; 2:CD003554.4 Shosteck H, Fairweather WR. Physician response rates to mail and
personal interview surveys. Public Opin Q 1979; 43:206–17.
Conflicts of interest: none declared.
Part of this work was presented in poster format at the British Society
for Dermatological Surgery section of the British Association ofDermatologists Annual Meeting in Manchester, July 2006.
Acute necrotic pancreatitis induced by severehypercalcaemia due to tacalcitol ointment
DOI: 10.1111/j.1365-2133.2006.07658.x
SIR, Acute necrotic pancreatitis results from severe aseptic
inflammation leading to tissue necrosis and abdominal pain.
Pancreatic necrosis can activate systemic inflammatory path-
ways and cause subsequent multiple organ failure with high
mortality.1 Gallstones and excessive alcohol intake account
for > 75% of cases of pancreatitis.2 Hypercalcaemia is a rare
cause of acute pancreatitis.3 Hypercalcaemia is induced by a
high parathyroid hormone (PTH) production, malignant
tumours with bone metastasis or paraneoplastic production of
PTH-related peptides. Rarely, excessive oral intake or topical
use of vitamin D has been identified to induce hyper-
calcaemia.4,5 However, hypercalcaemia due to topical use of
vitamin D-containing ointments is usually mild and is not
associated with severe organ dysfunction. We report a patient
with an excessive use of a vitamin D analogue-containing
ointment leading to acute hypercalcaemia and acute necrotic
pancreatitis.
A 28-year-old man was referred to the Department of
Dermatology for acute exacerbation of psoriasis [Psoriasis Area
and Severity Index (PASI) score of 68]. At admission he
denied systemic or topical treatment with vitamin D ana-
logues. Therapy with dithranol in alternation with tacalcitol
(Curatoderm�; Hermal, Reinbek, Germany) was initiated. The
latter treatment was limited to 30% of body surface area once
daily.
On the third day after admission he developed severe
abdominal pain and vomiting and was referred to the Depart-
ment of Gastroenterology. At this time the patient admitted
that he had already used a vitamin D analogue ointment
(tacalcitol) for 3 months at a mean dosage of about 30 g per
day. Thus, the cumulative dosage was approximately 2500–
3000 g, exceeding the manufacturer’s guidelines by eight to
10 times. No alcohol intake within recent days or symptoms
of an infection were reported. The patient did not take any
other medication or vitamin supplements.
Apart from the severe psoriasis and tenderness in the middle
abdomen, physical examination was unremarkable. The labora-
tory results showed an elevation of lipase (2340 U L)1, normal
13–60) and amylase activity (1186 U L)1, normal 13–53).
Serum calcium levels were greatly increased (3.45 mmol L)1,
normal 2.10–2.60; ionized calcium 1.85 mmol L)1, normal
1.15–1.30). PTH was totally suppressed (intact PTH
< 5 ng L)1), while 1,25-dihydroxyvitamin D3 (calcitriol)
levels were elevated (60 ng L)1, normal 16–57). Notably,
serum 25-hydroxyvitamin D3 (calcidiol) levels were extremely
increased (> 100 lg L)1, normal 9–38). Angiotensin-convert-
ing enzyme level was normal.
Computed tomography of the chest and abdomen 3 days
after the onset of symptoms showed acute pancreatitis with
pancreatic oedema and areas of necrosis but no evidence of
gallstones or obstructed bile ducts (Fig. 1). There were no
signs of lymphadenopathy suspicious for sarcoidosis.
Severe hypercalcaemia due to vitamin D intoxication was
considered to be responsible for the acute pancreatitis.
A treatment with isotonic saline infusions and furosemide
to achieve a high fluid turnover was started. However, as
calcium levels did not decrease significantly a 5-day course
of oral steroids (Decortin�; Merck, Darmstadt, Germany;
100 mg once daily) was initiated. This regimen led to a
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
576 Correspondence
normalization of laboratory parameters and clinical recovery.
After resolution of the pancreatitis, magnetic resonance cho-
langiopancreatography demonstrated an open biliary and pan-
creatic system without any evidence of stones or tumours
(Fig. 2).
Pancreatitis is an acute inflammation of the pancreas clinic-
ally characterized by abdominal pain and elevated pancreatic
enzyme activities.1 Hypercalcaemia is a rare cause of acute
pancreatitis.3 The pathophysiological mechanisms leading to
acute pancreatitis are not well understood. Recent evidence
suggests that calcium can precipitate in the pancreatic duct
and a calcium-dependent activation of trypsinogen occurs
within the parenchyma.6,7 Accordingly, calcium infusion in
rats leads to hyperamylasaemia and morphological alterations
of the pancreas.6
The major causes for hypercalcaemia are cancer and hyper-
parathyroidism.8 Hypercalcaemia can also be induced by high
concentrations of calcitriol or calcidiol due to an increase in
intestinal calcium absorption and bone resorption.8 Vitamin
D3 derivates (e.g. tacalcitol) have been used successfully in the
treatment of psoriasis.9 Ingestion of high doses of vitamin D
or topical application for the treatment of skin diseases has
repeatedly been shown to cause hypercalcaemia.4,5 However,
severe organ damage secondary to hypercalcaemia due to an
excessive topical application has yet not been reported. There-
fore our report of acute necrotic pancreatitis due to vitamin D
intoxication by an excessive use of vitamin D-containing oint-
ment seems of great clinical importance.
It is noteworthy that the PASI score was extremely high in
our patient, indicating a very severe disease where scaling
could have influenced drug absorption. Furthermore, the
cumulative application exceeded the manufacturer’s guidelines
by eight to 10 times and long-term exposure by two to three
times.10 This is particularly important as the amount of drug
absorption by the skin is often underestimated by patients and
ointments are considered as relatively harmless in comparison
with the oral preparation. Therefore, calcium levels should
be monitored in patients using vitamin D-containing medica-
tions with severe disease or who are likely to overdose the
treatment.
C . KNA CK S T EDT
R. WINOGRAD*
A. KOCH*
F . ABUZAHRA�C. TRAUTWE IN*
H.E . WASMUTH*
Medical Department I, *Medical Department III and
�Department of Dermatology,
University Hospital Aachen, RWTH Aachen,
Pauwelsstrasse 30, 52074 Aachen, Germany
Correspondence: Hermann E. Wasmuth.
E-mail: hwasmuth@ukaachen.de
References
1 Imrie CW. Acute pancreatitis: overview. Eur J Gastroenterol Hepatol1997; 9:103–5.
2 Kusnierz-Cabala B, Kedra B, Sierzega M. Current concepts on diag-nosis and treatment of acute pancreatitis. Adv Clin Chem 2003;
37:47–81.3 Brandwein SL, Sigman KM. Milk-alkali syndrome and pancreatitis.
Am J Med Sci 1994; 308:173–6.4 Jacobus CH, Holick MF, Shao Q et al. Hypervitaminosis D associated
with milk drinking. N Engl J Med 1992; 326:1173–7.5 Bourke JF, Berth-Jones J, Hutchinson PE. Hypercalcaemia with top-
ical calcipotriol. BMJ 1993; 306:1344–5.6 Mithofer K, Fernandez-del Castillo C, Frick TW et al. Acute hyper-
calcemia causes acute pancreatitis and ectopic trypsinogen activa-tion in the rat. Gastroenterology 1995; 109:239–46.
7 Ward JB, Petersen OH, Jenkins SA, Sutton R. Is an elevated concen-tration of acinar cytosolic free ionised calcium the trigger for acute
pancreatitis? Lancet 1995; 346:1016–19.
8 Ziegler R. Hypercalcemic crisis. J Am Soc Nephrol 2001; 12:S3–9.9 Lambert J, Trompke C. Tacalcitol ointment for long-term control
of chronic plaque psoriasis in dermatological practice. Dermatology2002; 204:321–4.
10 van de Kerkhof PCM, Berth-Jones J, Griffiths CEM et al. Long-termefficacy and safety of tacalcitol ointment in patients with chronic
plaque psoriasis. Br J Dermatol 2002; 146:414–22.
Conflicts of interest: none declared.
Fig 2. Magnetic resonance cholangiopancreatography showing the
gall bladder, common bile duct and pancreas. There is no evidence of
gall stones or compression due to tumours.
Fig 1. Computed tomography of the abdomen showing the pancreas
surrounded by traces of necrosis.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 577
Raised limb bands in infancy: a post-traumaticaetiology?
DOI: 10.1111/j.1365-2133.2006.07673.x
SIR, We read with great interest the article by Lateo et al.,1
recently published in this Journal, of four children with raised
limb bands. The authors argued that this entity could repre-
sent a new expression of amniotic band syndrome either
resulting from ‘exogenous’ constrictions by fibrous amniotic
bands or as the result of intrinsic developmental defects. We
report three further cases of raised limb bands and postulate
an acquired post-traumatic pathogenesis.
Patient 1. A 5-month-old girl born at term following an
uncomplicated pregnancy was referred for consultation regard-
ing linear lesions on the back of her lower legs that had first
appeared at the age of 2 months and were increasing in num-
ber. The patient’s mother noticed that the lesions were initial-
ly red and flat after wearing socks, and were still persistent.
While the redness faded, the eruption became progressively
palpable (Fig. 1a). The bands were asymptomatic and the
child was otherwise healthy. No notable trauma, except for
wearing socks, preceded their development. There was no
family history of similar lesions. Examination showed papular,
yellow, thin, linear, horizontal lesions on the mid-calves. They
were bilaterally symmetrical without any associated constric-
tion of the soft tissues or other limb abnormalities. Skin
biopsy revealed a dermal infiltrate of adipocytes along vessels
and eccrine ducts (Fig. 1b).
Patient 2. A 4-month-old boy born at term following an
uncomplicated pregnancy presented with two linear bands at
the posterior aspect of his legs. His mother reported red, flat
bands appearing below the elastic of his socks. Examination
revealed brown, raised lesions running horizontally on both
calves (Fig. 2) without any other associated abnormalities
especially on the lower limbs. No skin biopsy was performed.
There was no family history of similar lesions and there
was no evident trauma before the development of the
bands. Eighteen months later, the limb bands remained
unchanged.
Patient 3. An 18-year-old boy, with a past medical history of
mild mental retardation, was referred for consultation regard-
ing hyperpigmentated linear lesions on the back of his legs
and right thigh. The bands had been present since childhood.
Examination showed two brown, thin, raised, linear, hori-
zontal lesions on the mid-calves and one on the posterior
aspect of the right thigh; the patient also had a transverse
palmar gyrus. Skin biopsy showed an infiltrate of adipocytes
along capillaries in the upper dermis. Two years later, the
limb bands remained unchanged.
Since the first report provided by Meggit et al. in 2002,
13 cases (including the present ones) of raised limb bands
developing in infancy have been described.1–4 In addition,
two observations recently reported by Zhu et al.5 as ’congenital
curvilinear palpable hyperpigmentation’ may actually be the
same entity because of similarities of history, clinical presenta-
tion and evolution.
The cause of this eruption remains unknown although
amniotic band syndrome has been suspected because of asso-
ciated limb constrictions observed in four patients,1,2,4 shor-
tened toes in one patient2 and club foot in one patient.4
However, no oedema or constricting limb band and finger
abnormalities were present in our patients. Nevertheless, in
two cases the skin lesion was preceded by circular erythema,
horizontally localized on the mid-calves and strictly located
at the elastic band of the socks, clearly suggesting a
(a) (b)
Fig 1. Patient 1. (a) Thin, linear,
semicircular, palpable eruption running
horizontally on the mid-calf. (b) Skin biopsy
of the linear papules demonstrating a dermal
infiltrate of adipocytes along eccrine ducts and
vessels (haematoxylin and eosin; original
magnification ·100).
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
578 Correspondence
post-traumatic aetiology. To support this hypothesis, histology
of the skin lesions showed in two cases the ascent of adipo-
cytes into the upper dermis, commonly described in post-trau-
matic piezogenic papules of the feet. Therefore, we postulate
that postnatally acquired stricture could share the same trau-
matic mechanism as the compression described in the ’extrin-
sic’ model for pathogenesis of amniotic band syndrome and
may represent a new aetiology of raised limb bands.
M.M. MARQUE
B . GU I L LOT
G. L E GAL L I C
D . BE S S I S
Department of Dermatology,
University Hospital of Montpellier,
Montpellier, France
E-mail: myriammarque@yahoo.fr
References
1 Lateo SA, Taylor AEM, Meggit SJ. Raised limb bands developing in
infancy. Br J Dermatol 2006; 154:791–2.2 Meggitt SJ, Harper J, Lacour M, Taylor AEM. Raised limb bands
developing in infancy. Br J Dermatol 2002; 147:359–63.3 Russi DC, Irvine AD, Paller AS. Raised limb bands developing in
infancy. Br J Dermatol 2003; 149:436–7.4 Dyer JA, Chamlin S. Acquired raised bands of infancy: association
with amniotic bands. Pediatr Dermatol 2005; 22:346–9.5 Zhu IY, Fitzpatrick JE, Weston WL. Congenital curvilinear palpable
hyperpigmentation. J Am Acad Dermatol 2005; 53:162–4.
Conflicts of interest: none declared.
Human papillomavirus type 5 infectionin a patient with Hailey–Hailey diseasesuccessfully treated with imiquimod
DOI: 10.1111/j.1365-2133.2006.07667.x
SIR, Hailey–Hailey disease, also known as familial benign
pemphigus (FBP), is a rare autosomal dominantly inherited
blistering disease characterized by recurrent vesicles and
erosions, particularly involving flexural areas. The major
underlying pathological process in FBP is acantholysis of
keratinocytes caused by mutations in the genes encoding
the Golgi secretory pathway Ca2+-ATPase (SPCA1, ATP2C1),
affecting cell–cell adhesion. It is not uncommon for
patients with FBP to have secondary bacterial infection.
Nevertheless, FBP with human papillomavirus type 5 (HPV-5)
infection has not been reported in the literature. We report
a patient with FBP with concomitant HPV-5 infection suc-
cessfully treated with topical imiquimod. To our knowledge,
this is the first reported case of FBP associated with HPV-5
infection.
A 62-year-old Taiwanese man presented with a 15-year his-
tory of recurrent itching and oozing rashes involving the scro-
tum and perineum. Two years prior to his first visit to our
clinic, he started to notice asymptomatic papular eruptions at
the perianal area. At the time, he was diagnosed and treated
for tinea cruris and eczema. Nevertheless, the lesions did not
show any response to the prescribed topical antifungal agents
or topical corticosteroids.
On physical examination, multiple, small linear erosions on
an erythematous base were present at the scrotal area
(Fig. 1a). The perianal skin was macerated with a whitish
appearance. In addition, several discrete, pea- to bean-sized,
flat-topped, brownish hyperkeratotic papules with a tendency
to coalesce were observed at the perianal area (Fig. 1b).
To clarify the nature of the lesions, skin biopsy of the peri-
anal lesions was performed. The histopathological features
revealed irregular psoriasiform acanthosis with intraepidermal
clefting and acantholysis (Fig. 2a). Interestingly, cytopathic
changes including chromatin clumping and perinuclear
koilocytic features, and enlargement of keratinocytes, were
found in the spinous layer (Fig. 2b, c). The characteristic
clinical appearance, as well as biopsy, readily confirmed the
diagnosis of FBP, whereas the cytopathic changes as shown in
the histopathological analysis were suggestive of superimposed
cutaneous warts. Viral typing by nested polymerase chain
reaction (PCR) confirmed the pathogen to be HPV-5. With
reference to previously published methods,1 we used general
consensus primers MY09/MY11 for first PCR to amplify the
corresponding part of the HPV L1 gene, followed by nested
PCR primers GP+5/GP+6. Each PCR was carried out in
GeneAmp PCR system 9700 (Perkin-Elmer, Foster City, CA,
U.S.A.). HPV typing by DNA autosequence was analysed by
GenBank online BLAST server, which confirmed the pathogen
to be HPV-5.
Further investigation revealed a positive family history of
FBP. Due to the extensive nature of the lesions, topical 5%
imiquimod cream three times weekly was prescribed. Clinical
resolution of the lesions occurred within a month and side-
effects were minimal (Fig. 1c). Imiquimod was discontinued
after 1 month of treatment and no recurrence was observed
during a 12-month follow-up period.
The differential diagnoses of hyperkeratotic, flattened pa-
pules on the anogenital region in patient with FBP include
concomitant condylomata acuminata and the verrucoid variant
Fig 2. Patient 2. Brown, linear, horizontal and palpable bands on the
posterior aspect of the legs.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 579
of FBP. Verrucoid FBP typically has a flexural or genital distri-
bution.2 Rabinovitz and Patel have reported a case of isolated
perianal verrucoid familial benign chronic pemphigus which
was initially mistaken for condylomata acuminata.3 In
addition, Langenberg et al.4 have suggested that all verrucoid
genital lesions with the histological characteristics of Hailey–
Hailey disease may represent FBP. The morphological and
histopathological features of our patient were compatible with
verrucoid FBP. Nevertheless, the presence of HPV infection
confirmed by viral typing, as well as the good response to
topical imiquimod, favoured a diagnosis of FBP with con-
comitant HPV-5 infection, rather than a verrucoid FBP,
which has not been reported to have an association with HPV
infection.
Another interesting feature of our patient was that he
was infected with HPV-5, an oncogenic HPV commonly
(a)
(b)
(c)
Fig 1. (a) Multiple tiny, fissured erosions on an erythematous background were noted over the scrotum. (b) The perianal skin was brownish-
grey, with multiple dark brownish, flat papules scattered around the perianal area. (c) The perianal brown papules had resolved significantly at the
1-month post-treatment follow-up, leaving only mild pigmentation.
(a) (b) (c)
Fig 2. (a) Biopsy from the papular lesions showed acanthosis with suprabasal cleavage and acantholysis. (b) Characteristic ‘dilapidated brick wall’
appearance resulting from acantholytic keratinocytes. (c) Cytopathic changes with hypergranulosis, chromatin clumping and perinuclear halo
(arrowhead) were found in the overlying skin. Haematoxylin and eosin; original magnification: (a) · 100; (b) · 200; (c) · 400.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
580 Correspondence
associated with a rare genetic disease, epidermodysplasia
verruciformis (EV). The general population is usually not
affected by this particular virus due to genetic protection.5
Patients with EV were found to have a defective cell-mediated
immune response to HPV infection manifested by the inhi-
bition of natural cytotoxicity and the proliferation of
T lymphocytes against HPV-infected squamous cells in EV skin
lesions.5,6
Although considered specific to EV, HPV-5 has also been
found in several benign skin conditions such as psoriasis
vulgaris and in the epidermal repair process.7,8 It has been
proposed that following immunosuppressive treatment with
phototherapy or topical corticosteroids, psoriatic skin
may become the natural reservoir for HPV-5.7 However,
EV-associated HPVs, including HPV-5, are found as a latent
infection in the hair follicles and the normal skin of
immunosuppressed and, to a lesser extent, immunocompetent
non-EV individuals, so this association with psoriasis is
probably not specific.9–11 We propose that latent infection
with HPV-5 may become temporarily activated in diseases
or conditions involving wound repair processes where exten-
sive keratinocyte migration and proliferation occur.8 HPV-5
DNA would not be detectable in healed lesions once normal
cellular immunity against EV-associated papillomaviruses is
resumed in non-EV patients who are genetically resistant to
HPV-5.
Given that there is no known defect in cell-mediated im-
munity in patients with FBP, the repeated wound repair pro-
cesses in lesions of FBP, in combination with a compromised
local immunity caused by prolonged treatment with topical
corticosteroid, might have contributed to the development of
the HPV-5-associated lesions in our patient. An alternative ex-
planation is that the verrucoid variant of FBP is associated with
HPV-5 infection, but that this specific HPV type has not previ-
ously been adequately looked for as the primers used would
normally be expected to detect mucosal HPV types rather than
beta papillomaviruses. A larger study including more patients
with the verrucoid variant of FBP is necessary to substantiate
this possibility.
We used the histopathological findings of the skin
biopsy (Fig. 2), namely, minimal inflammation of the lesional
skin, as our guide to decide the treatment of choice. The
lack of immune response suggested that immunomodu-
latory agents might be beneficial in our patient by enhancing
his local immunity against HPV infection. We therefore
treated our patient with imiquimod 5% cream, which had
been proven to be clinically effective in treating anogenital
warts. In comparison with other treatment modalities such as
electrocautery, cryotherapy, chemodestruction or excision,
topical imiquimod application is less painful, with milder
tissue destruction.12 In our patient, three times weekly
application of imiquimod 5% cream brought about significant
clinical improvement within 1 month. Side-effects were
minimal.
We propose that conditions such as FBP or autoimmune
bullous diseases with clinically refractory wound healing may
be prone to EV-associated HPV infection. In such circum-
stances, topical immunomodulatory agents are a rational treat-
ment choice, although the exact therapeutic mechanism awaits
further exploration.
C-C . CHAN
H-Y. THONG*
Y-C. CHAN�Y-H. L I AO
Department of Dermatology,
National Taiwan University Hospital and
National Taiwan University College of Medicine,
7 Chung-Shan South Road, Taipei 100, Taiwan
*Department of Management Science and Engineering,
School of Engineering, Stanford University,
Stanford, CA, U.S.A.
�Department of Nursing, Taichung Armed Forces
General Hospital, Taichung, Taiwan
Correspondence: Y-H. Liao.
E-mail: yihua@ha.mc.ntu.edu.tw
References
1 Ko MJ, Chu CY. Disseminated human papillomavirus type 11infection in a patient with pemphigus vulgaris: confirmed by DNA
analysis. J Am Acad Dermatol 2004; 51:S190–3.2 Murakawa GJ, Kerschmann R, Berger T. Vulvar verrucous papules.
Arch Dermatol 1999; 135:204–5, 207–8.3 Rabinovitz AJ, Patel VJ. Isolated perianal verrucoid familial benign
chronic pemphigus. Cutis 1982; 30:660–2.4 Langenberg A, Berger TG, Cardelli M et al. Genital benign chronic
pemphigus (Hailey–Hailey disease) presenting as condylomas. J AmAcad Dermatol 1992; 26:951–5.
5 Majewski S, Malejczyk J, Jablonska S et al. Natural cell-mediatedcytotoxicity against various target cells in patients with epider-
modysplasia verruciformis. J Am Acad Dermatol 1990; 22:423–7.6 Cooper KD, Androphy EJ, Lowy D et al. Antigen presentation and
T-cell activation in epidermodysplasia verruciformis. J Invest Dermatol1990; 94:769–76.
7 Majewski S, Jablonska S. Possible involvement of epidermodys-plasia verruciformis human papillomaviruses in the immunopatho-
genesis of psoriasis: a proposed hypothesis. Exp Dermatol 2003;12:721–8.
8 Favre M, Majewski S, Noszczyk B et al. Antibodies to human papil-lomavirus type 5 are generated in epidermal repair processes.
J Invest Dermatol 2000; 114:403–7.9 Boxman IL, Berkhout RJ, Mulder LH et al. Detection of human
papillomavirus DNA in plucked hairs from renal transplantrecipients and healthy volunteers. J Invest Dermatol 1997; 108:712–
15.10 Boxman IL, Russell A, Mulder LH et al. Case–control study in a
subtropical Australian population to assess the relation betweennon-melanoma skin cancer and epidermodysplasia verruciformis
human papillomavirus DNA in plucked eyebrow hairs. TheNambour Skin Cancer Prevention Study Group. Int J Cancer 2000;
86:118–21.
11 Harwood CA, Surentheran T, Sasieni P et al. Increased risk of skincancer associated with the presence of epidermodysplasia verruci-
formis human papillomavirus types in normal skin. Br J Dermatol2004; 150:949–57.
12 Sauder DN. Immunomodulatory and pharmacologic properties ofimiquimod. J Am Acad Dermatol 2000; 43:S6–11.
Conflicts of interest: none declared.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 581
Perioral dermatitis in a patient withmyasthenia gravis following systemiccorticosteroid treatment
DOI: 10.1111/j.1365-2133.2006.07668.x
SIR, Perioral dermatitis is a common dermatosis presenting as a
persistent erythematous eruption composed of tiny papules
and papulopustules distributed symmetrically around the
mouth and over the chin. A narrow zone of sparing around
the vermilion border of the lips is a pathognomonic feature.
Additional lesions may arise on the nasolabial folds, glabella,
periocular skin and eyelids.1–5 Facial flushing and comedones
are notably absent. Classically, perioral dermatitis develops sec-
ondary to the use of topical corticosteroids or facial cosmetics.1
Young women (age 16–45 years) are most often affected,
although cases involving children and men have been repor-
ted.1,2 Treatment requires cessation of the offending agent with
or without the addition of antibiotics such as tetracycline or
erythromycin.1–5 Without adequate treatment, the disease
tends to run a persistent and fluctuating course. While perioral
dermatitis has been closely associated with topical corticoster-
oid use, the emergence of this condition in patients taking sys-
temic corticosteroids has rarely been reported in the literature.6
We report a 37-year-old woman with no significant past
medical history who was admitted following 2 days of gener-
alized muscle weakness, blurry vision, difficulty swallowing,
and voice changes. She was diagnosed with myasthenia gravis
(MG) following a positive edrophonium test, single fibre elec-
tromyography that demonstrated severe dysfunction at the
neuromuscular junction, and positive anti-MuSK antibody.
Antibodies against skeletal muscle and acetylcholine receptor
binding were negative. A total-body computed tomographic
scan revealed no signs of malignancy. She was treated with
oral prednisone 100 mg daily, pyridostigmine (Mestinon�;
Valeant, Costa Mesa, CA, U.S.A.), and was scheduled for five
rounds of plasmapheresis.
After 3 weeks of treatment with oral prednisone and pyrido-
stigmine, the dermatology department was consulted to evalu-
ate the patient for an asymptomatic eruption of 1–2-mm
erythematous papules distributed circumferentially around her
mouth and over her chin (Fig. 1). A small zone (2–3 mm) of
sparing was noted around her lip margin. She had minimal
involvement of the nasolabial folds, and no lesions of the
glabella, eyelids or periocular region. Physical examination also
revealed 23 small (< 1 mm), erythematous papulopustules
sparsely distributed across her upper back. No lesions were
observed on her neck, chest or extremities. She denied a history
of acne, topical corticosteroid or cosmetics use, or similar erup-
tions in the past. Based on this clinical presentation, the diagno-
sis of perioral dermatitis secondary to systemic corticosteroid
use was made. The patient was educated on the aetiology of
her dermatological condition and provided with treatment
options. She declined treatment of her perioral dermatitis at this
time, opting to wait for resolution of the disease as her corti-
costeroid dose was tapered over the coming months.
While numerous studies have linked perioral dermatitis with
topical corticosteroid use, only one other report, published in
this Journal by Adams et al. in 1982, specifically explores the
emergence of perioral dermatitis in five renal transplant
patients maintained on oral prednisolone.6 In our case, we
report a young woman who developed her first perioral erup-
tion 3 weeks after beginning oral prednisone 100 mg daily for
treatment of MG. Of note, the patients reported by Adams et al.
developed their eruptions after 7–13 months of systemic corti-
costeroids, whereas our patient’s lesions appeared in the first
month of steroid therapy, which is more consistent with the
time course of steroid-induced acneiform eruptions.
J .M. GOS S
K .M. NORD*
M.R. OLART E�M.E . GROS SMAN*
Columbia University, College of Physicians
and Surgeons, New York, NY, U.S.A.
Departments of *Dermatology and
�Neurology, Columbia University,
College of Physicians and Surgeons,
New York, NY, U.S.A.
Correspondence: Marc E. Grossman.
E-mail: marcmeg9@aol.com
References
1 Plewig G, Kligman AM. Perioral dermatitis. In: Acne and Rosacea, 3rdedn. Berlin: Springer-Verlag, 2000; 504–6.
2 Wilkinson DS, Kirton V, Wilkinson JD. Perioral dermatitis:a 12 year review. Br J Dermatol 1979; 101:245–57.
3 Cotterill JA. Perioral dermatitis. Br J Dermatol 1979; 101:259–62.4 Weber G. Rosacea-like dermatitis: contraindications or intolerance
reactions to strong steroids. Br J Dermatol 1972; 86:253–9.5 Hafeez ZH. Perioral dermatitis: an update. Int J Dermatol 2003;
42:514–17.6 Adams SJ, Davison AM, Cunliffe WJ, Giles GR. Perioral dermatitis in
renal transplant recipients maintained on corticosteroids and immu-nosuppressive therapy. Br J Dermatol 1982; 106:589–92.
Conflicts of interest: none declared.
Fig 1. A 37-year-old woman with perioral dermatitis appearing
3 weeks after the initiation of systemic corticosteroid treatment.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
582 Correspondence
Confluent and reticulated papillomatosissuccessfully treated with amoxicillin
DOI: 10.1111/j.1365-2133.2006.07676.x
SIR, Further to the recently published experience of the Mayo
Clinic in the treatment of confluent and reticulated papilloma-
tosis (CRP),1 we report a case of CRP treated successfully with
amoxicillin.
Confluent and reticulated papillomatosis is an unusual skin
disorder originally described by Gougerot and Carteaud. It
consists of a scaly, macular and papular eruption typically
affecting the seborrhoeic areas of young adults. As the
name suggests, lesions become confluent in the centre, coales-
cing to form a reticulated pattern at the periphery of affected
areas.
We describe a 29-year-old female who initially presented in
2004 with a 4-year history of an asymptomatic eruption
affecting the axillae, mons pubis, submammary (Fig. 1) and
inguinal folds. She had been treated unsuccessfully with
topical steroids and antifungal agents. Differential diagnoses
included a fungal eruption, pityriasis versicolor, Darier’s
disease and Dowling–Degos disease (reticulate pigmented
(a) (b)
Fig 2. Right axilla (a) before and (b) after
treatment.
Fig 1. Confluent and reticulated
papillomatosis affecting axillae, mons pubis
and submammary areas.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 583
anomaly of the flexures) but these were excluded by investi-
gations; skin scrapings for mycology were negative and a skin
biopsy showed mild chronic inflammation in the upper
dermis with some mild pigmentary incontinence. Wood’s
light examination was unremarkable. A skin swab and culture
of skin scrapings on chocolate agar were negative. Following
clinical and histological correlation a diagnosis of CRP was
made.
The patient was treated unsuccessfully with oral mino-
cycline 100 mg daily for 3 months, and thereafter all treat-
ment was stopped because the patient became pregnant.
During pregnancy, she was treated with amoxicillin 250 mg
(taken three times daily) for a coincidental lower respiratory
tract infection and noted dramatic improvement in her CRP.
Clinical examination confirmed complete clearance. A further
1-month course of amoxicillin 250 mg (taken three times
daily) was prescribed 3 months later for disease relapse.
Again, improvement was noted after approximately 1 week,
with clearance of the eruption within 3 weeks (Fig. 2),
confirming that the initial response was not coincidental.
Several theories for the cause of CRP have been suggested,
including an abnormal host response to Pityrosporum orbiculare
and more recently infection with the Dietzia strain of Actinomy-
cete.2 In conjunction with the microbiology team we attempted
to isolate Dietzia strain X from culturing skin scrapings on
chocolate agar but without success.
Various antibiotics have been reported to treat CRP, includ-
ing azithromycin, erythromycin and oral fusidic acid.3 A
recent retrospective analysis of 39 cases with CRP from
the Mayo Clinic reported complete clearance in 14 of
22 patients treated with minocycline therapy, partial
response in a further four of 22 patients and four cases
where a response to minocycline was not documented.1 Our
patient was initially treated with minocycline but this was not
beneficial.
To the best of our knowledge this is the first report of
successful treatment with amoxicillin in CRP.
R .F . DAV I S
K .E . HARMAN
Department of Dermatology, Leicester Royal Infirmary,
Leicester, U.K.
E-mail: rosiejohnston@doctors.org.uk
References
1 Davis MDP, Weenig RH, Camilleri MJ. Confluent and reticulate
papillomatosis (Gougerot–Carteaud syndrome): a minocycline-responsive dermatosis without evidence for yeast pathogenesis.
A study of 39 patients and a proposal of diagnostic criteria. Br JDermatol 2006; 154:287–93.
2 Natarajan S, Milne D, Jones AL et al. Deitzia strain X: a newlydescribed Actinomycete isolated from confluent and reticulated papillo-
matosis. Br J Dermatol 2005;153: 825–7.
3 Jang HS, Oh CK, Cha JH et al. Six cases of confluent and reticulatedpapillomatosis alleviated by various antibiotics. J Am Acad Dermatol
2001; 44:652–5.
Conflicts of interest: none declared.
Folliculotropic mycosis fungoides with CD30+large-cell transformation in a young woman:beneficial effect of bexarotene
DOI: 10.1111/j.1365-2133.2006.07672.x
SIR, Folliculotropic mycosis fungoides (MF) is a rare variant of
cutaneous T-cell lymphoma (CTCL). Folliculotropic MF has a
male/female ratio of 4–5 : 1. The sites of predilection are the
face, neck and upper trunk. Clinically, folliculotropic MF
shows acneiform lesions, including prominent lesional and
perilesional comedo-like eruptions, epidermal cysts, follicular
keratoses, erythematous patches and plaques. Sometimes there
may be additional mucinorrhoea and hair loss in affected
areas. Histologically, this subtype of MF is characterized by
atypical lymphocytes infiltrating predominantly the hair folli-
cles. Pautrier microabscesses may be present, as in nonfollicu-
lotropic MF.1,2 Mucinous degeneration of hair follicles may be
present. Folliculotropic MF has recently been suggested to be
more refractory to standard therapy and associated with a
worse prognosis as compared with classical MF.1 We report a
young woman with folliculotropic MF presenting a CD30+
large-cell transformation. She experienced full remission upon
treatment with low-dose bexarotene, administered for a time
period of 1 year.
At the age of 17 years our patient developed eczematous
lesions on her extremities, thought to be atopic dermatitis.
In addition, since the age of 19 years there was a progressive
development of acneiform lesions on her face with small and
larger cysts (Fig. 1a). At the age of 28 years she presented
with multiple poikilodermatous plaques containing small whi-
tish cysts. Skin biopsy showed the typical histological pattern
of folliculotropic MF (Fig. 1b). Immunohistologically the
atypical lymphocytes demonstrated a CD3+, CD4+ and CD8–
phenotype, with a few CD20+ B cells and CD30+ cells
admixed. Polymerase chain reaction analysis performed from
lesional skin showed a monoclonal rearrangement of the T-cell
receptor. Staging procedures did not reveal any lymph node
or organ infiltration of her lymphoma. Some months after
therapy with ultraviolet (UV) B 311 nm and systemic isotre-
tinoin (Roaccutane�; Roche, Mannheim, Germany) had been
started, she consecutively developed three fast-growing, ulcer-
ated cherry-sized nodules at her right elbow and at her chin
(Fig. 1c). Two nodules were excised and examined histologic-
ally. The epidermis was necrotic, covered with an overlying
crust. The dermis was filled with a dense sheet-like nodular
infiltrate, predominantly consisting of large CD3– cells, with
pale cytoplasm and prominent nucleoli, which stained positive
for CD30 (Fig. 1d). The diagnosis of CD30+ large-cell trans-
formation of folliculotropic MF was made. The patient’s whole
face and the CD30+ nodule on her chin were treated by elec-
tron beam irradiation (20 Gy). As the cysts persisted almost
unchanged in the facial area 3 months after radiotherapy
(Fig. 2a) as well as within the lesions on the trunk and
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
584 Correspondence
extremities, we initiated treatment with bexarotene (Targre-
tin�; Zeneus, Munich, Germany) 150–450 mg daily. After
18 weeks the first lesions showed healing. This treatment was
accompanied by a slow but steady further regression of cysts
and comedones, resulting in a nearly complete healing after a
period of 12 months (Fig. 2b) at a total dose of 129Æ98 g.
Follicular atrophy became apparent where the cystic lesions
had regressed. There was no relapse of follicular MF or
CD30+ large-cell transformed nodules during the whole treat-
ment period with bexarotene. The patient has been off therapy
for 4 months without signs of relapse.
Until now, folliculotropic MF, although a well recognized
variant of MF, has been reported only rarely. Possibly, due to
the intra- and perifollicular localization of the neoplastic
lymphocytes, follicular MF seems to be less responsive to
treatment modalities normally applied for classical MF.1 Some
authors reported a response to electron beam radiation, or
psoralen plus UVA alone or in combination with isotretinoin
and/or interferon alfa-2a.3–5 To date, to our knowledge only
two patients have been reported to be successfully treated with
oral bexarotene.6,7 Bexarotene is a selective retinoid X recep-
tor-binding molecule, which induces apoptosis in target cells.
At a dose of 300 mg m)2 daily, response rates of more then
50% are reported in early-stage CTCL.8
In our patient follicular MF underwent large-cell CD30+
transformation, presenting with rapidly growing nodules.
Transformation of MF is often associated with resistance to
therapy and poor prognosis.1 We treated our patient with local
radiation therapy, followed by therapy with bexarotene (initial
dose 150 mg daily, rising to 450 mg daily) and achieved
complete remission of plaques and cysts after 12 months.
New nodules were not encountered. It remains to be clarified
whether bexarotene, besides being active in folliculotropic MF,
will also inhibit the eruption of CD30+ transformed nodular
infiltrates in our patient. There are recent communications
indicating that transformation to a more aggressive CTCL
can occur in patients under therapy with bexarotene.9,10
C . MITT E LDORF
R . STADL ER*
H.P . B ERT SCH
C . NEUMANN
Department of Dermatology,
Goettingen University, von-Siebold-Str. 3,
37073 Goettingen, Germany
*Department of Dermatology,
Medical Centre Minden, Portastrasse 7–9,
32427 Minden, Germany.
E-mail: derhigoe@med.uni-goettingen.de
References
1 Kazakov DV, Burg G, Kempf W. Clinicopathological spectrum of
mycosis fungoides. J Eur Acad Dermatol Venereol 2004; 18:397–415.2 Flaig MJ, Cerroni L, Schuhmann K et al. Follicular mycosis fungoid-
es. A histopathologic analysis of nine cases. J Cutan Pathol 2001;28:525–30.
3 Leverkus M, Rose C, Brocker E-B, Goebeler M. Follicular cutaneousT-cell lymphoma: beneficial effect of isotretinoin for persisting
cysts and comedones. Br J Dermatol 2005; 152:176–98.4 Lacour JP, Castanet J, Perrin C, Ortonne JP. Follicular mycosis
fungoides. J Am Acad Dermatol 1993; 29:330–4.5 Willemze R, Jaffe ES, Burg G et al. WHO–EORTC classification for
cutaneous lymphomas. Blood 2005; 105:3768–85.
(a)
(c) (d)
(b)
Fig 1. Folliculotropic mycosis fungoides (MF) with CD30+ large-cell
transformation. (a) Initial clinical presentation with acneiform lesions
on the face, comprising small and larger cysts. (b) Scanning
magnification of a pretreatment skin biopsy showing the typical
histological pattern of folliculotropic MF, with epidermal cysts
(haematoxylin and eosin; original magnification · 50). Inset shows
epidermotropism of atypical lymphocytes in the cyst wall, forming
Pautrier microabscesses. (c) Fast growing, ulcerated cherry-sized
nodule on the chin. (d) Dense sheet-like nodular infiltrate,
predominantly consisting of large cells with pale cytoplasm and
prominent nucleoli which stained positive for CD30 (inset)
(haematoxylin and eosin; original magnification · 100).
(a) (b)
Fig 2. Folliculotropic mycosis fungoides with CD30+ large-cell
transformation. (a) Three months after electron beam irradiation of
the face (20 Gy), and before initiating therapy with bexarotene
(Targretin�). The cysts persisted almost unchanged in the facial area.
(b) After therapy with bexarotene 150–450 mg daily for 12 months,
total dose of 129Æ98 g. Nearly complete regression of cysts.
� 2006 The Authors
Journal Compilation � 2006 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 585
6 Apisarnthanarax N, Ha CS, Duvic M. Mycosis fungoides with fol-licular mucinosis displaying aggressive tumor-stage transformation:
successful treatment using radiation therapy plus oral bexarotenecombination therapy. Am J Clin Dermatol 2003; 4:429–33.
7 Shistik G, Scalf LA, Fenske N, Glass LF. Follicular mucosis fungoid-es: successful treatment with oral bexarotene. J Drugs Dermatol 2004;
3:301–4.8 Duvic M, Martin AG, Kim Y et al. Phase 2 and 3 clinical trial of oral
bexarotene (Targretin capsules) for the treatment of refractory orpersistent early-stage cutaneous T-cell lymphoma. Arch Dermatol
2001; 137:581–93.
9 Kreuter A, Altmeyer P. Rapid onset of CD8+ aggressive T-celllymphoma during bexarotene therapy in a patient with Sezary
syndrome. J Am Acad Dermatol 2005; 53:1093–5.10 Bouwhuis SA, Davis MDP, el-Azhary RA et al. Bexarotene treatment
of late-stage mycosis fungoides and Sezary-syndrome: developmentof extracutaneous lymphoma in 6 patients. J Am Acad Dermatol 2005;
52:991–6.
Conflicts of interest: none declared.
Lichen scrofulosorum caused byMycobacterium szulgai: a new cause of atuberculide reaction
DOI: 10.1111/j.1365-2133.2007.07683.x
SIR, A 26-year-old Ghanaian man presented with a 1-year his-
tory of an asymptomatic eruption. On examination there were
uniform 2–3-mm flesh-coloured, predominantly follicular
papules, some of which had central scale as well as a few tiny
pustules (Fig. 1). The papules were scattered on the face,
arms, dorsa of the hands, trunk and legs. There was no pal-
pable lymphadenopathy. He was otherwise completely well
and asymptomatic. Chest X-ray and blood tests including a full blood count
were normal. Histological examination of one of the papules
taken from the dorsum of the hand revealed a superficial
and deep granulomatous folliculitis, with well-formed
noncaseating epithelioid granulomas containing Langhans
giant cells which closely surrounded the hair follicle
(Fig. 2a,b). No acid-fast bacilli were seen on Ziehl–Neelsen
staining, and culture was negative. Amplification by poly-
merase chain reaction (PCR) from the skin biopsy indicated
the presence of mycobacterial DNA with the sequence of
the amplified fragment being that of the atypical myco-
bacterium Mycobacterium szulgai. A Mantoux test was strongly
positive with ulceration. Human immunodeficiency virus
serology was negative.
Treatment was commenced with standard quadruple anti-
tuberculous therapy (isoniazid, rifampicin, pyrazinamide and
ethambutol) and the eruption resolved completely within
6 weeks. After 2 months he ran out of medication, and the
rash recurred within several days. It resolved again when ther-
apy was re-instituted.
(a)
(b)
Fig 2. (a) Skin biopsy of a papular lesion (haematoxylin and eosin;
low power) showing perifollicular granulomatous inflammation and
overlying lichenoid change. (b) Skin biopsy of a papular lesion
(haematoxylin and eosin; high power) showing noncaseating
granulomatous infiltrate.
Fig 1. Clinical appearance of the papular eruption in this patient.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
586 Correspondence
The combination of the clinical features of a widespread
flesh-coloured, papular eruption, combined with the granulo-
matous folliculitis and a strongly positive Mantoux test, is
typical of the rare tuberculide reaction known as lichen
scrofulosorum. Tuberculides occur as the result of a vigorous
cell-mediated immune response in the skin to mycobacteria.1
Lichen scrofulosorum is the least frequently reported of
the tuberculide reactions,1 although it may be more common
than previously realised, as in a recent study of 511 patients
with tuberculosis, 39 of them had lichen scrofulosorum.2 It
generally occurs in children or young adults.1,2 Our patient
demonstrated the typical clinical features of flesh-coloured
perifollicular flat-topped or scaly papules, and pustules ran-
ging from 0Æ5 to 3 mm in diameter, particularly occurring
on the trunk.2 The histology is of noncaseating granulomas
surrounding hair follicles and sweat ducts.1 Lichen scrofuloso-
rum is usually associated with M. tuberculosis infection, gener-
ally of lymph nodes or bone rather than the lungs,1,2
although no internal focus of infection is found in 12–28%
of patients.2 Lichen scrofulosorum has also been reported to
occur along with other tuberculide reactions in some
patients.3 In addition to M. tuberculosis, lichen scrofulosorum
can be caused by other mycobacteria, including M. avium4 and
M. bovis (from bacille Calmette–Guerin vaccination).5 Lichen
scrofulosorum has not previously been reported in association
with M. szulgai infection. The response to treatment of tuber-
culous and nontuberculous forms of lichen scrofulosorum is
usually good, with resolution occurring within 1–4 months
of appropriate antimycobacterial treatment, as seen in our
patient.1 Untreated, lesions persist for months or years,
although spontaneous resolution without scarring may occur
eventually.2
In lichen scrofulosorum, mycobacteria are not visible on
microscopy of biopsied skin, and cannot be cultured from
the lesions.1,2 There are reports of identification of mycobac-
terial nucleic acid sequences from biopsied skin in erythema
induratum and papulonecrotic tuberculide using PCR.1,6 This
is the first case of the identification of mycobacterial nucleic
acid sequences from skin lesions in lichen scrofulosorum by
PCR: in two previous cases, PCR did not identify mycobacte-
rial sequences.3,6 The identification of mycobacterial nucleic
acid sequences by PCR despite negative microscopy and cul-
ture for mycobacteria from the skin lesions implies either
that the cell-mediated immune response in the skin keeps the
number of bacilli very low or that the PCR is identifying
fragments of dead mycobacteria only. Mycobacterium szulgai is a
slow-growing scotochromogenic mycobacterium widely dis-
tributed in the environment which rarely causes disease in
humans.7 It has been isolated from water, including aquaria.7
It can be distinguished from other mycobacteria by analysis
of its 16S ribosomal DNA sequence. Mycobacterium szulgai can
cause a lung disease similar to pulmonary tuberculosis, and
these patients are usually,8 but not always, immunosup-
pressed.9 It has also been reported to affect bones and
joints.1,8 Mycobacterium szulgai can cause a skin disease with
sporotrichoid spread of ulcerating lesions10 and subcutaneous
nodules.8 However, it has not previously been reported to
cause any tuberculide reactions. The mycobacterium is sensi-
tive to standard antituberculous agents,1,10 and sometimes
clarithromycin.10
This case highlights the value of PCR on skin biopsies for the
investigation of tuberculide reactions, and the commonality of
pathological responses which can be induced by a wide variety
of mycobacteria in the skin.
Acknowledgments
We thank Dr T. Harrison (Infectious Diseases Unit, St George’s
Hospital) for advice and assistance in managing the patient.
G .L . RO S S
H. CHONG*
T. COL LYNS�D.M. GASCOYNE-B INZ I�
R.P .E . SARKANY�
Skin & Cancer Foundation,
95 Rathdowne Street,
Carlton, Vic. 3053, Australia
Departments of *Cellular Pathology and
�Dermatology, St George’s Hospital,
Blackshaw Road,
London SW17 0QT, U.K.
�Department of Microbiology,
Leeds General Infirmary, Great George Street,
Leeds LS1 3EX, U.K.
Correspondence: Gayle Ross, 37 Union Road,
Surrey Hills, Vic. 3127, Australia.
E-mail: wiggills@yahoo.com.au
References
1 Yates VM, Rook GAW. Mycobacterial infections. In: Rook’s Textbook
of Dermatology (Burns DA, Breathnach SM, Cox NH, Griffiths CEM,eds), 7th edn, Vol. 2. Oxford: Blackwell Publishing, 2004; 28.20–
28.35.2 Singal A, Bhattacharya S. Lichen scrofulosorum: a prospective study
of 39 patients. Int J Dermatol 2005; 44:489–93.3 Park YM, Hong JK, Cho SH, Cho BK. Concomitant lichen scro-
fulosorum and erythema induratum. J Am Acad Dermatol 1988;38:841–3.
4 Komatsu H, Terunuma A, Tabata N, Tagami H. Mycobacterium aviuminfection of the skin associated with lichen scrofulosorum: report
of three cases. Br J Dermatol 1999; 141:554–7.5 Park YM, Kang H, Cho SH, Cho BK. Lichen-scrofulosorum-like
eruption localized to multipuncture BCG vaccination site. J Am AcadDermatol 1999; 41:262–4.
6 Torrelo A, Valverde E, Mediero I, Zambrano A. Lichen scrofuloso-rum. Pediatr Dermatol 2000; 17:373–6.
7 Abalain-Colloc ML, Guillerm D, Salaun M et al. Mycobacterium szulgaiisolated from a patient, a tropical fish and aquarium water. Eur J
Clin Microbiol Infect Dis 2003; 22:768–9.8 Cross GM, Guill MA, Aton JK. Cutaneous Mycobacterium szulgai infec-
tion. Arch Dermatol 1985; 121:247–9.9 Gur H, Porat S, Haas H et al. Disseminated mycobacterial
disease caused by Mycobacterium szulgai. Arch Intern Med 1984; 144:1861–3.
10 Kapur N, Schuster H, Parker N et al. Severe sporotrichoid infection
with Mycobacterium szulgai. Clin Exp Dermatol 2004; 29:337–9.
Conflicts of interest: none declared.
� 2007 The Authors
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Correspondence 587
Long-term efficacy of topical tacrolimuson oral lesions of chronic graft-versus-hostdisease
DOI: 10.1111/j.1365-2133.2006.07679.x
SIR, Chronic graft-versus-host disease (cGVHD) is a common
complication of allogeneic haematopoietic cell transplantation,
occurring about 100 days postallograft. cGVHD may involve
eyes, liver, intestine, skin and mucous membranes. The oral
mucosa is involved in 80% of patients with cGVHD, with
polymorphous lesions including erythema, erosions, ulcera-
tions, lichenoid striae, atrophy, superficial mucoceles and
xerostomia.1 Treatment of multiorgan cGVHD usually requires
immunosuppressive drugs, especially ciclosporin and cortico-
steroids, but oral cGVHD is often refractory to systemic treat-
ment. Therefore, adjunctive topical treatments may improve
clinical responses and prevent side-effects of systemic drugs,
especially when lesions are limited to the oral mucosa, and
can preserve the graft-versus-leukaemia effect of cGVHD. In
this way, several topical treatments such as corticosteroids
(prednisone, dexamethasone, budesonide), immunosuppres-
sants (ciclosporin, azathioprine) and phototherapy have been
used.2 The efficacy of topical tacrolimus on oral lesions of
cGVHD has recently been reported without evaluation of
recurrence after discontinuation of treatment.3,4 We report
three additional patients with oral and lip cGVHD resistant to
systemic treatments who were treated with tacrolimus oint-
ment, with a long period of follow up after discontinuation of
treatment.
Patient 1. A 41-year-old man allografted for acute myelogenous
leukaemia was treated for cutaneous, intestinal, ocular and oral
cGVHD. Systemic treatment included mycophenolate mofetil,
ciclosporin and budesonide, without local treatment for kera-
totic and ulcerative oral lesions (Fig. 1a). A topical treatment
with clobetasol propionate cream 0.5% and tacrolimus oint-
ment 0.1% three times daily was prescribed. One week later,
ulcers of the lip had resolved (Fig. 1b) while erosions of the
buccal mucosa persisted. However, the patient had not used
topical tacrolimus for the oral lesions as recommended. Two
months later, topical tacrolimus treatment was stopped due
to vomiting. Seven months later, oral and lip ulcers
relapsed despite treatment with clobetasol. Topical tacrolimus
was reintroduced and the lip lesions promptly regressed.
During the treatment, the blood level of tacrolimus was
undetectable.
Patient 2. A 58-year-old man had received allogeneic peripheral
blood progenitor cell transplantation for chronic lymphocytic
leukaemia. Five months after allograft, cGVHD occurred on
the skin, liver, eyes and mouth. Oral lesions were character-
ized by leucokeratosis, oral superficial mucoceles and ulcers of
the tongue, buccal mucosa and lips (Fig. 2a). Oral lesions
were refractory to systemic (corticosteroids, ciclosporin,
mycophenolate mofetil) and topical (prednisone, clobetasol)
treatments. The introduction of topical tacrolimus 0.1%
allowed a rapid clinical improvement in 1 month (Fig. 2b)
without detectable blood levels of tacrolimus. Topical treat-
ment was progressively withdrawn and after 1 year the oral
lesions had not relapsed (Fig. 2c).
Patient 3. A 61-year-old man had been treated for a chronic
myelogenous leukaemia by an allogeneic blood stem cell graft.
Three months after allograft, he developed liver, skin, ocular
and oral cGVHD that was treated with oral corticosteroids and
ciclosporin. Oral lesions consisted of leucokeratosis of the pal-
ate and buccal mucosa, xerostomia, and ulcers of the buccal
mucosa and lips. Topical clobetasol three times daily was pre-
scribed for 8 months, with poor results. This was replaced
with topical tacrolimus 0.1% three times daily. After 15 days
of treatment, the lip ulcers decreased in size. This treat-
ment was applied for 2 months and followed by a progressive
withdrawal over 1 month. One year later, the oral lesions
(a)
(b)
Fig 1. Patient 1. (a) Ulceration of the lip due to chronic
graft-versus-host disease. (b) After 1 week, the ulcers of the lip have
resolved with topical tacrolimus treatment.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
588 Correspondence
relapsed. A topical treatment including clobetasol and tacro-
limus was reintroduced, allowing total clearance after
1 month. During the treatment, the blood level of tacrolimus
was undetectable.
Oral lesions of cGVHD are often difficult to manage. Several
small studies2 have reported an improvement of cGVHD oral
lesions using topical therapies (prednisone, dexamethasone,
budesonide, ciclosporin, azathioprine, phototherapy) with
minor side-effects (local burning, bad taste, nausea). How-
ever, the results of these open-label trials with very small
groups must be interpreted with caution. Despite the appar-
ent benefits of adjunctive topical treatments, some patients
are not improved with combined systemic and topical treat-
ments.2 More recently, topical tacrolimus has been proposed
as an effective treatment of cutaneous and oral cGVHD.3–5
Tacrolimus is a macrolide with immunosuppressive properties
due to calcineurin inhibition. The mode of action of tacro-
limus which suppresses the production of local interleukin-2,
interferon-c and tumour necrosis factor-a, all involved in
cGVHD, could explain its efficacy.5 As observed on atopic
skin, topical tacrolimus could penetrate the oral epithelium6
and concentrate in mucosal connective tissue involved in
cGVHD. Furthermore, the cellular entry of tacrolimus is
independent of the glucocorticoid receptor. This could explain
the clinical benefit of tacrolimus in corticosteroid-resistant
patients. Blood levels of tacrolimus were undetectable in our
three patients, as in other studies in oral cGVHD or lichen
planus.7 This result strongly supports a local mechanism of
action.
Follow up after treatment has shown that two patients have
relapsed, after 7 and 12 months, respectively. This suggests
for the first time in cGVHD a benefit of topical tacrolimus in
terms of recurrence but, as has been shown in oral lichen
planus, relapse indicates a palliative and not a curative effect.7
Even though epidemiological studies have not shown an
increase of cutaneous cancer after topical use of tacrolimus the
Food and Drug Administration has recently issued public
health advice to inform about the potential cancer risk based
on a few case reports and animal studies.8 Further long-term
studies are necessary to assess the safety of topical tacrolimus,
especially in the treatment of oral cGVHD, an inflammatory
disease that can lead to squamous cell carcinoma.9,10
J . -C . FR I CA IN
V . S I B AUD*
N. SWETY ENGA�R. TABR I Z I�F . CAMPANA
A. TA I E B*
Departments of Odontology and
�Maxillofacial Surgery, Pellegrin University Hospital,
Bordeaux, France
*Department of Dermatology,
Saint Andre University Hospital, Bordeaux, France
�Department of Haematology,
Haut Leveque University Hospital, Pessac, France
E-mail: jean-christophe.fricain@biophys.u-bordeaux2.fr
References
1 Fricain JC, Sibaud V, Hafian H et al. Oral manifestations ofchronic graft-versus-host disease. Ann Dermatol Venereol 2005; 132:
1017–25.2 Imanguli MM, Pavletic SZ, Guadagnini JP et al. Chronic graft versus
host disease of oral mucosa: review of available therapies. Oral SurgOral Med Oral Pathol Oral Radiol Endod 2006; 101:175–83.
3 Sanchez AR, Sheridan PJ, Rogers RS. Successful treatment of orallichen planus like chronic graft-versus-host-disease with topical
tacrolimus: a case report. J Periodontol 2004; 4:613–19.
(a)
(b)
(c)
Fig 2. Patient 2. (a) Ulcer of the lip due to chronic graft-versus-host
disease. (b) Improvement of the lesion after 1 month of treatment
with topical tacrolimus. (c) One year after treatment with topical
tacrolimus, the lesion did not relapse.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 589
4 Eckardt A, Starke O, Stadler M et al. Severe oral graft-versus-host-disease following allogeneic bone marrow transplantation: highly
effective treatment with topical tacrolimus. Oral Oncol 2004;40:811–14.
5 Elad S, Or R, Resnick I et al. Topical tacrolimus – a novel treatmentalternative for cutaneous chronic graft-versus-host-disease. Transpl
Int 2003; 16:665–70.6 Gupta AK, Adamiak A, Chow M. Tacrolimus: a review of its use
for the management of dermatoses. J Eur Acad Dermatol Venereol 2002;16:100–14.
7 Olivier V, Lacour JP, Mousnier A et al. Treatment of chronic erosive
oral lichen planus with low concentrations of topical tacrolimus:an open prospective study. Arch Dermatol 2002; 10:1335–8.
8 Ormerod AD. Topical tacrolimus and pimecrolimus and the risk ofcancer: how much cause for concern?. Br J Dermatol 2005;
153:701–5.9 Demarozy F, Soligo D, Lodi G et al. Squamous cell carcinoma of
the oral cavity associated with graft versus host disease: report of acase and review of the literature. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 2005; 100:63–9.10 Szeto HC, Shek TWH, Lie AKW et al. Squamous cell carcinoma of
the tongue complicating chronic graft-versus-host-disease afterallogeneic hematopoietic stem cell transplantation. Am J Hematol
2004; 77:200–2.
Conflicts of interest: none declared.
Minocycline-induced pigmentation ofpre-existing capillaritis
DOI: 10.1111/j.1365-2133.2007.07680.x
SIR, Excellent absorption, long half-life and lipophilic proper-
ties inducing good tissue distribution have made minocycline
an effective therapeutic option in the treatment of acne vul-
garis. However, use of minocycline has been associated with
various forms of hyperpigmentation, incidence of which is
well reported in the dermatology and rheumatology literature.
We report a patient with minocycline-induced discoloration
of capillaritis, with no evidence of dyspigmentation elsewhere.
Such localized pigmentation at the site of pre-existing capilla-
ritis appears to be a unique phenomenon, not previously
reported.
A 36-year-old man with insulin-dependent diabetes mellitus
who had received a renal transplant was referred by the renal
physicians for management of worsening nodulocystic acne.
His immunosuppressive cocktail included azathioprine, predn-
isolone and tacrolimus. Dermatological examination showed
moderately severe, tender nodulocystic acne affecting the face,
chest and back. Asymptomatic capillaritis of both lower legs
was also noted. Treatment was commenced with oral mino-
cycline modified release capsules 100 mg daily (Minocin MR�;
Lederle, Maidenhead, U.K.) in conjunction with topical 0Æ01%
tretinoin gel (Retin-A�; Janssen-Cilag, High Wycombe, U.K.).
Eighteen months later, after several defaulted dermatology
appointments the patient presented with slate-blue discoloration
of both lower legs at sites of pre-existing capillaritis (Fig. 1). No
evidence of dyspigmentation was noted at other cutaneous or
mucosal sites, and the patient denied any other symptoms of
minocycline toxicity. As his acne had improved only slightly,
treatment with minocycline was discontinued. This led to a slow
but gradual resolution of the dyspigmentation.
Dyspigmentation is a recognized adverse effect of mino-
cycline which produces three distinct types of cutaneous
pigmentation: type I, blue-black pigmentation confined to
sites of scarring or inflammation on the face; type II,
blue-grey circumscribed pigmentation of normal skin of the
lower legs and forearms; and type III, diffuse muddy brown
pigmentation of normal skin accentuated in sun-exposed
areas.
Mouton et al.1 described a fourth type of minocycline-
induced pigmentation (MIP) in two patients with circum-
scribed blue-grey pigmentation within acne scars confined to
the back. MIP is not confined to the skin and mucosa, but has
also been noted to occur in bone,2 thyroid,3 atherosclerotic
plaques,4 subcutaneous fat5 and heart valves.6
Based on histochemistry, immunohistochemistry, electron
microscopic and energy-dispersive X-ray analyses, it has been
speculated that the pigment may be a drug metabolite–
protein complex chelated with calcium, or an insoluble
minocycline–melanin complex.1 Similar observations have
been made by other authors who noted electron-dense gran-
ules in MIP skin biopsy samples showing the presence of
larger quantities of iron and smaller quantities of sulphur and
calcium.5 Despite these findings, the exact pathogenesis of
MIP remains obscure, along with the explanation for the
observation of preferential involvement of specific tissue sites
with sparing of others. Pigmentation at the site of pre-existing
capillaritis in our patient may be explained by increased red
cell extravasation and perhaps increased minocycline depos-
ition in these areas. Occurrence of MIP on the lower legs may
Fig 1. Minocycline-induced blue-grey pigmentation at the site of
pre-existing capillaritis.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
590 Correspondence
be a type II variant, but the involvement of pre-existing capil-
laritis and sparing of normal skin appears to be unique.
V . MADAN
J .T . L E AR
Department of Dermatology,
Central Manchester and Manchester Children’s
University Hospital NHS Trust,
Manchester Royal Infirmary,
Oxford Road, Manchester M13 9WL, U.K.
E-mail: vishalmadan@doctors.org.uk
References
1 Mouton RW, Jordaan HF, Schneider JW. A new type of mino-
cycline-induced cutaneous hyperpigmentation. Clin Exp Dermatol2004; 29:8–14.
2 Pandit S, Hadden W. Black pigmentation of bone due to long-termminocycline use. Surgeon 2004; 2:236–7.
3 Birkedal C, Tapscott WJ, Giadrosich K et al. Minocycline-inducedblack thyroid gland: medical curiosity or a marker for papillary
cancer? Curr Surg 2001; 58:470–1.
4 Gerson DM, Robinson M. Black pigmentation of atherosclerotic pla-ques associated with chronic minocycline therapy. Cardiovasc Pathol
2006; 15:168–70.5 Fakhfakh AC, Humbert P, Aubin F. Cutaneous pigmentation induced
by minocycline: ultrastructural analysis and X-ray microanalysis.Ann Dermatol Venereol 1992; 119:975–9.
6 Sant’Ambrogio S, Connelly J, DiMaio D. Minocycline pigmentationof heart valves. Cardiovasc Pathol 1999; 8:329–32.
Conflicts of interest: none declared.
Anti-desmoglein-1 antibodies are prevalentin Tunisian patients with hydatidosis andleishmaniasis
DOI: 10.1111/j.1365-2133.2006.07687.x
SIR, Pemphigus is an autoimmune blistering disease mediated
by pathogenic antibodies directed against desmoglein (Dsg) 1
and Dsg3.1,2 In Tunisia, pemphigus has particular epidemio-
logical features. It occurs as an endemic form with an inci-
dence rate of 6Æ7 cases per million per year. Case series
suggest that pemphigus, especially the foliaceus form (PF), is
frequent among young Tunisian women with an incidence
rate reaching 20 cases per million in rural areas. This could be
related to a risk factor more prevalent and/or more often
expressed in these areas.3,4
We have reported previously that anti-Dsg1 antibodies were
prevalent in Tunisian healthy subjects, which argues for the
role of a still unknown environmental factor in the occurrence
of Tunisian endemic pemphigus.5 An epidemiological case–
control study demonstrated that several environmental factors
were significantly associated to Tunisian endemic pemphigus
such as traditional cosmetics, Turkish baths and cutting up
raw poultry.6 However, infectious factors have not been yet
investigated.
Recent data demonstrated that in endemic areas of Fogo
selvagem, anti-Dsg1 antibodies were found not only in
patients with PF, but also in patients with parasitic diseases,
thus suggesting a possible involvement of these microorgan-
isms in the aetiopathogenesis of pemphigus.7
In Tunisia, hydatidosis and leishmaniasis are two endemic
parasitoses that are predominant mainly in rural areas where a
higher incidence of Tunisian endemic pemphigus has also
been reported.8 Based on these observations, we aimed to
search for a link between these two parasitoses and Tunisian
pemphigus. For this purpose, we searched for antibodies
against Dsg1 and Dsg3 in sera samples of parasitic patients.
Dsg reactivity was compared with a group of Tunisian normal
subjects.
Serum samples were obtained from 58 patients with parasi-
tic disease, 35 with hydatidosis and 23 patients with visceral
leishmaniasis. We also obtained sera from 59 patients with PF
and 152, age-, sex- and origin-matched, Tunisian normal
individuals. Anti-Dsg1 and anti-Dsg3 antibodies were detected
by a commercial enzyme-linked immunosorbent assay (ELISA)
(MBL, Nagoya, Japan) using recombinant ectodomain of Dsg1
and Dsg3, respectively. The assay method was performed
according to the manufacturer’s instructions. The cut-off
values were set at 14 and 7 for anti-Dsg1 and anti-Dsg3
ELISAs, respectively. Index values ranging from the cut-off
value to 20 were considered as weakly positive. For all posi-
tive sera, we completed with (i) IgG subclass studies by ELISA,
using antihuman IgG1, IgG2, IgG3 and IgG4 murine mono-
clonal antibodies (Sigma, Steinheim, Germany); (ii) indirect
immunofluorescence on monkey oesophagus using a fluo-
rescein-conjugated antihuman IgG antibody (The Binding Site,
Birmingham, U.K.); and (iii) immunoblot analysis using
human epidermal extract as described.9 We also tested patients
with PF and controls for antileishmania and antihydatid fluid
antibodies using an ‘in house’ ELISA as described.10,11 Plates
were coated with hydatid fluid or soluble leishmania extract
and cut-off values were calculated as the mean plus 2 SD of
assay values obtained from Tunisian healthy subjects.
For Dsg1 ELISA, 55 (93Æ22%) patients with PF had positive
values, as expected and observed in other studies. Ten sera
from healthy individuals (6Æ57%) were also positive for anti-
Dsg1 antibodies. In the infectious disease group, we found
positive results in 14 of the 35 hydatidosis sera (40%) and five
of the 23 visceral leishmaniasis sera (21Æ7%). The prevalence
of anti-Dsg1 in these two groups was significantly higher than
that of the control group (P < 10)4 and P ¼ 0Æ04, respect-
ively). Index values were higher than 20 for 11 sera from the
hydatidosis group, and for two sera from the visceral leish-
maniasis group (Fig. 1a).
For Dsg3 ELISA, only three sera from patients with hydati-
dosis (8Æ5%) and four sera from patients with visceral leish-
maniasis (17Æ3%) were positive. The prevalence of antibodies
directed against Dsg3 was significantly higher (P ¼ 0Æ004) in
sera from the visceral leishmaniasis group than the control
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 591
group (3%). However, all index values were weakly positive
in the visceral leishmaniasis group (Fig. 1b).
IgG subclass studies showed that anti-Dsg1 antibodies found
in patients with hydatidosis were IgG2 in all cases. In two
patients, additional IgG1 and IgG3 anti-Dsg1 antibodies were
also detected. In visceral leishmaniasis patients, IgG3 was the
most common subclass, followed by IgG1 and IgG2. Similar sub-
class distribution was noted for anti-Dsg3 antibodies (Table 1).
Among all positive sera (for anti-Dsg1 and or anti-Dsg3)
from parasitic patients, only two sera, from a patient with
hydatidosis and a patient with visceral leishmaniasis, showed
intercellular staining on monkey oesophagus with a titre of 1:
10. By immunoblot analysis on epidermal extract, only one
serum from a patient with hydatidosis reacted with the
160-kDa band of Dsg1.
The prevalence of antibodies against parasitic antigens in sera
samples from patients with PF did not differ significantly from
the control group. For antihydatid fluid antibodies, similar prev-
alences (4%) were detected in patients with PF and in controls.
A higher prevalence of anti-soluble leishmania extract anti-
bodies was observed (10% in patients with PF vs. 5% in con-
trols); however, the difference was not statistically significant.
In this study, we demonstrated that a significant number
of patients with parasitic diseases, such as leishmaniasis and
hydatidosis, possess anti-Dsg1 and/or anti-Dsg3 antibodies
when tested against the ectodomain of desmogleins using
both highly specific and sensitive antibodies. These anti-
bodies belong to IgG1, IgG2 and IgG3 subclasses but were
never IgG4, which is the most common in Tunisian
patients with pemphigus.5 Recent data demonstrated that in
endemic areas of Fogo selvagem, anti-Dsg1 antibodies are
prevalent in patients with parasitic diseases, thus suggesting
a possible involvement of these microorganisms in the aeti-
opathogenesis of pemphigus.7 In this study, Diaz et al.7
found that 83% of patients with onchocerciasis, 43% of
patients with leishmaniasis and 58% with Chagas disease
have anti-Dsg1 antibodies. Epitope mapping studies revealed
that all positive sera recognized epitopes on the EC5 (extra-
cellular 5) domain of Dsg1. The authors hypothesized that
this domain might contain the initial epitope(s) that trig-
ger(s) the autoimmune response in pemphigus. In our
study, the majority of positive sera gave negative staining
by indirect immunofluorescence and immunoblot analysis.
Several explanations may account for the discrepancy
between results given by ELISA and other immunological
techniques. Firstly, the solid phase ELISA may be more sen-
sitive than other assays for detecting anti-Dsg antibodies.
Secondly, and as reported by Li et al.,12 antibodies against
anti-EC5 gave negative staining by indirect immunofluores-
cence due to the cryptic nature of the EC5 domain. We
can hypothesize that anti-Dsg1 antibodies detected in our
parasitic patients might be directed against cryptic, conform-
ational epitopes on the EC5 domain. Further studies using
an immunoprecipitation assay with recombinant proteins are
needed to confirm this point.
The second finding of this study is that pemphigus
patients had negative serological tests for hydatidosis and
leishmaniasis. Similar findings were also observed in Fogo
selvagem sera, which did not recognize parasitic antigens.
Diaz et al. hypothesized that saliva of the haematophagous
vector containing cadherin proteins could be the sensitizing
antigens and not the parasite itself.7 Although there is no
haematophagous vector for hydatidosis, this parasite could
use ‘cadherin-like’ molecules in the initial phase of invasion
Fig. 1. Analysis of sera from patients with pemphigus foliaceus (n),
hydatidosis (h),visceral leishmaniasis (e) and normal subjects (s) in
ELISA using the recombinant ectodomain of desmoglein (Dsg) 1 (a)
and of Dsg3 (b). The red line shows the cut-off value. The mean
index value of each group is indicated.
Table 1 Reactivity of IgG subclasses with desmoglein 1 and 3 in
patients with hydatidosis and visceral leishmaniasis
IgG subclasses
Hydatidosis
(n ¼ 35)
Visceral leishmaniasis
(n ¼ 23)
Dsg1 ELISAIgG 14 5
IgG1 1 (7%) 1 (20%)IgG2 14 (100%) 1 (20%)
IgG3 1 (7%) 2 (40%)IgG4 0 (0%) 0 (0%)
None 0 (0%) 1 (20%)Dsg3 ELISA
IgG 3 4IgG1 1 (33%) 0 (0%)
IgG2 3 (100%) 1 (25%)IgG3 0 (0%) 3 (75%)
IgG4 0 (0%) 0 (0%)None 0 (0%) 0 (0%)
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
592 Correspondence
(oncosphere stage) to cross the intestinal barrier. It is of note
that many arguments are in line with a potential role of
hydatidosis in the aetiopathogenesis of pemphigus. Firstly,
these two diseases are endemic in rural areas and occur more
frequently in women. Interestingly, contact with ruminants,
which are hosts for Echinococcus granulosis, was found to be a
risk factor for Tunisian endemic pemphigus.6 Secondly,
hydatidosis seems to offer an adequate microenvironment for
IgG4 isotype switching.13
In conclusion, our results confirm that anti-Dsg1 antibodies
are prevalent in patients with parasitic diseases, suggesting a
possible link of these environmental factors to Tunisian endemic
pemphigus. Additional experiments and follow-up of subjects
involved in the present study are needed to confirm a direct link
between these parasitoses and Tunisian endemic pemphigus.
Acknowledgments
This work was supported by a grant from Le Comite Mixte
Pour La Cooperation Universitaire Franco-Tunisenne. We thank
Pr Louzir H for kindly providing leishmania extract.
M. KAL L E L S E L LAM I*
M. Z I TOUN I*
W. TOMB AR I*
M. BEN AYED�O. AB IDA�
L . LAADHAR*
M. MOKN I�B. FEZ ZA§
H. TURK I–
I . MOKHTAR**
A. BEN OSMAN�R. KAMOUN MOHAMED§
P . JO LY��F . TRON��
D. G I L B E R T��H. MASMOUD I�
S . MAKN I*
(MEMBER S O F TH E ‘FRANCO-TUN I S I AN GROUP OF SURV EY AND
RE S E ARCH ON TUN I S I AN ENDEM I C P EMPH IGU S ’ )
*Laboratoire d’Immunologie and
�Service de Dermatologie,
Hopital La Rabta 1007, Tunis, Tunisia
�Laboratoire d’Immunologie,
Hopital Habib Bourguiba, Sfax, Tunisia
§Service de Dermatologie,
Hopital Charles Nicolle, Tunis, Tunisia
–Service de Dermatologie,
Hopital Hedi Chaker, Sfax, Tunisia
**Service de Dermatologie,
Hopital Habib Thameur, Tunis, Tunisia
��INSERM U519, Institut Federatif
de Recherche Multidisciplinaire
sur les Peptides (IFR23), Faculte Mixte
de Medecine et de Pharmacie, Rouen, France
Correspondence: M. Kallel Sellami
E-mail: maryam_kallel@yahoo.com
References
1 Koulu L, Kusumi A, Steinberg MS et al. Human autoantibodies
against a desmosomal core protein in pemphigus foliaceus. J ExpMed 1984; 160:1509–18.
2 Ogawa MM, Hashimoto T, Konohana A et al. Immunoblot analysisof Brazilian pemphigus foliaceus antigen using different antigen
sources. Arch Dermatol Res 1990; 282:84–8.3 Morini JP, Jomaa B, Gorgi Y et al. Pemphigus foliaceus in young
women. An endemic focus in the Sousse area of Tunisia. ArchDermatol 1993; 129:69–73.
4 Bastuji Garin S, Souissi R, Blum L et al. Comparative epidemiologyof pemphigus in Tunisia and France: unusual incidence of pemphi-
gus foliaceus in young Tunisian women. J Invest Dermatol 1995;104:302–5.
5 Kallel Sellami M, Ben Ayed M, Mouquet H et al. Anti-desmoglein 1antibodies in Tunisian healthy subjects: arguments for the role of
environmental factors in the occurrence of Tunisian pemphigusfoliaceus. Clin Exp Immunol 2004; 137:195–200.
6 Bastuji Garin S, Turki H, Mokhtar I et al. Possible relation ofTunisian pemphigus with traditional cosmetics: a multicenter case–
control study. Am J Epidemiol 2002; 155:249–56.7 Diaz LA, Arteaga LA, Hilario-Vargas J et al. Anti-desmoglein-1 anti-
bodies in onchocerciasis, leishmaniasis, and Chagas disease suggest
a possible etiological link to Fogo selvagem. J Invest Dermatol 2004;123:1045–51.
8 Ben Rachid MS, Ben Ammar R, Redissi T et al. Geography of majorparasitosis in Tunisia. Arch Inst Pasteur Tunis 1984; 61:17–41.
9 Hashimoto T, Ogawa MM, Konohana A, Nishikawa T. Detection ofpemphigus vulgaris and pemphigus foliaceus antigens by immuno-
blot analysis using different antigen sources. J Invest Dermatol 1990;94:327–31.
10 Maalej IA, Chenik M, Louzir H et al. Comparative evaluation ofELISAs based on ten recombinant or purified Leishmania antigens
for the serodiagnosis of Mediterranean visceral leishmaniasis. Am JTrop Med Hyg 2003; 68:312–20.
11 Rickard MD, Honey RD, Brumley JL, Mitchell GF. Serological diag-nosis and post-operative surveillance of human hydatid disease.
The enzyme-linked immunosorbent assay (ELISA) using variousantigens. Pathology 1984; 16:211–15.
12 Li N, Aoki V, Hans-Filho G et al. The role of intramolecular epitopespreading in the pathogenesis of endemic pemphigus foliaceus
(Fogo selvagem). J Exp Med 2003; 197:1501–10.13 Aceti A, Pennica A, Teggi A et al. IgG subclasses in human hydatid
disease: prominence of the IgG4 response. Int Arch Allergy Immunol1993; 102:347–51.
Conflicts of interest: none declared.
Needle-free anaesthesia prior to botulinumtoxin type A injection treatment of palmarand plantar hyperhidrosis
DOI: 10.1111/j.1365-2133.2007.07691.x
SIR, Botulinum toxin type A (BTX-A) has emerged as a new
technique to treat intractable cases of focal hyperhidrosis
(HH).1 Injections on the palms and soles, although effective,
are very painful and need some form of anaesthesia. Peripheral
nerve blockade, the standard method of anaesthesia used, may
have serious drawbacks,2 particularly when used repeatedly.
Needle-free anaesthesia may offer an alternative solution.3 The
technique involves the use of needle-free injection of 0Æ02–
0Æ04 mL of 2% lidocaine without adrenaline prior to BTX-A
injection with needle. The mixture of lidocaine and BTX-A
does not jeopardize toxin potency.4
The device used to achieve needle-free anaesthesia is
the MED-JET� MBX (Medical International Technologies,
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 593
Montreal, QC, Canada; Fig. 1), which has the advantage over
similar devices such as the Dermojet� (Robbins Instruments,
Chatham, NJ, U.S.A.), of providing an adjustable range of vol-
umes and pressures necessary to control the penetration depth.
For palmar HH, the volume is adjusted to 0Æ02 mL per spurt
(range 0Æ01–0Æ1 mL) and the pressure system set to 120 p.s.i.
A first shot of lidocaine is discharged at the centre of the palm
and if a superficial skin weal fails to appear, the pressure is
steadily raised until a weal is obtained. Once the weal is
formed, injections are evenly distributed at 1Æ5–2-cm intervals
over the whole palmar surface including the fingers. For plan-
tar HH, higher volumes (0Æ04 mL) and pressure may be
required to produce an anaesthetic weal. On average, 50 sites
per hand or 30 sites per foot are injected in this manner.
BTX-A is injected through a 31 gauge needle preferably after
each sequence of four or five lidocaine weals (Fig. 2). Injec-
tions are carried out intradermally rather than subcutaneously
in an attempt to reduce the incidence of significant muscular
weakness.5 Universal precautions including full-face shield or
face mask and protective eyeglasses are taken to avoid contam-
ination caused by potential blood splatter. The device has
recently been approved by Health Canada, and U.S. Food and
Drug Administration approval is pending.
Between August 2004 and July 2005, 16 patients have been
treated with this technique. Clinical data were collected
through a questionnaire and are summarized in Table 1. All
Fig 2. Botulinum toxin type A (BTX-A)
injection with 31 gauge needle follows each
sequence of formation of four or five
lidocaine weals with the needle-free device.
Fig 1. The MED-JET� MBX model by Medical
International Technologies Inc. (Montreal,
Canada), a subsidiary of Medical International
Technology U.S.A.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
594 Correspondence
patients found the needle-free anaesthesia tolerable and accep-
ted to be reinjected if their treatment was effective.
The injected dose varied between 50 and 200 units of
BTX-A (Botox�; Allergan Inc., Irvine, CA, U.S.A.) recon-
stituted in preserved saline (3–5 mL per vial of 100 units).
Initially 50 or 100 units of BTX-A were injected according
to the size of the injected site and later increased up to a
total of 200 units per site if a satisfactory result was not
obtained. All patients gave written informed consent prior to
commencement of the BTX-A injections. All patients were eli-
gible candidates to receive BTX-A injections after failing to
respond to topical preparations6 and/or iontophoresis.
Patient 1 experienced an unexpected remission of her axil-
lary and plantar HH, following her palmar treatment with
BTX-A. Patients 4 and 6 complained of severe muscle weak-
ness of their hands that persisted for 26 and 12 weeks,
respectively. The time lapse between the injection of BTX-A
and its onset of action was 6 weeks in patients 11 and 15.
Patient 16 was needle phobic: he requested to be treated
with BTX-A directly on his left hand. BTX-A was reconstituted
in 1Æ5 mL preserved saline and the volume per spurt was
adjusted to 0Æ03 mL. This setting provided 2 units of BTX-A
per spurt for a total dose of 100 units. After failing to respond
to this dosage, he was given an additional 50 units of BTX-A
on his left hand, and 150 units on his right hand, this time
with needle-free anaesthesia prior to BTX-A injection with
needle. His treatment remained effective even 6 months after
his initial injections.
The average duration of the effect of BTX-A was 8 months
in this group. When the effect of BTX-A started to fade away,
topical aluminium chloride preparations were used to try to
extend the interval between BTX-A injections. Other side-
effects were limited to mild ecchymoses that faded away in a
week or two.
The majority of recent research has demonstrated less
painful injection with needle-free jet devices compared with
traditional needle delivery.7 Needle-free devices are used to
inject all types of liquid: anaesthetics, corticosteroids and even
BTX-A.
Dermojet� has been successfully used to inject BTX-A for
plantar HH8 but the authors found the method inadvisable for
palmar HH because of possible injury to the superficial palmar
nerves or vessels. Using smaller volumes and lower pressure
would certainly control the desired penetration depth and mini-
mize this type of injury. Further studies using objective evalu-
ation methods are warranted on a larger number of patients.
A . BENOHAN I ANDermatology Division, Department of Medicine,
University of Montreal,
Montreal, Quebec, Canada
E-mail: info@benohanian.com
References
1 Lowe N, Campanati A, Bodokh I et al. The place of botulinum toxin
type A in the treatment of focal hyperhidrosis. Br J Dermatol 2004;151:1115–22.
2 Hayton MJ, Stanley JK, Lowe NJ. A review of peripheral nerveblockade as local anaesthesia in the treatment of palmar hyperhidro-
sis. Br J Dermatol 2003; 149:447–51.3 Benohanian A. Surgical pearl: use of needle-free anesthesia in the
treatment of palmar hyperhidrosis with botulinum A toxin. J Am
Acad Dermatol 2005; 52:1073–4.4 Gassner HG, Sherris DA. Addition of an anesthetic agent to
enhance the predictability of the effects of botulinum toxin type Ainjections: a randomized controlled study. Mayo Clin Proc 2000;
75:701–4.5 Heckmann M, Schaller M, Plewig G, Ceballos-Baumann A. Optimi-
zing botulinum toxin therapy for hyperhidrosis. Br J Dermatol 1998;138:553–4.
Table 1 Demographic details and results of 16 patients treated with botulinum toxin type A for palmar or plantar hyperhidrosis
Patient
Sex/age
(years)
Injection
date Site
Units
per side
Efficacy
in months
Muscle weakness
duration (weeks)
Willingness to
be reinjecteddespite muscle
weakness
1 F/46 6 Aug 04 Hands 200 12 2 Yes2 M/44 10 Aug 04 Hands 200 15 0
3 M/31 10 Aug 04 Hands 200 15 04 F/45 10 Aug 04 Hands 160 15 26 Yes
5 F/24 3 Sep 04 Hands 100 Failure 06 F/45 28 Sep 04 Hands 150 4 12 No
7 M/33 28 Sep 04 Hands 150 3 2 Yes8 F/27 5 Oct 04 Hands 100 13 0
9 M/24 5 Oct 04 Hands 100 5 010 M/30 23 Nov 04 Hands 50 12 0
11 F/32 14 Dec 04 Hands 50 11 012 F/53 1 Feb 05 Feet 100 8 0
13 M/33 1 Feb 05 Feet 100 7 0
14 F/42 1 Mar 05 Hands 100 3 12 Yes15 F/44 5 Apr 05 Feet 100 4 0
16 M/32 14 Jul 05 Hands 150 > 5 2 Yes
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 595
6 Benohanian A. Antiperspirants and deodorants. Clin Dermatol 2001;19:398–405.
7 Ellis GL, Owens A. The efficacy and acceptability of using a jetinjector in performing digital blocks. Am J Emerg Med 1993; 11:648–
50.8 Vadoud-Seyedi J. Treatment of plantar hyperhidrosis with botulinum
toxin type A. Int J Dermatol 2004; 43:969–71.
Conflicts of interest: none declared.
Increased human papillomavirus type 31 DNAload in a verrucous high-grade intraepithelialneoplasia of a human immunodeficiencyvirus-infected patient with extensive bowenoidpapulosis
DOI: 10.1111/j.1365-2133.2007.07690.x
SIR, Bowenoid papulosis (BP) represents a multifocal intraepi-
thelial neoplasia primarily located in the anogenital region of
predominantly young adults.1 Although BP shows clinically
benign-looking papular lesions, histopathological findings
reveal features of a squamous cell carcinoma (SCC) in situ, and
high-risk human papillomaviruses (HPVs), mainly HPV 16,
are regularly found in the lesions. BP usually has a benign
course and tends to regress spontaneously, in contrast to
Bowen’s disease (BD), which develops into SCC after a certain
duration of time. Malignant transformation has so far only
sporadically been reported in BP. Immunosuppression, espe-
cially human immunodeficiency virus (HIV) infection, greatly
increases the risk for premalignant and invasive HPV-related
conditions.2
We report a 57-year-old HIV-infected heterosexual man
with extensive genital BP. He was diagnosed as HIV-1 positive
in May 2005, presenting with a severely compromised immuno-
logical and virological HIV status (CD4+ cells 67 lL)1; HIV
RNA 244 000 copies mL)1). At first physical examination, he
revealed widespread brownish papules with a scaly or smooth
surface located on the mons pubis, genitals, inguinal folds,
buttocks, and the inner aspects of the thighs (Fig. 1a). Con-
comitant Candida albicans infection of the intertriginous areas
was present. Furthermore, a wart-like, verrucous nodule at the
perineal area, 8 · 8 mm in diameter, was detected that dif-
fered clinically from the other BP lesions (Fig. 1b). Histopatho-
logical examination of the nodule revealed acanthosis,
parakeratosis, and atypical cells within the entire epidermis
consistent with SCC in situ. Immunohistochemical staining of
the nodule using anti-p16 monoclonal antibody demonstrated
strong and diffuse immunoreactivity in both the nuclei and
the cytoplasm. p16 staining of the nodule reached the upper
layers of the epidermis (Fig. 2) and exceeded that of the BP
lesions (Table 1). HPV typing for 37 different low- and high-
risk HPVs from the two different types of lesions (the verru-
cous nodule shown in Fig. 1b and the widespread brownish
papules shown in Fig. 1a) showed identical high-risk HPV
types in both types of lesions (HPV 31 and 52). The low-risk
HPV 84 was present only in the brownish papules (Table 1).
Further genital low- or high-risk HPV types were not detected.
Interestingly, HPV 31 loads (quantitated by real-time polymer-
ase chain reaction with type-specific primers and probes and
expressed as HPV DNA copies per b-globin gene copy3) sig-
nificantly differed in the two types of lesions: HPV 31 load of
the verrucous nodule was two orders of magnitude higher
than that in the brownish papules (Table 1). Semiquantitative
viral load determination for HPV 52 and HPV 84 revealed
similar loads in the two brownish papules, and a lower HPV
52 load in the warty nodule compared with the brownish
papules (Table 1). The nodule was surgically removed and all
other lesions were treated with cautery fulguration. Imiqui-
mod 5% cream therapy was offered to prevent recurrences,
but was declined by the patient. He finally refrained from
therapy and was lost to follow up.
Fig 1. (a) Widespread brownish papules with
a scaly or smooth surface located on the mons
pubis, genitals, inguinal folds, and inner
aspects of the thighs. (b) Singular, wart-like
nodule located on the perineum.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
596 Correspondence
In the past, BP was mainly considered to be a benign
condition despite distinctive histopathological findings of
intraepithelial neoplasia, and no malignant transformation
was observed in the long-term follow up of a large collective
of patients with BP.4 However, development of SCC from BP
was reported recently, and most of these patients had signs
of immunosuppression. Although HPV 16 is found in the
majority of patients with BP, five cases of HPV 31-associated
BP have been reported in the literature so far, and two of
them transformed into SCC.5,6 Interestingly, elevated amounts
of HPV 31 were detected in the SCC as compared with other
BP lesions in the case of a woman with pancytopenia.6 This
finding is in line with our present patient, in whom an
exceedingly high HPV 31 DNA load was found in a singular
warty lesion as compared with the surrounding BP lesions.
Furthermore, immunohistochemical analysis of p16, a marker
that demonstrates HPV oncogene expression independent of
the infecting HPV type,7 revealed clearly more p16 positivity
within the epidermis of the warty lesion (Table 1). Besides
integration (which could occur alongside the high HPV 31
load), increased HPV oncogene expression could be
explained by mutations in the E6/E7 promotor or simply by
the large numbers of viral genomes present (elevated gene
dose). It is tempting to speculate that SCC transformation
could have occurred without treatment in this patient, and
that the elevated HPV 31 load is related to the advanced
grade of intraepithelial neoplasia. For cervical intraepithelial
neoplasia (CIN) an association between increased high-risk
HPV DNA loads and advanced CIN is established.8 Moreover,
our virological findings could corroborate the possibility that
both BP and BD are part of a spectrum of the same disease.
Considering the clinical features of the verrucous nodule one
might speculate that a common genital wart developed
within an area of dysplasia. However, no HPV types associ-
ated with genital warts were detectable within the lesion
(Table 1). We recently found elevated high-risk HPV DNA
loads in cervical high-grade squamous intraepithelial lesions
of HIV-positive women, compared with HIV-negative
women.3 Moreover, we observed high incidences of anal
intraepithelial neoplasia in HIV-positive homosexual men.2
Accordingly, all HIV-infected patients should be carefully
Fig 2. Immunohistochemical analysis of the squamous cell carcinoma
in situ (warty nodule) using anti-p16 monoclonal antibody
demonstrates a strong and diffuse immunoreactivity for p16 in both
the nuclei and cytoplasm, including also the upper layers of the
epidermis (original magnification · 40).
Table 1 Patient’s virological and histological features
Site of the lesion Clinical feature HPV typesaHPV 31
DNA loadb,cHPV 52
DNA loadcHPV 84
DNA loadc Histologydp16
staininge
Mons pubis Brownish papule 31, 52, 84 26b
1 : 10 000c1 : 10 000c 1 : 10c II 50%
Thigh (left) Brownish papule 31, 52, 84 51b
1 : 1000c1 : 10 000c 1 : 10c I 20%
Perineal area Warty nodule 31, 52 5735b
1 : 1 000 000c1 : 1000c – III 70%f
HPV, human papillomavirus. aHPV typing was performed as described previously.2,9 Probes used for enzyme immunoassay (EIA) hybridiza-
tion were described previously.10 bQuantitative HPV DNA load determination was performed by real-time polymerase chain reaction (PCR)with type-specific primers and probes as described previously.3 HPV 31 DNA load is expressed as HPV 31 DNA copies per b-globin gene
copy. cSemiquantitative HPV DNA load determination was performed by serial dilution (from 1 : 10 to 1 : 100 000 000) of extractedsample DNA in Tris–ethylenediamine tetraacetic acid buffer containing human placental DNA (40 ng lL)1). HPV group-specific PCR2 of the
diluted samples was performed and PCR products were hybridized to type-specific probes.9,10 The last dilution that gave a positive signal inthe EIA-based hybridization9 with type-specific probes10 for HPV 31, 52 and 84, respectively, is stated. dGrade of intraepithelial neoplasia.ep16 staining results are expressed as the percentage of positive cells per field. fReaching the upper third of the epithelium.
� 2007 The Authors
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Correspondence 597
screened for HPV-related disorders, including BP, and should
be treated as early as possible, as they have a higher risk of
malignant transformation, widespread disease, and frequent
recurrences.
Acknowledgments
We thank Monika Junk, Barbara Panz and Sabine Richter
for excellent technical assistance. This study was supported
by the Federal Ministry of Education and Research, German
Competence Network HIV/AIDS, Grant No. 01 KI 0501.
A . KREUT ER*
N.H. BROCKMEY ER*
H. P F I S T E R�P . ALTMEY ER*
U. WIE LAND�FOR THE GERMAN COMPE T ENC E
NETWORK HIV/AIDS
*Department of Dermatology and Allergology,
Ruhr-University Bochum, Gudrunstr.
56, 44791 Bochum, Germany
�Institute of Virology, University of Cologne,
Cologne, Germany
E-mail: a.kreuter@derma.de
References
1 Wade TR, Kopf AW, Ackerman AB. Bowenoid papulosis of the
genitalia. Arch Dermatol 1979; 115:306–8.2 Kreuter A, Brockmeyer NH, Hochdorfer B et al. Clinical spectrum
and virologic characteristics of anal intraepithelial neoplasia in HIVinfection. J Am Acad Dermatol 2005; 52:603–8.
3 Weissenborn SJ, Funke AM, Hellmich M et al. Oncogenic humanpapillomavirus DNA loads in human immunodeficiency virus-posi-
tive women with high-grade cervical lesions are strongly elevated.J Clin Microbiol 2003; 41:2763–7.
4 Patterson JW, Kao GF, Graham JH, Helwig EB. Bowenoid papulo-sis: a clinicopathologic study with ultrastrucural observations. Cancer
1986; 57:823–36.5 Yoneta A, Yamashita T, Jin HY et al. Development of squamous cell
carcinoma by two high-risk human papillomaviruses (HPVs),a novel HPV-67 and HPV-31 from bowenoid papulosis. Br J Dermatol
2000; 143:604–8.6 Hama N, Ohtsuka T, Yamazaki S. Elevated amount of human papil-
lomavirus 31 DNA in a squamous cell carcinoma developed frombowenoid papulosis. Dermatology 2004; 209:329–32.
7 Sano T, Oyama T, Kashiwabara K et al. Expression status ofp16 protein is associated with human papillomavirus oncogenic
potential in cervical and genital lesions. Am J Pathol 1998; 153:1741–8.
8 Snijders PJ, Hogewoning CJ, Hesselink AT et al. Determination ofviral load thresholds in cervical scrapings to rule out CIN 3 in
HPV 16, 18, 31 and 33-positive women with normal cytology.
Int J Cancer 2006; 119:1102–7.9 Jacobs MV, Snijders PJ, van den Brule AJ et al. A general primer
GP5+/GP6(+)-mediated PCR-enzyme immunoassay method forrapid detection of 14 high-risk and 6 low-risk human papillo-
mavirus genotypes in cervical scrapings. J Clin Microbiol 1997; 35:791–5.
10 van den Brule AJ, Pol R, Fransen-Daalmeijer N et al. GP5+/6+ PCRfollowed by reverse line blot analysis enables rapid and high-
throughput identification of human papillomavirus genotypes.J Clin Microbiol 2002; 40:779–87.
Conflicts of interest: none declared.
Telangiectatic cutaneous metastasis fromcarcinoma of the prostate
DOI: 10.1111/j.1365-2133.2006.07696.x
SIR, Carcinoma of the prostate is the most common cancer in
men, but is only rarely associated with metastases to the skin.1
Cutaneous metastases, when they occur, are usually nodular.2
Telangiectatic cutaneous metastases are very rare and are usu-
ally described in patients with breast cancer. There is a single
report in the literature of a telangiectatic cutaneous metastasis
from prostate cancer.3 We describe a second patient develop-
ing a telangiectatic cutaneous metastasis 12 years after diagno-
sis with prostate cancer.
An 83-year-old man with a 12-year history of prostate can-
cer metastatic to bone presented with a 3-month history of a
slowly enlarging patch on his left chest wall. The lesion was
mildly pruritic and occasionally associated with a burning sen-
sation. He denied any trauma or application of topical agents
to the area and had not had any similar lesions in the past. He
had received various treatments for his prostate cancer, includ-
ing goserelin acetate, bicalutamide, flutamide and radiation
therapy to the pelvis. At presentation, he was receiving palli-
ative radiation therapy and pamidronate.
The patient appeared weak and cachectic. On physical
examination he had a nontender, nonblanching, violaceous
and telangiectatic patch located anterior to the left axilla on
the chest wall (Fig. 1a). There were no other skin findings.
Pertinent laboratory studies included a prostate-specific anti-
gen (PSA) level of 227 ng L)1 (normal range, 0–4 ng mL)1),
which had risen from 95Æ8 ng mL)1 in the 2 months pre-
ceding his presentation with the skin lesion. A full blood
count and chemistry profile were within normal limits. A skin
biopsy from the patch showed intravascular aggregates of PSA-
positive adenocarcinoma within ectatic papillary dermal blood
vessels (Fig. 1b, c). The patient died from disease 6 months
after presenting with his cutaneous metastasis.
In the U.S.A., carcinoma of the prostate is the most com-
mon form of cancer in men and the second most common
cause of cancer-related mortality. It is estimated that 300 000
new cases are detected each year, of which roughly 41 000
(13Æ7%) will ultimately prove to be fatal.1 Carcinoma of the
prostate preferentially metastasizes to bone, lung, liver and
adrenal glands, whereas skin metastasis is considered excep-
tional and accounts for fewer than 1% of cutaneous metasta-
ses.2 In a retrospective study of 4020 patients with metastatic
disease, Lookingbill et al.4 found that 420 (10%) had cutane-
ous metastases. Most arose from primary tumours in the lung,
large intestine and oral cavity. None of the cutaneous metasta-
ses was of prostatic origin.4
When prostate carcinoma does metastasize to the skin, the
metastases usually appear as multiple or solitary nodules on
the abdominal skin, in the suprapubic area, and on the
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
598 Correspondence
anterior aspect of the thighs.2,5,6 Less frequently, the metasta-
ses involve other sites and may not have the typical nodular
morphology (Table 1).3,5,7–23 A telangiectatic cutaneous meta-
stasis from prostatic carcinoma has been described only once
in the literature. More frequently, metastases with this mor-
phology arise from the breast, the parotid gland, or in associ-
ation with apocrine carcinoma.3,24–27
Clinically, telangiectatic cutaneous metastases may demon-
strate telangiectasia, or erythema, or show purpuric papules,
or lymphangioma circumscriptum-like pseudovesicles.24–27
The single reported case of telangiectatic metastatic prostate
cancer presented as a plaque resembling angiosarcoma of the
scalp.3 Histological examination demonstrates ectatic, often
engorged, and predominantly superficial dermal blood vessels
that contain erythrocytes and aggregates of neoplastic cells
that frequently appear ‘free-floating’ within the vascular
lumina.24,26,28 Metastases from a prostatic primary carcinoma
have the typical histological features of prostatic adenocarci-
noma and are immunoreactive for both PSA and prostatic acid
phosphatase.2
Table 1 Atypical clinical presentations of cutaneous metastatic prostatic carcinoma
First author, year, reference Morphology Site Atypical features
Azana, 19937 Nodule Neck
Bluefarb, 19578 Nodules Abdomen Zosteriform distributionBoswell, 20053 Violaceous plaque Forehead Resembling angiosarcoma
Cox, 19949 Inflammatory papules, plaques Thigh, inguinal Resembling cellulitis; histopathology:lymphatic infiltration
Landow, 198010 Nodules Cutaneous lip, scalpLandow, 198010 Nodule Supraclavicular
Marcoval, 199811 Papulonodules Nipple and periareolar Resembling mammary Paget’s diseaseMueller, 20045 Nodular plaques Chest
Ng, 200012 Inflammatory plaque Thigh Resembling cellulitis; histopathology:lymphatic infiltration
Offidani, 199713 Nodules Scalp, chestPeison, 197114 Nodule Scalp Resembling sebaceous cyst
Pieslor, 198615 Nodule Umbilicus Resembling Sister Joseph’s noduleRazvi, 197516 Nodules Nose, abdomen
Reingold, 196617 Nodule ScalpRonchese, 194018 Nodules Scalp Resembling turban tumours
Rossetti, 199119 Nodule Chest Resembling basal cell carcinomaSchellhammer, 197320 Nodules Scalp
Sharma, 200521 Papulonodules Face, neck, chest Multiple nodules resemblingtrichoepitheliomas
Stahl, 198022 Ulcerated nodules Dorsal hands, forearms Resembling pyodermaWhitmore, 198623 Indurated, erythematous plaque Chest Resembling morphoea
(a) (b) (c)
Fig 1. (a) Clinical appearance of a nontender, nonblanching, violaceous and telangiectatic patch located anterior to the left axilla on the chest
wall. (b) Prostate-specific antigen-positive tumour masses within dilated blood vessels (original magnification · 200). (c) Tumour and thrombus
within dilated blood vessel (haematoxylin and eosin; original magnification · 200).
� 2007 The Authors
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Correspondence 599
Prostate carcinoma may metastasize via four mechanisms:
local extension from an underlying tumour, implantation
within a surgical scar, lymphatic spread and haematogenous
spread.29 Cutaneous metastases at sites distant from the pri-
mary tumour are thought to result from haematogenous
dissemination.29 Our case, with intravascular aggregates of
PSA-positive tumour cells, seems to corroborate this hypoth-
esis. It is possible that the patch of telangiectatic carcinoma
represents in-transit metastasis that would have preceded
eventual involvement of more distant sites such as the face
or scalp.
Cutaneous metastasis of prostate carcinoma, regardless of
how it manifests, is a marker of advanced disease and por-
tends a grave prognosis, with most patients dying from their
disease within 1 year of presentation of cutaneous findings.29
Acknowledgments
We are grateful to Dr Beverly Faulkner-Jones for proofreading.
S . REDDY
R .H. BANG*
M.E . CONTR E RA S*
Department of Dermatology, University of Texas
Health Science Center, San Antonio, TX, U.S.A.
*Department of Dermatology, University of
New Mexico, Albuquerque, NM, U.S.A.
Correspondence: R.H. Bang.
E-mail: drmagicmd@aol.com
References
1 Boring CC, Squires TS, Tong T et al. Cancer statistics, 1994. CA
Cancer J Clin 1994; 44:7–26.2 Duran EP, Paradela A, Farina MC. Cutaneous metastases from pro-
static carcinoma. J Surg Oncol 1996; 62:144–7.3 Boswell JS, Davis MDP. Violaceous plaque on the forehead clinically
resembling angiosarcoma: cutaneous metastasis in a patient withprostatic adenocarcinoma. J Am Acad Dermatol 2005; 53:744–5.
4 Lookingbill DP, Spangler N, Helm KF. Cutaneous metastases inpatients with metastatic carcinoma: a retrospective study of 4020
patients. J Am Acad Dermatol 1993; 29:228–36.5 Mueller TJ, Wu H, Greenberg RE et al. Cutaneous metastases from
genitourinary malignancies. Urology 2004; 63:1021–6.6 Steinkraus V, Lange T, Abeck D et al. Cutaneous metastases from
carcinoma of the prostate. J Am Acad Dermatol 1994; 31:665–6.7 Azana JM, de Misa RF, Gomez MI et al. Cutaneous metastases from
prostatic cancer. J Dermatol 1993; 20:786–8.
8 Bluefarb SM, Wallk S, Gecht M. Carcinoma of the prostate withzosteriform cutaneous metastases. Arch Dermatol 1957; 76:402–7.
9 Cox SE, Ponciano D, Cruz JR. A spectrum of inflammatory metasta-sis to skin via lymphatics: three cases of carcinoma erysipeloides.
J Am Acad Dermatol 1994; 30:304–7.10 Landow RK, Rhodes DW, Bauer M. Cutaneous metastases: report of
two cases of prostate cancer. Cutis 1980; 26:399–401, 409.11 Marcoval J, Moreno A, Jucgla A et al. Prostatic adenocarcinoma with
cutaneous metastases overlying oestrogen-induced gynaecomastia.Clin Exp Dermatol 1998; 23:119–20.
12 Ng CS. Carcinoma erysipeloides from prostate cancer presenting ascellulitis. Cutis 2000; 65:215–16.
13 Offidani A, Simonetti O, Cellini A et al. Skin metastases from prostatecancer associated with malignant melanoma. Cutis 1997; 59:278–80.
14 Peison B. Metastasis of carcinoma of the prostate to the scalp. Simula-tion of a large sebaceous cyst. Arch Dermatol 1971; 104:301–3.
15 Pieslor PC, Hefter LG. Umbilical metastases from prostatic carci-noma – Sister Joseph nodule. Urology 1986; 27:558–9.
16 Razvi M, Firfer R, Berskson B. Occult transitional cell carcinoma ofthe prostate presenting as skin metastasis. J Urol 1975; 113:734–5.
17 Reingold IM. Cutaneous metastases from internal carcinoma. Cancer1966; 19:162–8.
18 Ronchese F. Metastases of the scalp mimicking turban tumours.Arch Dermatol Syph 1940; 41:639–48.
19 Rossetti RB, Villaca Neto CM, Paschoal LH, Burnier Junior M.
Cutaneous metastasis originating from prostate adenocarcinoma.Int J Dermatol 1991; 30:363.
20 Schellhammer PF, Milsten R, Bunts RC. Prostatic carcinoma withcutaneous metastases. Br J Urol 1973; 45:169–72.
21 Sharma R, Chandra M. Cutaneous metastases from carcinoma ofthe prostate: a case report. Dermatol Online J 2005; 11:24.
22 Stahl D, Veien NK. Cutaneous metastases simulating other derma-toses. Cutis 1980; 26:282–4.
23 Whitmore WF Jr. Stage A prostate cancer. J Urol 1986; 136:883.24 Lin JH, Lee JY, Chao SC, Tsao CJ. Telangiectatic metastatic breast
carcinoma preceded by en cuirasse metastatic breast carcinoma. Br JDermatol 2004; 150:523–4.
25 Dobson CM, Tagore V, Myint AS, Memon A. Telangiectatic meta-static breast carcinoma in face and scalp mimicking cutaneous
angiosarcoma. J Am Acad Dermatol 2003; 48:635–6.26 Zanca A, Ferracini U, Bertazzoni MG. Telangiectatic metastasis from
ductal carcinoma of the parotid gland. J Am Acad Dermatol 1993;28:113–14.
27 Kiyohara T, Kumakiri M, Kawami K et al. Apocrine carcinoma ofthe vulva in a band-like arrangement with inflammatory and tel-
angiectatic metastasis via local lymphatic channels. Int J Dermatol2003; 42:71–4.
28 Pickard C, Callen JP, Blumenreich M. Metastatic carcinoma of thebreast: an unusual presentation mimicking cutaneous vasculitis.
Cancer 1987; 59:1184–6.29 Powell FC, Venencie PY, Winkelman RK. Metastatic prostate
carcinoma manifesting as penile nodules. Arch Dermatol 1984;120:1604–6.
Conflicts of interest: none declared.
Isolated recurrence of vesicobullousincontinentia pigmenti in a schoolgirl
DOI: 10.1111/j.1365-2133.2006.07700.x
SIR, The inflammatory vesicobullous first stage of incontinentia
pigmenti (IP) can recur, albeit rarely. It has previously been
described only in infants and young children under the age of
18 months and it has usually been preceded by a systemic
infection.
A 5-year-old schoolgirl presented with a 2-day history of a
linear rash on her right leg extending from the medial knee to
the buttock and consisting of vesicobullous lesions arising on
inflamed skin (Fig. 1). There was no history of anteceding
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
600 Correspondence
systemic illness or trauma. In addition, there were symmetrical
linear white atrophic streaks on her trunk and limbs, as a con-
sequence of her neonatal involvement with the same rash.
Swabs for bacterial and viral culture were negative and
C-reactive protein was normal. She was known to have
maternally inherited IP and had first presented 2 days after
birth with a whorled vesicobullous erythematous eruption on
both legs and the trunk. She had subsequently remained well,
with no complications.
A diagnosis of isolated recurrence of vesicobullous IP was
made. The rash was treated with once daily application of a
potent steroid and settled after a fortnight, in keeping with
the usual course in which the acute skin lesions last between
2 weeks and 4 months.1 Unilateral presentation of cutaneous
IP is rare and recurrence in such a distribution has not been
reported. The vesicobullous nature of the lesions and their
rapid resolution distinguished the rash from the differential of
lichen striatus, in which lesions are usually papular, reach
their maximal extent in 2 weeks and tend to resolve after
3–6 months.
In IP the mutated nuclear essential modulator (NEMO)
gene encodes a nonfunctional protein that is unable to acti-
vate nuclear factor-jB, which protects against tumour necro-
sis factor (TNF)-induced apoptosis. Most cases of IP have a
single acute episode in which most NEMO mutated cells are
destroyed. Subsequent infections, associated with the produc-
tion of TNF-a, can trigger apoptosis in residual NEMO
mutated cell lines.1 Reactivation can also occur after a phys-
ical insult such as laser2 or liquid nitrogen therapy.3 Previ-
ously reported cases of recurrence can be divided into two
groups: (i) infants and children under the age of 18 months,
with vesicobullous lesions in previously spared skin, with
five of the eight reported cases associated with a preceding
trigger; and (ii) school-age children or adults, with areas of
whorled erythema, without vesiculation, localized to previ-
ously affected areas, less predictably associated with an ante-
ceding trigger (Table 1).
Our patient uniquely re-presented with asymmetrical
vesicobullous lesions of IP at the age of 5 years in the absence
of a systemic illness. It is difficult to understand how her sig-
nificant number of residual NEMO mutated cells had been
Table 1 Reported cases of reactivation of
incontinentia pigmenti
Study Age (years)
Number of
recurrences
Preceding systemic
trigger
Presence of
vesicles
Bodak et al.1 < 1 One Yes YesBodak et al.1 < 1 Two Yes Noa
Bodak et al.1 < 1 Several Yes (all) YesBodak et al.1 < 1 Several Some Yes
van Leeuwen et al.4 < 1 One No YesSahn and Davidson5 < 1 Several No Yes
Llombart et al.6 1Æ5 Several Yes Noa
Patrizi et al.7 1Æ5 One No Yes
This case 5 One No YesBodak et al.1 7 Several Some Noa
Pfau and Landthaler8 7 Several Yes (all) Noa
Barnes9 Adult One No Noa
Bessems et al.10 Adolescence Several No Noa
aWhorled demarcated erythema only.
Fig 1. Vesicobullous eruption arising on inflamed skin of right
medial thigh.
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Correspondence 601
spared apoptosis for so long, and the mechanism responsible
for triggering the vesicobullous eruption at this late age.
We report this case to highlight the possibility of unilateral
reactivation of the vesicobullous stage of IP several years after
the initial presentation, in the absence of a systemic illness,
and to emphasize the importance of obtaining a neonatal
and family history to explain a vesicobullous eruption in
childhood.
S . DARN E
A . J . CARM I CHA E L
Department of Dermatology, The James Cook
University Hospital, Middlesbrough TS4 3BW,
U.K.
E-mail: sharmela@doctors.org.uk
References
1 Bodak N, Hadj-Rabia S, Hamel-Teillac D et al. Late recurrenceof inflammatory first-stage lesions in incontinentia pigmenti.
Arch Dermatol 2003; 139:201–4.2 Nagase T, Takanashi M, Takada H, Ohmore K. Extensive vesiculo-
bullous eruption following limited ruby laser treatment for incon-tinentia pigmenti: a case report. Australas J Dermatol 1997; 58:155–7.
3 Gatalano RA, Lopatynsky M, Tasman WS. Treatment of proliferativeretinopathy associated with incontinentia pigmenti. Am J Ophthalmol
1990; 110:701–2.
4 van Leeuwen RI, Wintzen M, van Praag MCG. Incontinentia pig-menti: an extensive second episode of a ‘first-stage’ vesicobullous
eruption. Pediatr Dermatol 2000; 17:70.5 Sahn EE, Davidson LS. Incontinentia pigmenti: three cases with
unusual features. J Am Acad Dermatol 1994; 31:852–7.6 Llombart B, Garcia L, Monteagudo C et al. Incontinentia pigmenti:
a case with an unusual course. J Eur Acad Dermatol 2005; 19:394–6.
7 Patrizi A, Neri I, Guareschi E, Cocchi G. Bullous recurrent eruptionof incontinentia pigmenti. Pediatr Dermatol 2004; 21:613–14.
8 Pfau A, Landthaler M. Recurrent inflammation in incontinentiapigmenti of a seven-year-old child. Dermatology 1995; 191:161–3.
9 Barnes CM. Incontinentia pigmenti. Report of a case with persistentactivity into adult life. Cutis 1978; 22:621–4.
10 Bessems PJM, Jagtman BE, van de Staak WJB et al. Progressivepersistent hyperkeratotic lesions in incontinentia pigmenti. Arch
Dermatol 1998; 124:29–30.
Conflicts of interest: none declared.
Infantile systemic hyalinosis: a case reportand mutation analysis in a Chinese infant
DOI: 10.1111/j.1365-2133.2006.07701.x
SIR, Infantile systemic hyalinosis (ISH; MIM 236490) is a rare
autosomal recessively inherited disease characterized by the
deposition of amorphous hyaline material in various tissues.
In 2003, Hanks et al.1 and Dowling et al.2 first reported muta-
tions in the gene encoding capillary morphogenesis protein 2
(CMG2) to be the cause of ISH. We present a Chinese patient
with ISH with two heterozygous mutations in CMG2.
This patient was the first child of healthy nonconsanguine-
ous Chinese parents. He was born at term by caesarean section
with a body weight of 2650 g. One month after birth he
developed violaceous papules on his face and refractory diar-
rhoea. Flexion contracture of the large joints gradually
appeared, and as a result he was lying in a leech-like position.
The bone of his left leg fractured after intensive physiotherapy
at 6 months of age. Violaceous plaques and hypertrophy of
the skin in his anogenital area spread slowly after 10 months
of age. Gingival hypertrophy began from 11 months. There
was no similar disease in his parents’ families.
Physical examination showed papules and nodular rashes
without clear demarcation on his face, back and anogenital area.
The consistency of the skin lesions varied from soft to hard.
There were also livid-red macules over bony prominences such
as the elbow and spine. His skin was generally oedematous and
thick. Pigmentation over the ankles and the metacarpal joints of
both hands, flexion contracture of large joints and hypertrophic
gingivae were quite characteristic (Fig. 1). His intelligence was
within normal range.
Screening for inheritable metabolic diseases including
amino acids in urine and plasma as well as serum immuno-
globulins was normal. Other blood biochemistry examinations
including serum vitamin A and 1,25-(OH)2 vitamin D3 were
within normal limits. His hands showed diffuse osteoporosis,
thin cortices and soft tissue swelling on X-ray examination.
(b)
(c)
Fig 1. The 20-month-old boy with infantile systemic hyalinosis
showing (a) violaceous nodules over his spinal prominences,
violaceous plaques in the napkin area, joint contracture of extremities,
and a leech-like body position; (b) hypertrophic gingivae; and
(c) a swollen and erythematous appearance of the metacarpo-
phalangeal and interphalangeal joints.
� 2007 The Authors
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602 Correspondence
A skin biopsy was taken from a lesion on his back. In the
dermis, we found plenty of amorphous hyaline matrix with
scattered fibroblasts and telangiectasia on haematoxylin and
eosin staining. On periodic acid–Schiff staining, the dermis
showed many oval or polygonal cells of a chondroid appear-
ance embedded in an abundant, amorphous and eosinophilic
matrix (Fig. 2).
Electron microscopic examination of the skin biopsy speci-
men showed fibroblasts with enlarged rough endoplasmic
reticulum containing dense particles, and intracytoplasmic and
membrane-bound vesicles of various sizes. These vesicles also
contained fine granular and fibrillar materials. In some places,
the basement membrane zone was discontinuous. Much
fibrillogranular material was located among collagen fibres.
The 17 coding exons of CMG2 were amplified from geno-
mic DNA samples by polymerase chain reaction, and directly
sequenced.1 A heterozygous 304–305insA mutation in exon 4
of CMG2 was found in the patient and his mother. A heterozy-
gous 1074delT mutation in exon 13 of CMG2 was found in
the patient and his father (cDNA sequence NM_058172 in
GenBank was used for nucleotide numbering, counting from
the A of the translational initiation codon ATG). These muta-
tions were not detected in 50 normal controls. The 304–
305insA allele results in a truncated protein of only 103
amino acid residues including two abnormal amino acid resi-
dues at the carboxyl terminus, and the 1074delT allele codes a
truncated protein of 407 amino acid residues including 49
abnormal amino acid residues at the carboxyl terminus. Alter-
natively, mutant CMG2 transcripts were absent in our patient’s
tissues due to nonsense-mediated mRNA decay. The two
mutations are of recessive inheritance as his parents, each car-
rying one mutant allele, were normal.
Two distinct syndromes due to mutations in CMG2 have
been described to date: ISH and juvenile hyaline fibromatosis.
ISH has an earlier onset, a more severe course and the wide-
spread deposition of hyaline material throughout the skin,
gastrointestinal tract, endocrine glands and muscles.3 Diagnosis
is made based on the clinical features such as cutaneous
lesions, gingival hyperplasia, osteoporosis and joint contrac-
ture as well as the findings of amorphous hyaline material in
the dermis.3,4
The protein encoded by CMG2 is a transmembrane protein
with a von Willebrand factor type A (vWA) domain in its
extracellular region.5 Genotype–phenotype analyses suggested
that in-frame and missense mutations within the cytoplasmic
domain result in a mild phenotype of juvenile hyaline fibro-
matosis, whereas mutations located in the vWA domain cause
a more severe form of the disease. In this case, the hetero-
zygous 304–305insA mutation causes premature translation
termination located in the vWA domain, whereas the hetero-
zygous 1074delT mutation results in abnormal amino acid
residues and premature translation termination confined to the
cytoplasmic domain. A homozygous 1074delT mutation was
previously found in two families.1 Therefore, the 1074 site
may be a mutation hotspot in the gene. However, this is the
first case of ISH caused by compound heterozygous mutations
in CMG2, and the 304–305insA mutation is a new finding not
reported in previous papers.
Y-C . HUANG
Y-Y. X IAO
Y-H. ZHENG
W. JANG
Y-L . YANG*
X- J . ZHU
Departments of Dermatology and
*Pediatrics, Peking University First Hospital,
Beijing 100034, China
Correspondence: Xue-Jun Zhu.
E-mail: zhuxj@public.bta.net.cn
Y-C.H. and Y-Y.X. contributed equally to this work.
References
1 Hanks S, Adams S, Douglas J et al. Mutations in the gene encoding
capillary morphogenesis protein 2 cause juvenile hyaline fibro-matosis and infantile systemic hyalinosis. Am J Hum Genet 2003;
73:791–800.2 Dowling O, Difeo A, Ramirez MC et al. Mutations in capillary mor-
phogenesis gene-2 result in the allelic disorders juvenile hyalinefibromatosis and infantile systemic hyalinosis. Am J Hum Genet 2003;
73:957–66.
Fig 2. (a) Biopsy of a skin lesion reveals an
amorphous hyaline matrix in the dermis with
telangiectasia (haematoxylin and eosin;
original magnification · 100); (b) oval or
polygonal cells of a chondroid appearance in
the dermis (periodic acid–Schiff; original
magnification · 400).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 603
3 Landing BH, Nadorra R. Infantile systemic hyalinosis: report of fourcases of a disease, fatal in infancy, apparently different from juvenile
systemic hyalinosis. Pediatr Pathol 1986; 6:55–79.4 Keser G, Karabulut B, Oksel F et al. Two siblings with juvenile
hyaline fibromatosis: case reports and review of the literature.Clin Rheumatol 1999; 18:248–52.
5 Bell SE, Mavilla A, Salazar R et al. Differential gene expression duringcapillary morphogenesis in 3D collagen matrices: regulated expres-
sion of genes involved in basement membrane matrix assembly, cellcycle progression, cellular differentiation and G-protein signaling.
J Cell Sci 2001; 114:2755–73.
Conflicts of interest: none declared.
Scytalidium infection associated withdyskeratosis congenita
DOI: 10.1111/j.1365-2133.2006.07702.x
SIR, Scytalidium dimidiatum (the anamorphic form of Nattrassia man-
giferae, formally known as Hendersonula toruloidea), a saprobic
mould, was first recognized as a human pathogen in 1970,1
with clinical manifestations similar to those of dermatophyto-
sis.2 These nondermatophytes are infrequently diagnosed
because the routine mycological medium used for isolation of
dermatophytes, Sabouraud’s dextrose agar (SDA), contains
cycloheximide, which inhibits their growth.1 Several cases of
S. dimidiatum have been reported in connexion with coexistent
systemic diseases such as chronic active hepatitis, systemic
lupus erythematosus and diabetes mellitus, as well as with var-
ious skin diseases such as eczema, pruritus, lichen simplex,
lichen planus, psoriasis, leprosy and urticaria.2,3 This is the
first report of onychomycosis caused by S. dimidiatum in a
patient with dyskeratosis congenita (DC).
A 16-year-old Indian boy, a field worker by profession,
presented to the dermatology outpatient clinic in July 2005
with a 6-year history of toenail and fingernail dystrophy.
Yellow–brown discoloration of the toenails was noticed ini-
tially, followed by laterodistal dystrophy of the big toe and
subsequently other toenails (Fig. 1). Microscopic examination
and fungal culture of nail scrapings performed elsewhere were
negative for dermatophyte and nondermatophyte moulds. He
was treated unsuccessfully with oral griseofulvin and oral fluc-
onazole but, by September 2001, all his fingernails had also
become similarly affected (Fig. 1).
Direct light microscopy of nail scrapings using 20%
potassium hydroxide solution revealed fungal hyphae, brown in
colour, a rare finding, with little variation in hyphal width.
Culture of nail scrapings was performed on SDA contain-
ing 0Æ005% chloramphenicol with and without 0Æ05%
cycloheximide. Scytalidium dimidiatum was isolated after 2 weeks
of incubation at 25 �C on three occasions on cycloheximide-
free SDA. Positive culture was confirmed by gross colony mor-
phology and colour: pale-olivaceous grey with scant aerial
mycelium and a pale mousy grey undersurface (Fig. 2). Micro-
scopic observations of a lactophenol–cotton blue mount pre-
pared from colonies revealed abundant hyphal loops with little
variation in hyphal width. There were scanty, long cylindrical to
barrel shaped, light brown arthrospores (3 · 2 lm) (Fig. 2).
Further examination of the patient revealed reticulate
pigmentation covering his entire body present since child-
hood, nail dystrophy (Fig. 1), and marked leucoplakia on the
tongue and oral mucosa. He had sparse hair on the lateral half
Fig 1. Clinical appearance: (a) reticulate
hyperpigmentation; (b) palmar
hyperkeratosis; (c,d) onychodystrophy.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
604 Correspondence
of the eyebrows, scarring alopecia, and hyperkeratosis of both
palms (Fig. 1). The presence of other features such as dental
caries, marked epiphora and subnormal intelligence further
supported the diagnosis of DC. Respiratory, cardiovascular and
abdominal examinations were normal. Blood counts were
within normal limits. Chest X-ray and abdominal ultrasound
revealed no abnormality. Enzyme-linked immunosorbent assay
for human immunodeficiency virus types 1 and 2 was nega-
tive. Skin biopsy revealed a sparse superficial perivascular
lymphocytic infiltrate with numerous melanophages within
the papillary dermis. The papillary dermis was slightly thick-
ened and showed delicate fibroplasia and mucin. The over-
lying epidermis showed focal vacuolar change in the basal
layer and infiltration of the interface by lymphocytes. The epi-
dermis was flattened in places (Fig. 3).
Our patient was given oral isotretinoin for DC. He was
commenced on oral itraconazole 200 mg twice daily for
7 days each month and topical terbinafine cream, without any
improvement in 6 months.
Scytalidium dimidiatum infection has been reported from South
Asia, Africa, the West Indies and South America.2 Our patient
was infected by a strain resembling the slow-growing type
(form 3), which occurs predominantly in individuals from the
Indian subcontinent.2
DC is a rare inherited disease characterized by a triad of
clinical manifestations including abnormal skin pigmentation,
nail dystrophy and mucosal leucoplakia.4 Along with the
above diagnostic triad our patient also presented with palmar
hyperkeratosis, which was negative for fungus on microscopy
and culture, and dental caries, both of which have been repor-
ted with high frequency in patients with DC.4 Bone marrow
suppression, which presents as anaemia in half of patients
with DC in their second or third decade,4 was not observed in
Fig 3. Photomicrograph showing superficial perivascular lymphocytic
infiltrate with numerous melanophages within papillary dermis
and focal vacuolar changes in the basal layer of the epidermis
(haematoxylin and eosin).
Fig 2. (a) Surface and (b) undersurface of
Scytalidium dimidiatum culture on Sabouraud’s
dextrose agar. (c) Lactophenol–cotton blue
mount showing abundant hyphal loops.
(d) Arthroconidia of S. dimidiatum (Indian
subcontinental strain).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 605
our patient. Nail dystrophy in DC involves the fingernails first,
with longitudinal fissuring, thinning, atrophy, distortion, and
rarely nail loss.5 In onychomycosis due to S. dimidiatum the
changes are thickening, discoloration and laterodistal inva-
sion.3 The nail changes in our patient may have resulted from
the combined effect of both Scytalidium infection and DC.
Among the antifungals, griseofulvin, ketoconazole and fluc-
onazole are ineffective against S. dimidiatum, while there are
isolated reports of efficacy of oral itraconazole, intravenous
amphotericin B, topical amorolfine and topical ciclopiroxol-
amine.6 Terbinafine has in vitro activity but no clinical effect.7
It is important to study the efficacy of new antifungal drugs
(voriconazole, ravuconazole, posaconazole and caspofungin)8
in Scytalidium infection and to develop specific therapy for this
orphan disease. Correct diagnosis of Scytalidium infection is
necessary to avoid lengthy and potentially toxic antifungal
treatment. Furthermore, in a patient with DC it may be worth-
while to evaluate the nail changes with regard to the possibil-
ity of associated fungal infection.
Acknowledgments
We thank Dr Uday Khopkar, Dermatologist, K.E.M Hospital,
Mumbai for his expert opinion on histopathology.
J . GARG
R . T I L AK
A.K . GULAT I
S . S INGH*
P . PRAKA SH
A. GARG
Departments of Microbiology and
*Dermatology, Institute of Medical Sciences,
Banaras Hindu University,
Varanasi, UP, India
Correspondence: Atul Garg.
E-mail: atulgargbhu@rediffmail.com
References
1 Gentles JC, Evans EGV. Infections of the feet and nails with Henderson-ula toruloidea. Sabouraudia 1970; 8:72–5.
2 Sigler L, Summerbell RC, Poole L et al. Invasive Nattrassia mangiferaeinfections: case report, literature review, and therapeutic and
taxonomic appraisal. J Clin Microbiol 1997; 35:433–40.3 Hay RJ, Moore MK. Clinical features of superficial fungal infections
caused by Hendersonula toruloidea and Scytalidium hyalinum. Br J Dermatol1984; 110:677–83.
4 Kraemer KH. Heritable diseases with increased sensitivity to cellularinjury. In: Fitzpatrick’s Dermatology in General Medicine (Freedberg IM,
Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI, FitzpatrickTB, eds), 5th edn, Vol. 2. New York: McGraw-Hill, 1999; 1858–
9.5 Bagby GC, Lipton JM, Sloand EM, Schiffer CA. Marrow failure.
Hematology 2004; 1:318–36.6 Goon ATJ, Seow CS. Three cases of Nattrassia mangiferae (Scytalidium
dimidiatum) infection in Singapore. Int J Dermatol 2002; 41:53–5.7 Clayton YM. Relevance of broad-spectrum and fungicidal activity of
antifungals in the treatment of dermatomycoses. Br J Dermatol 1994;130 (Suppl. 43):7–8.
8 Lecha M, Effendy I, Chauvin MF et al. Treatment options – develop-ment of consensus guidelines. J Eur Acad Dermatol Venereol 2005; 19
(Suppl. 1):25–33.
Conflicts of interest: none declared.
Pityriasis rubra pilaris and hypothyroidism.Efficacy of thyroid hormone replacementtherapy in skin recovery
DOI: 10.1111/j.1365-2133.2006.07697.x
SIR, A 24-year-old man, without any relevant medical history,
presented with progressive erythroderma that had begun
3 months previously. His skin lesions were initially localized
on the forearms but a rapid diffuse extension had occurred.
Psoriasis had been suspected, and he had undergone psoralen
plus ultraviolet A therapy, in association with topical steroids,
without any improvement. At admission to our department,
he presented with erythroderma with rare areas of normal
skin on the limbs. Follicular keratotic papules and orange
palmoplantar keratoderma were observed and the face and
scalp were severely affected. We noticed eyebrow depilation
and marked alopecia associated with apathy of the patient
(Fig. 1a). Laboratory studies showed an inflammatory syn-
drome. Blood count and serum IgE level were normal. Human
immunodeficiency virus (HIV), hepatitis B virus and hepatitis
C virus serologies were negative. Lipid determination showed
a borderline high level of cholesterol at 5Æ87 mmol L)1 (desir-
able < 5Æ3; high > 6), without hypertriglyceridaemia. Skin
biopsy showed an alternation of parakeratosis and ortho-
hyperkeratosis especially in follicular orifices. In the dermis
there was a moderate lymphocytic infiltrate. The histopatholo-
gical and clinical evidence led to the diagnosis of pityriasis
rubra pilaris (PRP).
In view of the eyebrow depilation, diffuse alopecia and
dyslipidaemia, hypothyroidism was suspected. This was con-
firmed by high serum levels of thyroid-stimulating hormone
(TSH; 25 lU mL)1; normal 0Æ35–3Æ5), T3 (2 pmol L)1; nor-
mal 2Æ5–5) and T4 (8Æ6 pmol L)1; normal 10–20). The hypo-
thyroidism was considered to be autoimmune, with elevation
of antithyroglobulin antibodies. Vitamin A and serum carotene
levels were normal.
Oral thyroid hormone replacement therapy was begun
(L-thyroxine with progressive dose increases), associated with
topical corticosteroids and emollients. This led to dramatic
improvement of skin lesions after only 1 month of treatment
(Fig. 1b), while the patient had previously been erythroder-
mic for 6 months without any improvement. In parallel, we
noted hair regrowth and recovery of functions, while his thy-
roid status remained elevated (TSH 8Æ6 lU mL)1). Topical
corticosteroids were stopped in 1 month. The patient was still
in complete remission after more than 1 year of follow up
and his serum TSH level was normal.
Our patient showed severe classical adult PRP (type I)1 and
concomitant autoimmune hypothyroidism. Thyroid hormone
replacement allowed a rapid and complete remission of PRP
without any additional treatment. This unusual association has
been reported only twice but is probably not fortuitous as it
may be explained by the physiopathology of PRP. The first
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
606 Correspondence
previously reported case was mentioned with neither details
nor reference in a literature review.2 The second case reported
was of erythrodermic PRP in a 4-year-old child, associated
with severe nonautoimmune hypothyroidism.3 As in our case,
complete remission of PRP was obtained after 3 months of
thyroid hormone replacement.
This dramatic skin improvement under thyroid hormone
replacement therapy is remarkable and argues against a fortuit-
ous association. While spontaneous remission of PRP is fre-
quent (80%), it occurred after several months or even after
1–3 years, especially in erythrodermic types.4 Systemic treat-
ments (retinoids, methotrexate) are usually necessary to accel-
erate the remission, and topical treatments such as topical
corticosteroids are generally less effective.5 In our patient,
improvement was rapidly obtained after a course of L-thyrox-
ine, without addition of any other systemic drug, suggesting
the direct effect of this treatment on PRP.
The pathogenesis of PRP is not clear, although a dis-
order of vitamin A metabolism has been suggested.6
Firstly, the histological appearance of PRP is quite similar
to that of hypovitaminosis A. Secondly, PRP has been
successfully treated in the past by high doses of vitamin
A.7 As thyroid hormone deficiency inhibits the transform-
ation of carotene to vitamin A, it may be responsible for
the occurrence of PRP in this patient. Our patient had
normal levels of carotene and vitamin A, but these serum
levels do not strictly reflect their reserves, especially in
the liver. Thus, an abnormality of vitamin A metabolism,
induced by hypothyroidism, could explain the occurrence
of PRP in association with hypothyroidism, and its recov-
ery after thyroid hormone replacement.
PRP associated with hypothyroidism may be a particular
subtype of PRP (such as the previously described association
between HIV and PRP8). This association, although unusual,
has to be made known because of the spectacular effect of
thyroid hormone replacement. Hypothyroidism must be sus-
pected when PRP occurs in association with asthenia and
apathy, as in our patient.
V . ORLA ND IN I
O. COGRE L
M.S . DOUTRE
C . BEY LOT
M. BEY LOT-BARRY
Department of Dermatology,
Hopital Haut-Leveque, avenue de Magellan,
33604 Pessac, France
E-mail: vincent.orlandini@chu-bordeaux.fr
References
1 Griffiths WAD. Pityriasis rubra pilaris. Clin Exp Dermatol 1980; 5:105–12.
2 Gross DA. Pityriasis rubra pilaris. Report of a case and analysis ofthe literature. Arch Dermatol 1969; 99:710–16.
3 Tunnessen WW, Nieburg PI, Voorhess ML. Hypothyroidism and pit-yriasis rubra pilaris. Response to thyroid hormone. J Pediatr 1976;
88:456–8.4 Griffiths WAD. Pityriasis rubra pilaris: the problem of its classifica-
tion. J Am Acad Dermatol 1992; 26:140–2.5 Chaabouni M. Treatment of pityriasis rubra pilaris. Ann Dermatol
Venereol 1999; 126:836.6 Finzi AF, Altomare G, Bergamaschini L, Tucci A. Pityriasis rubra
pilaris and retinol-binding protein. Br J Dermatol 1981; 104:253–6.7 Griffiths WAD. Vitamin A and pityriasis rubra pilaris. J Am Acad
Dermatol 1982; 7:555.8 Sanchez-Regana M, Fuentes CG, Creus I et al. Pityriasis rubra pilaris
and HIV infection: a part of the spectrum of HIV-associated follicu-lar syndrome. Br J Dermatol 1995; 133:818–19.
Conflicts of interest: none declared.
(a) (b)
Fig 1. (a) Severe erythema of the face and
eyebrow depilation. (b) Comparative
photograph after 1 month of thyroid
hormone replacement therapy.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 607
Nicorandil-induced peristomal ulcers:is nicorandil also associated withgastrointestinal fistula formation?
DOI: 10.1111/j.1365-2133.2006.07698.x
SIR, Further to the recently published report by Claeys et al.1 of
two patients with perivulval and perianal ulceration induced
by nicorandil, we present a series of four cases of nicorandil-
induced peristomal ulceration in patients with a previous his-
tory of diverticular disease requiring gastrointestinal surgery.
To our knowledge this is a previously unreported association.
Patient 1 was an 83-year-old woman who presented with
painful vulval and peristomal ulceration. She had a defunction-
ing colostomy following the development of a colovaginal fis-
tula in 2004; a barium enema performed prior to the surgery
demonstrated diverticular disease. In 2005 she presented with
vaginal discharge and was found to have several vesicovaginal
fistulae; however, these were managed conservatively because
of medical comorbidity. She had a history of ischaemic heart
disease and had been taking nicorandil since 2001. Examina-
tion revealed three vulval ulcers and a well-defined sloughy
peristomal ulcer. There were no clinical features of pyoderma
gangrenosum (Fig. 1). Nicorandil was discontinued and alter-
native antianginal therapy (isosorbide mononitrate) com-
menced. At 2 months, the ulcers had healed without any
additional treatment.
Patient 2 was an 82-year-old woman seen with a 2-week
history of painful peristomal ulceration. She had a history of a
colovaginal fistula secondary to diverticular disease that had
been treated 1 year previously with a Hartman’s procedure
(resection of part of the colon with formation of a colos-
tomy). At the time of surgery she also had drainage of an
abscess in the left groin that had since failed to heal. She was
on multiple medications for ischaemic heart disease, hyperten-
sion and also osteoarthritis, including nicorandil 20 mg twice
daily. On examination she had a sloughy peristomal ulcer and
an area of ulceration in the left groin. After consultation with
her general practitioner the nicorandil was discontinued and
the ulcers were treated topically with sulcrafate powder. At
1 month review there was considerable improvement and by
10 months the ulcers had healed.
Patient 3 was a 59-year-old man who 3 months prior to pre-
sentation had had an ileostomy performed for presumed fistulat-
ing diverticular disease. Two months postsurgery he had
developed painful peristomal ulceration that had not responded
to treatment with topical triamcinolone (Adcortyl in Orabase�;
Bristol-Myers Squibb, Uxbridge, U.K.). He had a previous his-
tory of rheumatoid arthritis, ischaemic heart disease and asthma,
and had been taking nicorandil 20 mg twice daily. He had two
shallow peristomal ulcers. The ulcers were treated topically with
sulcrafate powder and the nicorandil was discontinued. At
3 month review the ulcers had completely healed.
Patient 4 was a 63-year-old woman with ischaemic heart
disease on nicorandil 20 mg daily who developed perianal
ulcers and then a colovesical fistula in 2005 necessitating a
defunctioning colostomy. She was seen in 2006 with peristo-
mal and perianal ulcers that healed within 3 months after
stopping nicorandil and changing to isosorbide mononitrate.
The initial healing of the peristomal ulcer was complicated by
prominent overgranulation treated with corticosteroid (Syna-
lar� gel; GP Pharma, Lidlington, U.K.).
The association between nicorandil and oral or anal ulceration
is well known.2,3 There are also a few reports of gastrointestinal
ulceration linked to nicorandil therapy, including a report of a
patient with multiple ulcers of the small and large intestine that
resolved on stopping nicorandil and a report of ulceration and
perforation of the terminal ileum possibly due to nicorandil.4,5
Interestingly, two of our patients had cutaneous ulceration prior
to the development of fistulae requiring stoma formation. The
theoretical link between fistulating bowel disease and nicorandil
therapy is currently being investigated.
The mechanism of nicorandil-induced mucosal ulceration is
as yet unknown; however, various hypotheses have been sug-
gested including a direct effect of the drug or its metabolites
on the mucosa, and a vascular steal phenomenon.6 In some
patients dose reduction may be sufficient to promote ulcer
healing; however, some will require cessation of the drug and
institution of alternative antianginal therapy. Recognition of
the potential association of nicorandil and peristomal ulcer-
ation and fistulating gastrointestinal disease is important, as
these conditions have high morbidity.
S . OGDEN
Y . MUKA SA
C .C . LYON*
I .H. COUL SON
Department of Dermatology, Burnley General
Hospital, Burnley BB10 2PQ, U.K.
*Department of Dermatology, York District
Hospital, York YO31 8HE, U.K.
E-mail: stephanie.ogden@ntlworld.com
References
1 Claeys A, Weber-Muller F, Trechot P et al. Cutaneous, perivulvar andperianal ulcerations induced by nicorandil. Br J Dermatol 2006;
155:494–6.2 Healy CM, Smyth Y, Flint SR. Persistent nicorandil induced oral
ulceration. Heart 2004; 90:e38.Fig 1. Peristomal ulcer (patient 1).
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
608 Correspondence
3 Katory M, Davies B, Kelty C et al. Nicorandil and idiopathic analulceration. Dis Colon Rectum 2005; 48:1442–6.
4 Egred M, Andron M, Morrison WL. Nicorandil may be associatedwith gastrointestinal ulceration. BMJ 2006; 332:889.
5 King PM, Suttie SA, Jansen JO, Watson AJM. Perforation of the ter-minal ileum: a possible complication of nicorandil therapy. Surgeon
2004; 2:56–7.6 Watson A, Al-Ozairi O, Fraser A et al. Nicorandil associated anal
ulceration. Lancet 2002; 360:546–7.
Conflicts of interest: none declared.
Variability in the clinical pattern of cutaneousside-effects of drugs with systemic symptoms:does a DRESS syndrome really exist?
DOI: 10.1111/j.1365-2133.2006.07704.x
SIR, In a recent study Peyriere et al.1 stated that the existence of a
clinical entity, known under various names including HSS (anti-
convulsant hypersensitivity syndrome), DRESS (drug reaction
with eosinophilia and systemic symptoms), DIDMOHS (drug-
induced delayed multiorgan hypersensitivity syndrome) and
DIHS (drug-induced hypersensitivity syndrome) cannot be
denied but that its definition, clinical and biological pattern, and
limits must be more accurately reappraised. We can fully
endorse that a gold standard is lacking, as is also repeatedly sta-
ted in the literature. Also the lack of consensus on nosology is
obvious, but this is minor if there is agreement on what are the
main characteristics of the ‘syndrome’. Although it is generally
accepted that a syndrome by its nature comprises a variable
combination of symptoms, the acronym DRESS is questioned as
eosinophilia need not necessarily be present in this syndrome.
The diversity of cutaneous adverse drug reactions is nearly
infinite. It makes sense to isolate syndromes, rather than to
consider the whole as a continuum, if it helps in finding ori-
ginal clinical patterns, courses, causes, mechanisms and treat-
ment. From long discussions between experts from different
countries in recent medical meetings on drug hypersensitivity
it appears that whatever the denomination, HSS/DRESS is
characterized by a variable combination of: (i) drug-induced
immunological background; (ii) later onset than other drug
reactions; (iii) longer duration than common ‘drug rashes’;
(iv) multiorgan involvement; (v) lymphocyte activation (node
enlargement, lymphocytosis, atypical lymphocytes); (vi) eosino-
philia; and (vii) frequent virus reactivation.
HSS/DRESS is specifically complicated because besides its
rather variable presentation it is a diagnosis by exclusion. Its
main features such as rash, fever and organ involvement can also
be attributed to a wide range of other causes such as infections,
and to concomitant and pre-existing diseases. Hence each symp-
tom should always be thoroughly investigated for its relation to
the syndrome. Not all symptoms and signs are always recog-
nized, and asymptomatic systemic involvement such as eosino-
philia and atypical lymphocytes are often not determined or are
determined too late, leading to their under-reporting. In add-
ition, partly due to the relatively long latency after initiation and
the long duration after cessation of the culprit drug, the symp-
toms are often not recognized as drug related. General aware-
ness of HSS/DRESS is very important due to the severity and
life-threatening potential of this type of drug reaction.
The RegiSCAR study group (as its predecessors EuroSCAR
and SCAR) is performing a prospective study of severe cutan-
eous adverse reactions (SCAR) in Austria, France, Germany,
Israel, Italy and the Netherlands, in order to investigate their
risk factors and mechanisms based on a large multinational
registry. Former projects of the group dealt with the spec-
trum of Stevens–Johnson syndrome/toxic epidermal necrolysis
(SJS/TEN)2 and acute generalized exanthematous pustulosis
(AGEP).3 Crucial to these studies has been a clear case defini-
tion. The combination of a scoring system and judgement of
cases by a review committee (blinded for possible risk factors)
has proven effective for validation in SJS/TEN and AGEP. The
group’s intention to extend investigations to HSS/DRESS raised
the need for an equally reliable approach for those cases.
RegiSCAR has collected cases of HSS/DRESS since 2002.
Patients are actively detected through a hospital network cov-
ering about 170 million inhabitants, using selected inclusion
criteria (Table 1). Information on reported cases of HSS/
DRESS is obtained by trained local interviewers using standard-
ized questionnaires, comprising elaborate questions on drug
use, morphology and extent of the rash, involvement of
lymph nodes and other organs, laboratory and clinical param-
eters to judge organ involvement as attributable to HSS/
DRESS, and course of the disease. Where possible, clinical pic-
tures and results of histological examination in the active
phase of the eruption are collected. Interviews take place at
the acute stage of the disease with a follow up at 8 ± 2 weeks
and 1 year, if the patient’s informed consent for participation
in a cohort is obtained. In addition, blood samples are taken
for immunological and genetic research.
Due to the complexity and variability of HSS/DRESS, inter-
pretation of clinical findings and laboratory data by an unorgan-
ized reviewing process would not have produced consistent
Table 1 Inclusion criteria for potential case of HSS/DRESS inRegiSCAR
HospitalizationReaction suspected to be drug related
Acute skin rasha
Fever above 38 �Ca
Enlarged lymph nodes at at least two sitesa
Involvement of at least one internal organa
Blood count abnormalitiesLymphocytes above or below the laboratory limitsa
Eosinophils above the laboratory limits (in percentage or
absolute count)a
Platelets below the laboratory limitsa
aThree or more required.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Correspondence 609
results. Based on information from the literature and clinical ex-
perience of the review committee we reached consensus on a
scoring system for our study which would allow for reproduci-
bly classifying cases as definite, probable, possible or no case.
Thorough case assessment was done on the basis of clinical pic-
tures and analysis of the collected data by experienced clinicians,
as this cannot be replaced by a scoring system alone, but will
always need professional judgement. An overview of the scoring
system is given in Table 2. To prevent bias, the review commit-
tee was blinded to the suspected drugs.
Although we are aware that virus reactivation may play a
role in the syndrome, we do not count the related organ in
case of a positive virus serology. However, it is still a matter
of debate whether reactivation of several herpesviruses in the
course of the disease is part of the syndrome or should be
interpreted as a complication, resulting in a more protracted
and relapsing disease.4,5
In pharmacovigilance, data are often only received retro-
spectively, whereas we most often see the patient in the acute
stage of the disease and systematically collect far more detailed
data, permitting us better to judge the presented symptoms.
Moreover, the advantages of the scale of our multinational
study over a national one, as proposed by Peyriere et al.,1 in a
rare syndrome such as HSS/DRESS, are obvious.
We anticipate our case definition and system of validation
will lead to a reliable identification of cases of HSS/DRESS for
further studies of pharmacoepidemiological and genetic risk
factors, as well as the immunological background. We expect
to be able to answer several long-standing questions after fur-
ther case enrolment in 12–18 months.
S .H. KARDAUN
A. S I DOROF F*
L . VAL EYR I E -AL LANORE�S . HAL E VY�
B.B . DA V IDOV I C I�M. MOCKENHAUPT§
J .C . ROU J E AU�
Center for Blistering Diseases,
Department of Dermatology,
University Medical Center Groningen,
University of Groningen, Hanzeplein 1,
9713 GZ Groningen, the Netherlands
*Department of Dermatology and Venereology,
Medical University of Innsbruck,
Innsbruck, Austria
�Department of Dermatology, Hopital
Henri Mondor, University of Paris XII,
Creteil, France
�Department of Dermatology,
Soroka University Medical Center,
Ben-Gurion University of the Negev,
Beer-Sheva, Israel
§Dokumentationszentrum schwerer
Hautreaktionen (dZh),
Department of Dermatology, University Medical Center,
Freiburg, Germany
E-mail: s.h.kardaun@derm.umcg.nl
Table 2 Scoring system for classifying HSS/DRESS cases as definite, probable, possible or no case
Score )1 0 1 2 Min. Max.
Fever ‡ 38Æ5 �C No/U Yes )1 0
Enlarged lymph nodes No/U Yes 0 1Eosinophilia No/U 0 2
Eosinophils 0Æ7–1Æ499 · 109 L)1 ‡ 1Æ5 · 109 L)1
Eosinophils, if leucocytes < 4Æ0 · 109 L)1 10–19Æ9% ‡ 20%
Atypical lymphocytes No/U Yes 0 1Skin involvement )2 2
Skin rash extent (% body surface area) No/U > 50%Skin rash suggesting DRESS No U Yes
Biopsy suggesting DRESS No Yes/UOrgan involvementa 0 2
Liver No/U YesKidney No/U Yes
Lung No/U YesMuscle/heart No/U Yes
Pancreas No/U Yes
Other organ No/U YesResolution ‡ 15 days No/U Yes )1 0
Evaluation of other potential causesAntinuclear antibody
Blood cultureSerology for HAV/HBV/HCV
Chlamydia/mycoplasmaIf none positive and ‡ 3 of above negative Yes 0 1
Total score )4 9
U, unknown/unclassifiable; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus. aAfter exclusion of other explanations:
1, one organ; 2, two or more organs. Final score < 2, no case; final score 2–3, possible case; final score 4–5, probable case; final score > 5,definite case.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
610 Correspondence
References
1 Peyriere H, Dereure O, Breton H et al. Variability in the clinical pat-tern of cutaneous side-effects of drugs with systemic symptoms:
does a DRESS syndrome really exist? Br J Dermatol 2006; 155:422–8.2 Auquier-Dunant A, Mockenhaupt M, Naldi L et al. Correlations
between clinical patterns and causes of erythema multiforme majus,Stevens–Johnson syndrome, and toxic epidermal necrolysis: results of
an international prospective study. Arch Dermatol 2002; 138:1019–24.3 Sidoroff A, Halevy S, Bavinck JN et al. Acute generalized exanthema-
tous pustulosis (AGEP) – a clinical reaction pattern. J Cutan Pathol2001; 28:113–19.
4 Kano Y, Hiraharas K, Sakuma K, Shiohara T. Several herpesvirusescan reactivate in a severe drug-induced multiorgan reaction in the
same sequential order as in graft-versus-host disease. Br J Dermatol2006; 155:301–6.
5 Seishima A, Yamanaka S, Fujisawa T et al. Reactivation of humanherpesvirus (HHV) family members other than HHV-6 in drug-
induced hypersensitivity syndrome. Br J Dermatol 2006; 155:344–9.
Conflicts of interest: none declared.
Book review
DOI: 10.1111/j.1365-2133.2006.07722.x
Textbook of Pediatric Dermatology, 2nd edn. J. Harper, A. Oranje
& N. Prose (editors). Oxford: Blackwell Publishing, 2005;
2272 pp. ISBN: 9781405110464. Price £350.00.
This extensive, two-volume text represents an international
collaboration of the world leaders in the field of paediatric
dermatology. It is meticulously and systematically organized
in sections and subsections based around disorders with com-
mon or related pathogenesis, modes of clinical presentation
and diseases presenting within specific body sites. As such it is
highly user friendly, allowing the reader not only to find
comprehensive details of specific disorders but also to retrieve
information to aid in the differential diagnosis and investiga-
tion of more ill-defined clinical presentations.
There have been many advances in the field of paediatric
dermatology since the first edition was published in 2000, par-
ticularly in the field of molecular genetics and in the under-
standing of the pathomechanisms of disorders. The second
edition has been extensively updated to reflect this knowledge
and the impact it has had on disease classification. There are
nine additional chapters and many new contributing authors.
While common clinical problems such as eczema and skin
infections are given significant coverage, much rarer disorders
are not short changed and as such this is an excellent refer-
ence text. Indeed, the largest section is devoted to the full
breadth of genetic skin disease and is a particular strength of
this book. There is extensive use of colour photography which
is generally of high quality. There has been a much greater
emphasis in the second edition on the practical aspects of dis-
ease management with useful algorithms and protocols in
many chapters which will undoubtedly be of great benefit to
the practising dermatologist.
This is a state-of-the-art textbook which no dermatology
department should be without and I suspect that most dermato-
logists with any paediatric practice will want to own a copy.
D.K .B . ARMSTRONGRoyal Hospital, Belfast BT12 6BA, U.K.
News and Notices
DOI: 10.1111/j.1365-2133.2007.07788.x
Congress: 2nd International Congress on Psoriasis
Dates: JUNE 21–24, 2007
Venue: Paris, Palais des congres, FRANCE
Web site: http://www.pso2007.com
Contact:
PSO 2007 c/o MCI
24 rue Chauchat
75009 Paris
FRANCE
Phone: +33 (1) 53 85 82 59
Fax: +33 (1) 53 85 82 83
Email: pso2007info@mci-group.com
Main topics:
– Scoring and monitoring the severity of the disease
– Management of the severe clinical manifestations
– Psoriasis in children and pregnancy
– Difficult to treat localisations
– Topical Treatment: what to choose and how to use
– Risk management and treatment optimisation: combination
and rational strategies
– Phototherapies: what to choose and how to use
– Alternative treatment
– Biologics
– Extracutaneous manifestations in Psoriasis
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
Book review 611
North West Vascular Anomalies Consensus Workshop
Wednesday 23rd May 2007
Call for Abstracts
A Scientific workshop to develop a consensus in the treatment
of vascular malformations throughout the United Kingdom.
Venue – Crowne Plaza Hotel Liverpool.
For further information abstract forms or registration forms,
please visit http://www.vascularanomalies.org.uk or contact
Janet.hughes@rlc.nhs.uk; Cath.gorst@rlc.nhs.uk
Royal Liverpool Children’s NHS Trust Eaton Road Liverpool
L12-2AP.
Corrigenda
DOI: 10.1111/j.1365-2133.2007.07789.x
Familial cutaneous collagenomas resulting from a novelmutation in LEMD3. Br J Dermatol 2007, Hershkovitzet al.
In the above mentioned article1 there was an error in one
of the author names. D.B. Amitai should have appeared as
B. Amitai.
The authors apologise for these errors.
References
1 Hershkovitz D, Amitai B, Sprecher E. Familial cutaneous collageno-mas resulting from a novel mutation in LEMD3. Br J Dermatol 2007;
156:375–7.
DOI: 10.1111/j.1365-2133.2007.07790.x
Validation of the U.K. Working Party diagnostic cri-teria for atopic eczema in a Xhosa-speaking Africanpopulation. Br J Dermatol 2007, Chalmers et al.
In the above mentioned article1 there were some errors in
the reference list.
Reference 2 should have appeared as follows and not as
was published:
2 Williams HC, Burney PG, Hay RJ et al. The UK Working
Party’s Diagnostic Criteria for Atopic Dermatitis. I. Deriva-
tion of a minimum set of discriminators for atopic derma-
titis. Br J Dermatol 1994;131:383–96.
Reference 15 should have appeared as follows and not as
was published:
15 Williams HC, Burney PG, Pembroke AC, Hay RJ. The
UK Working Party’s Diagnostic Criteria for Atopic Derma-
titis. III. Independent hospital validation. Br J Dermatol
1994;131:406–16.
The authors apologise for these errors.
References
1 Chalmers DA, Todd G, Saxe N, Milne JT, Tolosana S, Ngcelwane PN,
Hlaba BN, Mngomeni LN, Nonxuba TC, Williams HC. Validation ofthe U.K. Working Party diagnostic criteria for atopic eczema in a
Xhosa-speaking African population. Br J Dermatol 2007; 156:111–16.
DOI: 10.1111/j.1365-2133.2007.07791.x
Ciclosporin use in toxic epidermal necrolysis. Br JDermatol 2007, Danby.
In the above mentioned article1 there was an error in one
of the corresponding email addresses.
ljmderm@aol.com should have appeared as fwdljm@
tds.net.
The author apologises for this error.
References
1 Danby FW. Ciclosporin use in toxic epidermal necrolysis. Br J Dermatol
2007; 156:390–1.
� 2007 The Authors
Journal Compilation � 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp575–612
612 Corrigenda
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