Rituximab in refractory autoimmune bullous diseases

Transcription

1 Clinical dermatology Review article doi: /j x Rituximab in refractory autoimmune bullous diseases E. Schmidt, N. Hunzelmann,* D. Zillikens, E.-B. Bröcker and M. Goebeler Departments of Dermatology, University of Wu rzburg; *University of Cologne; University of Schleswig-Holstein, Campus Lu beck; and University of Heidelberg, University Medical Center Mannheim, Germany Summary Treatment of autoimmune blistering diseases consists of systemic glucocorticosteroids usually in combination with additional immunosuppressants such as azathioprine and mycophenolate mofetil or immunomodulators such as dapsone, antibiotics, intravenous immunoglobulins, and immunoadsorption. In some patients, these treatment regimens are not sufficient to control disease activity and or lead to intolerable adverse events. Rituximab, originally developed for the treatment of non- Hodgkin s lymphoma, is an anti-cd20 humanized monoclonal antibody leading to transitory B-cell depletion. For this indication, rituximab is widely employed, and severe side-effects rarely observed. Subsequently, the B-cell-depleting effect of rituximab has been exploited successfully in various autoimmune disorders, including autoimmune blistering diseases. Here, we review the effect of rituximab in such diseases. To date, application of rituximab has been reported in 26 treatmentresistant patients with the vulgaris, foliaceus, and paraneoplastic variants of pemphigus as well as in bullous pemphigoid and epidermolysis bullosa acquisita. All but a single patient showed clinical improvement with reduction of lesion formation. In about a third, a clinical remission requiring further immunsuppressive medication was achieved, and in about a quarter, complete remission was induced. In addition, the mode of action and adverse events of rituximab as well as adjuvant immunosuppressive treatments, and the effect on levels of circulating autoantibodies in these patients are discussed. Introduction Autoimmune bullous diseases are usually treated with systemic glucocorticosteroids, frequently in combination with other immunosuppressants, such as azathioprine, mycophenolate mofetil, methotrexate, and cyclophosphamide, or immunomodulators such as dapsone, antibiotics, and high-dose intravenous immunoglobulins (reviewed in Goebeler et al 1 ). Bullous pemphigoid (BP), linear IgA disease, pemphigoid gestationis, anti-p200 Correspondence: Enno Schmidt, MD, PhD, Department of Dermatology, University of Würzburg, Josef-Schneider-Str. 2, D Würzburg, Germany. schmidt_e@klinik.uni-wuerzburg.de Conflict of interest: none declared. Accepted for publication 10 February 2006 pemphigoid, and lichen planus pemphigoides can usually be controlled by standard regimens. In contrast, treatment of pemphigus, mucous membrane pemphigoid and epidermolysis bullosa acquisita (EBA) is often challenging, and only a few randomised controlled therapeutic trials are available for these diseases. 1 In the two latter conditions, intolerably high doses of systemic corticosteroids are frequently required and severe corticosteroid-related side-effects may develop (reviewed in Goebeler et al. 1 ). For these patients, new treatment modalities are needed. Rituximab is a monoclonal humanized antibody directed against the B-cell-specific, cell-surface antigen CD20. Originally developed for the treatment of B-cell malignancies, it has been successfully used off-label in a large panel of autoimmune diseases (reviewed in Virgolini and Marzocchi 2 ). Ó 2006 Blackwell Publishing Ltd Clinical and Experimental Dermatology, 31,

2 Mode of action Rituximab binds to CD20-expressing B lymphocytes, including (i) early B cells in the bone marrow, (ii) autoantigen-specific memory B cells, and (iii) mature B cells already sensitized to antigen, which may subsequently transform into antibody-producing plasma cells. In contrast, it does not interact with stem cells or plasma cells (reviewed in Virgolini and Marzocchi 2 ). Interestingly, the natural ligand of CD20 has not yet been identified, and CD20 knockout mice do not show an apparent phenotype. 3 In malignant B cells, rituximab was shown to inhibit proliferation by complementand antibody-mediated cytotoxicity, and apoptosis. The modulation of the CD20-mediated signal transduction pathways in non-hodgkin s lymphoma has recently been reviewed (Jazirehi and Bonavida 4 ). By these mechanisms, CD20-positive B cells are depleted from the circulation. This mode of action also appears attractive for the treatment of autoimmune diseases, particularly when pathogenically relevant autoantibodies are present, as in pemphigus. In addition to the removal of CD20-positive B cells that may later develop into autoantibody-producing plasma cells, other B-lymphocyte functions are likely to be disturbed. This can be concluded from the fact that a strong transitory reduction of B lymphocytes is achieved in nearly every patient, irrespective of the clinical response. 5 7 The degree of B-cell depletion may, however, be variable, and was reported to depend on both serum levels of rituximab and polymorphisms of the receptor FccRIIIa in patients with systemic lupus erythematosus. 7 It may be hypothesized that rituximab also affects (i) autoantigen processing and presentation of B cells, (ii) help for autoreactive T cells, and (iii) production of T-cell-modulating cytokines. The pathogenic role of autoreactive T cells in pemphigus has been studied intensively, and desmoglein 3-specific T cells were demonstrated to release interleukins 4, 6, and 10 upon stimulation (reviewed in Hertl and Veldman 8 ). Furthermore, rituximab may also exert its action by removal of autoreactive memory B lymphocytes. 2 These additional effects that do not directly interfere with autoantibody production may explain the good clinical response to rituximab in Th 1 cell-mediated rheumatoid arthritis. 6 Efficacy in autoimmune blistering diseases To date, 26 patients with autoimmune bullous disorders were reported to have been treated with rituximab, including 18 patients with pemphigus vulgaris (PV), 4 with paraneoplastic pemphigus (PNP), 2 with pemphigus foliaceus (PF), and 1 each with BP and EBA (Table 1). The administration protocol for rituximab was adopted from treatment schedules of lymphoma patients. Usually, four courses of rituximab were given intravenously at a dose of 375 mg m 2 body surface at weekly intervals. Interestingly, a single infusion of 375 mg m 2 and even of 100 mg m 2 might be sufficient to achieve both B-cell depletion and clinical responses, as reported in patients with systemic lupus erythematosus. 9 In all patients, except 1 with PNP who received 8-weekly rituximab infusions only, 10 adjuvant immunosuppressants were employed, including cyclophosphamide alone in 1 and systemic glucocorticosteroids in the remaining 24 patients (Table 1). Of these 24 patients, 8 were treated with systemic corticosteroids alone, and in the remaining 16 patients, systemic corticosteroids were combined with mycophenolate mofetil (8 patients), cyclophosphamide (4), and azathioprine and methotrexate (2) (Table 1). In two patients with PNP, there was complete remission (defined as clinical remission and no further therapy necessary) of the pemphigus lesions, and in one, a partial remission (defined as healing of more than 50% of lesions), whereas in the remaining PNP patient, pemphigus lesions progressed. In all patients, the B-cell lymphoma responded in parallel with the pemphigus lesions A fifth patient with haematological malignancy (B-cell precursor acute lymphocytic leukaemia), received rituximab for BP that he had developed together with a glucocorticosteroid-refractory graftversus-host disease following an unrelated cord-blood transplantation. In addition to rituximab, weekly infusions of the anti-cd25 antibody daclizumab (1 mg kg body weight) were administered. 14 This patient died in clinical remission 12 months after the last rituximab infusion while still on mycophenolate mofetil and weekly daclizumab. Complete remission was induced in 5 (25%) of the 20 patients with PV and PF, a clinical remission (defined as healing of all lesion but further therapy required) in 6 (30%), and a partial remission in 9 (45%) (Table 1). The patient with EBA (Fig. 1) showed complete remission. 15 Healing of lesions started between 1 and 10 weeks and clinical remission occurred between 1 and 9 months after the first rituximab infusion. No relationship between the intensity of immunosuppression and clinical response was obvious; e.g., in patients with complete remission, low, moderate, and high prednisolone doses with or without further immunosuppressants were used (Table 1). With the limited data available so far, the 504 Ó 2006 Blackwell Publishing Ltd Clinical and Experimental Dermatology, 31,

3 Table 1 Rituximab in autoimmune bullous diseases: outcome, adverse events, and adjuvant therapies. No. Diagnosis Age (years) Adjuvant therapy* Outcome Follow-up (months) Adverse events Ref 1 PNP 61 Rituximab 375 mg m 2 every 8 weeks PR 3 None 2 PNP 73 Moderate-dose pred CR 12 None 3 PNP 64 Cyclophosphamide (2 mg kg day) + low-dose pred PD 4 Fatal bacterial sepsis 4 PNP 74 Moderate-dose pred PR 2 None 5 PF 56 Single pulse dexamethasone (3x100 mg) CR 37 None 6 PV 30 Mycophenolate mofetil + moderate-dose pred PR 11 Bacterial sepsis 7 PV 54 Mycophenolate mofetil + high-dose pred PR 3 None 8 PV 54 Low-dose pred PR 5 None 9 PV 53 Cyclophosphamide (100 mg day) + moderate-dose pred CliR 10 None 10 PV 34 Azathioprine + moderate-dose pred + PR 9 Bacterial pneumonia 11 PV 42 Mycophenolate-mofetil + cyclosporine + high-dose pred PR 16 Bacterial arthritis 12 PV 20 Moderate-dose pred CliR 9 None 13 PV 51 Moderate-dose pred + cyclophosphamide (2.5 mg kg) CR 18 None 14 PV 37 Low-dose pred + cyclophosphamide (2.0 mg kg) CliR 5 Fatal Pneumocystis carinii pneumonia 15 PV 47 Cyclophosphamide (1.6 mg kg) PR 9 None 16 PV 39 High-dose pred CR 10 None 17 PV 14 Mycophenolate mofetil + low-dose methylpred CR 31 Hypogammaglobulinaemia 18 PV 55 Low-dose methylpred PR 12 None 19 PV 60 Mycophenolate mofetil + low-dose pred CliR 24 None 20 PV 26 Mtx 10 mg week + low-dose pred PR 10 None 21 PV 27 Mtx 20 mg week + low-dose pred PR 10 None 22 PF 67 Mycophenolate mofetil + low-dose pred CR 18 None 23 PV 57 Mycophenolate mofetil + low-dose pred CliR 36 None 24 PV 17 High-dose pred + CliR 17 None 25 BP 10 Mycophenolate mofetil + moderate-dose methylpred CliR 13 Fatal bacterial sepsis + daclizumab (1 mg kg week) 26 EBA 46 Azathioprine + high-dose pred + colchicine CR 19 Deep venous thrombosis *Prednisolone dose: low, <50 mg day; moderate, >50<100 mg day; high, >100 mg day; PNP and PV patients are presented in order of publication date; associated with CD20-positive non-hodgkin lymphoma; additional rituximab infusions (375 mg m 2 ) were administered: in patient no. 10, a second course of four infusions at weekly intervals after 6 months following a relapse, in patient no. 24, regular infusions every 4 8 weeks; associated with chronic graft-versus-host disease following unrelated cord blood transplantation due to B-cell precursor acute lymphocytic leukaemia. BP, bullous pemphigoid, EBA, epidermolysis bullosa acquisita; MTX, methotrexate; pred, prednisolone; PF, pemphigus foliaceus; PNP, paraneoplastic pemphigus; PV, pemphigus vulgaris; CliR, clinical remission (healing of all lesions); CR, complete remission (clinical remission and no further therapy needed); PR, partial remission (healing of > 50% of lesions), PD, progressive disease. requirement for additional immunosuppression remains to be explored. To reduce disease activity until rituximab exerts its effect, adjuvant immunosuppression that may be tapered according to the clinical response is advocated. In two PV patients, additional rituximab infusions were administered: in one who suffered from a relapse 6 months after the first application of rituximab, a second course of four rituximab infusions again resulted in a partial remission. 16 In the other patient infusions at 4- to 8-weekly intervals led to a clinical remission 17. Therefore, it appears feasible to explore the use of repeated rituximab infusions during the course of the disease to sustain clinical improvement in these patients. Autoantibody levels during treatment with rituximab In 21 of the reported pemphigus patients, circulating autoantibody levels have been investigated by ELISA using recombinant desmoglein 1 and or 3 as target antigens (n ¼ 11) or indirect immunofluorescence on monkey oesophagus (n ¼ 10). As expected, in the majority of patients (n ¼ 15), serum levels of autoantibodies paralleled disease activity. Interestingly, although clinical improvement was seen in six patients, autoantibody levels remained unchanged or decreased by only one titre step or < 30%, respectively It may be speculated that in these patients long-lived plasma cells are responsible for the sustained autoantibody Ó 2006 Blackwell Publishing Ltd Clinical and Experimental Dermatology, 31,

4 Figure 1 (a, b) Disseminated erosions and blisters on the back of patient no. 26 with epidermolysis bullosa acquisita before rituximab had been initiated. In the same patient, 11 weeks after the first rituximab infusion, lesions had healed leaving postinflammatory hyperpigmentation (c) and milia on the upper back (d). 15 production and or that mechanisms apart from reducing B cells that may later develop into antibodyproducing plasma cells (see above) account for the clinical effect. Similar observations have been made in patients with systemic lupus erythematosus, in whom serum anti-double-stranded DNA antibody titres remained largely unchanged despite good clinical responses. 2 It may be argued, however, that in contrast to autoimmune bullous diseases, the pathogenic relevance of autoantibodies in systemic lupus erythematosus is less evident. In some patients, in addition to autoantibodies, total IgG and or IgG levels to recall antigens such as varicella zoster and herpes simplex viruses, were monitored during the course of the disease. In contrast to autoantibody titres, levels of both total IgG and IgG to recall antigens did not change. 15,16,22 24 This observation is in line with previous reports on rheumatoid arthritis and systemic lupus erythematosus. 2,6,7 In one patient who received regular rituximab infusions over 17 months, IgG levels to recall antigens decreased over time. 17 The reason for the apparently higher susceptibility of autoantigen-specific CD20-positive B lymphocytes towards rituximab compared with other immunoglobulin-producing B cells remains elusive. It may be speculated that in autoimmune diseases, CD20- positive B lymphocytes are involved in the generation of short-lived plasma cells synthesizing pathogenic autoantibodies, whereas in situations such as infection immunity, immunoglobulin production is sustained by long-lived plasma cells. Adverse events Since its approval for the treatment of non-hodgkin s lymphoma in 1997, rituximab has been used in more than patients. 2 Apart from infusion-related events, adverse events including opportunistic infections are rare. 2,6 In patients with autoimmune bullous disorders, infusion-related side-effects, such as headache, fever, chills, urticaria, pruritus and hypotension were usually mild and could be controlled or prevented by premedication with paracetamol (acetaminophen) and antihistamines (Table 2). However, both life-threatening and fatal opportunistic infections have been reported following rituximab therapy, particularly when Time Medication 0 h Intravenous sodium chloride 0.9% or Ringer s solution, 1000 ml; infusion velocity: 500 ml h 1 h Oral paracetamol (acetaminophen), 1000 mg; intravenous clemastine, 2 mg 2 h Intravenous rituximab (MabTheraä), 375 mg m 2 body surface; infusion velocity: 50 mg h. Intravenous sodium chloride 0.9% or Ringer s solution, 1000 ml; infusion velocity: 250 ml h 3 h When no adverse effects have occurred, increase rituximab infusion velocity by 50 mg h every 30 minutes to a maximum of 400 mg h Table 2 Recommended treatment protocol for patients with refractory autoimmune blistering diseases with rituximab. After initiation of rituximab infusion, there should be hourly control of blood pressure, pulse, body temperature, and respiratory rate. During rituximab infusion, bedside emergency medication (e.g. intravenous clemastine, ranitidine, prednisolone, adrenaline, and theophylline) should be available in case of anaphylaxis. 506 Ó 2006 Blackwell Publishing Ltd Clinical and Experimental Dermatology, 31,

5 combined with immunosuppressants. 2,6 In patients with autoimmune blistering diseases, serious adverse events, including fatal outcomes, were observed in eight patients (31%) and tended to affect patients with underlying malignancy and or combination immunosuppressive regimens (Table 1). This rate of serious adverse events is considerably higher than the 17% of 18 patients with systemic lupus erythematosus and 9% of 161 patients with rheumatoid arthritis. 6,9 In a minority of patients, a transient hypogammaglobulinaemia was observed for 6 9 months, which resolved in parallel with the raise of peripheral B lymphocytes. 5 In contrast, in a patient with juvenile pemphigus, a decrease of total IgG to 60% of its normal value developed 10 months after the first rituximab injection, and has now lasted for more than 2 years. 24 Conclusion and future perspectives Rituximab appears to be a rational and effective treatment regimen in refractory autoimmune bullous disorders. In all of the reported patients with autoimmune bullous diseases except one with PNP, at least a partial remission could be induced and in about a quarter of them, rituximab led to a complete clinical remission with healing of all skin lesions. However, about a third of patients suffered from severe treatment-related side-effects, mostly due to systemic infections. Further studies are needed that should determine (i) the optimal dosage and number of rituximab infusions required to induce remission; (ii) adequate adjuvant immunosuppressive medication, including initial dosage and tapering mode; (iii) subgroups of patients with other treatment-resistant autoimmune bullous diseases such as mucous membrane pemphigoid, likely to benefit from rituximab; (iv) risk factors for developing severe adverse reactions; and (v) the benefit of repeated rituximab infusion as maintenance therapy. At present, rituximab may be applied in patients with autoimmune bullous diseases that have been resistant for more than 3 months to at least two treatment regimens including a combination therapy of systemic glucocorticosteroids (at a minimum dose of 1.0 mg kg body weight prednisolone equivalent) with another immunosuppressant such as mycophenolate mofetil or azathioprine. So far, patients with refractory pemphigus and possibly EBA appear to be particularly suitable for rituximab therapy. The patient s informed written consent should be obtained, after explaining its off-label use, mechanisms of action, potential adverse effects, including severe systemic infections, and other therapeutic options. A protocol for rituximab infusions is detailed in Table 2. Systemic glucocorticosteroids (initially mg kg body weight) in combination with azathioprine, mycophenolate mofetil, and methotrexate, respectively, appear to be adequate adjuvant immunosuppressive medication. Care should be taken to detect and treat systemic infections as early as possible. In order to minimize systemic infection, immunosuppressive medication may be tapered as soon as clinical improvement occurs. In addition to the clinical picture, regular monitoring of autoantibody levels has been found useful as guidance to taper the adjuvant immunosuppressive medication. Learning points Rituximab, a humanized anti-cd20 monoclonal antibody that depletes B cells, is increasingly used off-label for refractory autoimmune diseases. Rituximab has been successfully administered in patients with otherwise treatment-resistant autoimmune bullous diseases such as paraneoplastic pemphigus, pemphigus vulgaris and pemphigus foliaceus. Of the patients treated, 90% showed significant clinical improvement, and in about 25% of these, complete remission was induced. Infusion-related adverse effects such as headache, fever, chills, urticaria, pruritus and hypotension are usually mild and can be controlled by premedication with paracetamol (acetaminophen) and antihistamines. Serious adverse events, mainly systemic infections including fatal outcomes, were observed in about a third of patients with autoimmune blistering disorders receiving rituximab. As long as controlled studies are not available, we suggest considering rituximab only in patients with autoimmune bullous diseases that have been resistant for more than 3 months to at least two standard treatment regimens. Adjuvant treatment may include systemic glucocorticosteroids (initially mg kg body weight) in combination with a steroid-sparing immunosuppressant and should be tapered off as early as possible. Care should be taken to detect systemic infections as early as possible. Ó 2006 Blackwell Publishing Ltd Clinical and Experimental Dermatology, 31,

6 References 1 Goebeler M, Sitaru C, Zillikens D. Bullous autoimmune disorders: Therapy. J Dtsch Dermatol Ges 2004; 2: Virgolini L, Marzocchi V. Rituximab in autoimmune diseases. Biomed Pharmacother 2004; 58: O Keefe TL, Williams GT, Davies SL, Neuberger MS. Mice carrying a CD20 gene disruption. Immunogenetics 1998; 48: Jazirehi AR, Bonavida B. Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-cd20 mab) in non-hodgkin s lymphoma: implications in chemosensitization and therapeutic intervention. Oncogene 2005; 24: Quartier P, Brethon B, Philippet P et al. Treatment of childhood autoimmune haemolytic anaemia with rituximab. Lancet 2001; 358: Edwards JC, Szczepanski L, Szechinski J et al. Efficacy of B cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med 2004; 350: Anolik JH, Campbell D, Felgar RE et al. The relationship of FcgammaRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus. Arthritis Rheum 2003; 48: Hertl M, Veldman C. T-cellular autoimmunity against desmogleins in pemphigus, an autoantibody-mediated bullous disorder of the skin. Autoimmun Rev 2003; 2: Looney RJ, Anolik JH, Campbell D et al. B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I II dose-escalation trial of rituximab. Arthritis Rheum 2004; 50: Heizmann M, Itin P, Wernli M et al. Successful treatment of paraneoplastic pemphigus in follicular NHL with rituximab: report of a case and review of treatment for paraneoplastic pemphigus in NHL and CLL. Am J Hematol 2001; 66: Borradori L, Lombardi T, Samson J et al. Anti-CD20 monoclonal antibody (rituximab) for refractory erosive stomatitis secondary to CD20 (+) follicular lymphomaassociated paraneoplastic pemphigus. Arch Dermatol 2001; 137: Schadlow MB, Anhalt GJ, Sinha AA. Using rituximab (anti- CD20 antibody) in a patient with paraneoplastic pemphigus. J Drugs Dermatol 2003; 2: Rossum MM, Verhaegen NT, Jonkman MF et al. Follicular non-hodgkin s lymphoma with refractory paraneoplastic pemphigus: case report with review of novel treatment modalities. Leuk Lymphoma 2004; 45: Szabolcs P, Reese M, Yancey KB et al. Combination treatment of bullous pemphigoid with anti-cd20 and anti-cd25 antibodies in a patient with chronic graft-versushost disease. Bone Marrow Transplant 2002; 30: Schmidt E, Benoit S, Bröcker EB et al. Successful adjuvant treatment of recalcitrant epidermolysis bullosa acquisita with anti-cd20 antibody rituximab. Arch Dermatol 2006; 142: Dupuy A, Viguier M, Bedane C et al. Treatment of refractory pemphigus vulgaris with rituximab (anti-cd20 monoclonal antibody). Arch Dermatol 2004; 140: Kong HH, Prose NS, Ware RE, Hall RP 3rd. Successful treatment of refractory childhood Pemphigus vulgaris with anti-cd20 monoclonal antibody (rituximab). Pediatr Dermatol 2005; 22: Cooper HL, Healy E, Theaker JM, Friedmann PS. Treatment of resistant pemphigus vulgaris with an anti-cd20 monoclonal antibody (rituximab). Clin Exp Dermatol 2003; 28: Herrmann G, Hunzelmann N, Engert A. Treatment of pemphigus vulgaris with anti-cd20 monoclonal antibody (rituximab). Br J Dermatol 2003; 148: Morrison LH. Therapy of refractory pemphigus vulgaris with monoclonal anti-cd20 antibody (rituximab). JAm Acad Dermatol 2004; 51: Arin MP, Engert A, Krieg T, Hunzelmann N. Anti-CD20 monoclonal antibody (rituximab) in the treatment of pemphigus. Br J Dermatol 2005; 153: Goebeler M, Herzog S, Bröcker EB, Zillikens D. Rapid response of treatment-resistant pemphigus foliaceus to the anti-cd20 antibody rituximab. Br J Dermatol 2003; 149: Espana A, Fernandez-Galar M et al. Long-term complete remission of severe pemphigus vulgaris with monoclonal anti-cd20 antibody therapy and immunophenotype correlations. J Am Acad Dermatol 2004; 50: Schmidt E, Herzog S, Bröcker EB, Zillikens D, Goebeler M. Long-standing remission of recalcitrant juvenile pemphigus vulgaris after adjuvant therapy with rituximab. Br J Dermatol 2005; 153: Salopek TG, Logsetty S, Tredget EE. Anti-CD20 chimeric monoclonal antibody (rituximab) for the treatment of recalcitrant, life-threatening pemphigus vulgaris with implications in the pathogenesis of the disorder. J Am Acad Dermatol 2002; 47: Virgolini L, Marzocchi V. Anti-CD20 monoclonal antibody (rituximab) in the treatment of autoimmune diseases: successful result in refractory pemphigus vulgaris. Report of a case. Haematologica 2003; 88: ELT Wenzel J, Bauer R, Bieber T, Tüting T. Successful rituximab treatment of severe pemphigus vulgaris resistant to multiple immunosuppressants. Acta Derm Venereol 2005; 85: Ó 2006 Blackwell Publishing Ltd Clinical and Experimental Dermatology, 31,