Ch.C. Zouboulis

The Human Sebocyte Culture Model Provides New Insights into Development and Management of Seborrhoea and Acne

Seborrhoea and acne are exclusively human diseases and sebaceous gland differentiation is species specific. Therefore, fundamental research on human sebaceous cell function and control requires human in vitro models. The human sebocyte culture model, introduced in 1989, has been used in several studies to elucidate sebaceous gland activity and its regulation at the cellular level. Cultured human sebocytes have been shown to preserve important sebocytic characteristics, although they undergo an incomplete terminal differentiation in vitro. In vitro synthesis of free fatty acids without bacterial involvement and marked interleukin 1α expression at the mRNA and protein levels with no further induction by lipopolysaccharides lead to the assumption that human sebocytes may initiate acne lesions by an intrinsic mechanism. Androgens affected sebocyte activity in vitro in a manner dependent on the localization of the sebaceous glands. In vitro stimulation of sebocyte proliferation by androgens could be completely abolished by spironolactone. Cultured sebocytes strongly expressed type 1 5α-reductase and metabolized testosterone to androstenedione, 5α-androstanedione, 5α-dihydrotestosterone, androsterone and 5α-androstanediol, whereas the levels of 5α-reductase activity were probably not feedback regulated. 4,7β-Dimethyl-4-aza-5αcholestan-3-one, a type 1 5α-reductase inhibitor, induced an early, marked down-regulation of 5α-reductase activity in human sebocytes in vitro, while hydrofinasteride, a type 2 inhibitor, required 103-fold higher concentrations to induce similar effects. Stimulation of sebocyte proliferation by insulin, thyroid-stimulating hormone and hydrocortisone indicates that the hormonal control of the sebaceous gland could be a complex mechanism. Retinoids inhibited sebocyte proliferation in a dose-dependent manner and down-regulated lipid synthesis and sebocyte differentiation in vitro. Isotretinoin was the most potent compound. On the other hand, vitamin A was found essential for sebocyte activity and differentiation in vitro and could be partially substituted by synthetic retinoids. The inhibitory effect of isotretinoin on sebocyte proliferation was barely affected by the presence of vitamin A. The low persistent isotretinoin levels or, more likely, the considerably elevated tretinoin concentrations detected in human sebocytes after treatment with isotretinoin in vitro may be responsible for the inhibitory effect of this compound on sebocyte activity.

Oral Retinoids in the Treatment of Seborrhoea and Acne

Isotretinoin is an extremely effective drug if given systemically in severe forms of seborrhoea and acne, being the only retinoid with potent sebostatic properties. Its unique activity on the sebaceous gland still remains unclear since isotretinoin barely binds to cellular retinoic-acid-binding proteins and to retinoic acid receptors. Its bioavailability is approximately 25% and can be increased by food 1.5–2 times; after 30 min, the drug is detectable in the blood and maximal concentrations are reached 2–4 h after oral intake. The major metabolites of isotretinoin in blood are 4-hydroxy- and 4-oxo-isotretinoin, while several glucuronides are detectable in the bile. 4-Oxo-isotretinoin is present in plasma in a 2- to 4-fold higher concentration 6 h after a single dose. Steady-state concentrations appear after 1 week. The half-life elimination rate of the parent compound ranges from 7 to 37 h while that of some metabolites does so from 11 to 50 h. Isotretinoin crosses the placenta and is recognized as a strong teratogenic compound. About 10–30% of the drug is metabolized via its isomer tretinoin. Excretion of isotretinoin occurs after conjugation with the faeces or after metabolization with the urine. The epidermal levels of isotretinoin are rather low and no progressive accumulation, either in serum or in the skin, is found. After discontinuation of therapy, isotretinoin disappears from serum and skin within 2–4 weeks. Isotretinoin is the most effective drug in reducing sebaceous gland size (up to 90%) by decreasing proliferation of basal sebocytes, suppressing sebum production and inhibiting sebocyte differentiation in vivo. The molecular basis for its antisebotrophic activity has not been fully elucidated. Isotretinoin also exhibits anti-inflammatory activities. Systemic isotretinoin is today the regimen of choice in severe seborrhoea, since it reduces sebocyte lipid synthesis by 75% with daily doses as low as 0.1 mg/kg after 4 weeks. Patients who have received oral isotretinoin therapy for seborrhoea do not usually experience a relapse for months or years. In severe acne, a 6- to 12-month treatment with isotretinoin 1 mg/kg/day reduced to 0.5 or 0.2 mg/kg/day according to the response is recommended (cumulative dose of >120 mg/kg). Contraception is essential during isotretinoin treatment in women of childbearing age 1 month before, during and for 3 months after discontinuation of treatment.

The 5α-Recluctase System and Its Inhibitors

5α-Reductase, the enzyme system that metabolizes testosterone into dihydrotestosterone, occurs in two isoforms. The type 1 isozyme is composed of 259 amino acids, has an optimal pH of 6-9 and represents the ‘cutaneous type’; it is located mainly in sebocytes but also in epidermal and follicular keratinocytes, dermal papilla cells and sweat glands as well as in fibroblasts from genital and non-genital skin. The type 2 isozyme is composed of 254 amino acids, has an optimal pH of about 5.5 and is located mainly in the epididymis, seminal vesicles, prostate and fetal genital skin as well as in the inner root sheath of the hair follicle and in fibroblasts from normal adult genital skin. The genes encoding type 1 and type 2 isozymes are found in chromosomes 5p and 2p, respectively, and each consists of 5 exons and 4 introns. During the last decade, several steroid analogues and non-steroid agents have been developed to interfere with 5α-reductase activity. Finasteride, which has a higher affinity for the type 2 isozyme, is the first 5α-reductase antagonist clinically introduced for treatment of benign prostate hyperplasia. The clinical evaluation of finasteride or other 5α-reductase inhibitors in the field of dermatology has been very limited; in particular, those that selectively bind to type 1 isozyme (e.g. MK-386, LY191704) may be regarded as candidates for treatment of androgen-dependent skin disorders such as seborrhoea, acne, hirsutism and/or androgenetic alopecia.

Growth control of melanoma cells and melanocytes by cytokines.

Aberrant proliferation of tumor cells characterizes cancer growth. Investigations of cellular growth control mechanisms have contributed to our understanding of carcinogenesis and to the identification of compounds with specific antitumor activity. Many cytokines have been found to act on melanoma tumors, either produced by the tumor cells themselves or by infiltrating host cells. Purified cytokines allowed direct comparison of the growth response between normal human melanocytes and malignant melanoma cells. The present paper summarizes results of a series of our own experiments not yet published and data from a review of the recent literature. Proliferation of normal human melanocytes is enhanced by several cytokines, including basic fibroblast growth factor (bFGF), melanoma growth stimulatory activity (MGSA), hepatocyte growth factor (HGF), and mast cell growth factor (MGF). Melanoma cells are additionally stimulated by epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) and nerve growth factor (NGF). Tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta 1 (TGF-beta 1), and interleukin (IL)-6 are all potent inhibitors of melanocyte growth, but they are less effective on melanoma cells or even stimulate their growth. Interferon (IFN)-alpha and IFN-gamma inhibited proliferation of melanoma cells but not of melanocytes, whereas IFN-beta showed antiproliferative effects in both cell types. These findings suggest an alteration in growth control mechanisms during melanocyte transformation and possibly play a role in melanoma pathogenesis.

A multicentre, single‐blind, randomized comparison of a fixed clindamycin phosphate/tretinoin gel formulation (Velac®) applied once daily and a clindamycin lotion formulation (Dalacin T®) applied twice daily in the topical treatment of acne vulgaris

Background  A successful phase III pilot study compared the efficacy and safety of a fixed clindamycin 1%/tretinoin 0·025% gel formulation (CTG; Velac® gel) applied once daily and a clindamycin 1% lotion formulation (CLN; Dalacin T® lotion) applied twice daily in the treatment of moderate to severe acne vulgaris. Objectives We aimed to follow up this study.Methods The two treatment regimens were compared in a multicentre, single‐blind, randomized 12‐week investigation of patients with moderate to severe acne vulgaris. Results At week 12, the mean percentage reduction in non‐inflamed lesions (open and closed comedones) was greater in the CTG group compared with the CLN group (P = 0·05). Absolute reductions in open and closed comedones were also greater in the CTG group, consistent with the comedolytic activity of tretinoin. There was a significantly greater absolute reduction in inflamed lesions (pustules, papules and nodules) from baseline to both end‐point (last observed efficacy outcome; P = 0·043) and week 12 (P = 0·018) in the CTG group compared with the CLN group. Evaluation of the calculated overall acne severity score, considering all five lesion subtypes, demonstrated a significantly greater mean percentage reduction in the CTG group compared with the CLN group, both at end‐point (P = 0·01) and at week 12 (P < 0·01). The more subjective assessment of overall acne severity according to the Cook scale also demonstrated a significantly greater mean reduction in the CTG group than the CLN group after 12 weeks of therapy (P = 0·007). CTG had a more rapid effect on the onset of improvement compared with CLN; a 50% reduction in total lesion counts by day 60 was found in 77% of patients on CTG compared with 56% receiving CLN (P = 0·003). This was largely due to the reduction in open comedone counts (P = 0·0006). For all other variables, CTG was at least as effective as CLN. Both treatments were well tolerated. Conclusions A single daily topical application of Velac® gel was superior to Dalacin T® lotion applied twice daily in reducing overall acne scores, and was faster acting. The simpler dosing regimen of Velac® gel and its rapid effect are likely to have a positive effect on both patient compliance and cost.

Principles of cutaneous cryosurgery : An update

Skin diseasesIntroductionCryosurgery is the well-aimed and controlled destructionof diseased tissue by application of cold (table 1). It hasbeen shown to be effective and efficient in various skindiseases, provides high cure rates and good cosmetic resultswith a few contra-indications and low incidence of compli-cations [1].History of CryosurgeryThe first physician who used freezing to treat a dermato-logical disease was C. Gerhardt, a German dermatologistfrom Jena [2]. In 1885, he published a paper on the treat-ment of cutaneous tuberculosis with cold. Gerhardt had builta system, in which the lesions were covered with ice blad-ders for 3 h twice a day. Four patients considerably im-proved after a 2- to 4-week treatment. In 1899, A.C. White,an American dermatologist from New York, used for thefirst time liquefied air to treat various skin disorders, such asverrucae vulgares, naevi, precancerous lesions and tumours.In the year 1905, M. Juliusberg, a dermatologist fromBerlin, introduced the term ‘cryotherapy’ for the treatmentof skin lesions with cold. He applied the first cryospray, asmall balloon filled with carbon dioxide released in spurts.Modern cryosurgery was born in the 1960s after liquidnitrogen became available, and closed-circuit devices work-ing with liquid nitrogen, freon gas and nitrogen protoxidehave been developed by the American neurosurgeons I.S.Cooper and A.S. Lee and their Italian colleagues V.A.Fasano, G. Broggi, T. de Nunno and P. Baggiore. Nowa-days, numerous sophisticated devices have been developedand commercialized, not only to preserve and deliver cryo-gens, but also to monitor temperatures in and underneaththe treated lesions, thus allowing a controlled and reliablecryosurgery of the diseased skin.CryobiologyThe biological changes that occur during and after cryo-surgery have been studied in vitro and in vivo and arecaused by reduction of tissue temperature and consequentfreezing [3–6]. Tissue injury is induced by cell freezing andby the vascular stasis that develops in the tissue after thaw-ing. The cryoreaction is, therefore, characterized by the

Mycophenolate Mofetil Is Ineffective in the Treatment of Mucocutaneous Adamantiades-Behçet’s Disease

Background: Cyclosporine A and azathioprine are effective on mucocutaneous lesions in Adamantiades-Behçet’s disease. Mycophenolate mofetil (MMF) is a drug resembling their activity but with comparably negligible adverse reactions. Objective: A prospective clinical proof-of-principle study was conducted to investigate the effectiveness and toxicity of MMF in mucocutaneous Adamantiades-Behçet’s disease. Methods: Thirty patients were to be treated with MMF 2 g/day p.o. for 6 months, in combination with prednisolone 30 mg/day p.o. during the first month of treatment. Inefficacy was followed by an increase in MMF dose to 3 g/day. The primary efficacy variable was the decrease in the disease activity index (DAI) according to a modified variant of the Iran Behçet’s Disease Dynamic Activity Measure system. Results: The study was interrupted due to inefficacy of the compound after the intermediate evaluation of the first 6 patients (aged 37.0 ± 7.7 years with disease duration of 10.0 ± 8.9 years) as required by the ethical committee. Although an improvement of the DAI from 5.2 ± 3.5 to 1.3 ± 0.5 was found after the first month of combination treatment, withdrawal of prednisolone led to quick relapses with a new index increase (3.0 ± 3.5). The treatment was discontinued in 3 patients after 3 months, in 2 patients after 4 months and in another one after 5 months due to deterioration of the disease. Introduction of interferon α2a (3 × 9 million IU 3×/week s.c.) in 3 patients decreased the activity index from 4.0 ± 1.0 to 0.0 ± 0.0. No adverse effects were detected under MMF treatment. Conclusion: MMF (2–3 g/day) is unable to control the signs of mucocutaneous Adamantiades-Behçet’s disease.

Polymorphisms in the Human Cytochrome P-450 1A1 Gene (CYP1A1) as a Factor for Developing Acne

Cytochromes P-450 are a supergene family of enzymes involved in the metabolism of a wide range of endogenous and foreign compounds. The existing genetic variations of the distinct isozymes lead to interindividually different metabolic capacity. Since vitamin A, endogenous retinoids and their natural metabolites are morphogenic for the sebaceous gland, we investigated the polymorphisms of cytochrome P-450 1A1, as being one of the most active isozymes involved in their interconversion. From the known mutations, two were investigated: an additional cleavage site for MspI in the 3′-flanking region identified as a thymine-to-cytosine transition 1,194 bp downstream of exon 7 (m1) and an adenine-to-guanine transition at position 4889 in exon 7 (m2). We studied 96 acne patients for ml and m2 mutations by restriction fragment length polymorphism and allele-specific polymerase chain reaction, respectively, and compared the results with 408 reference individuals. No statistically significant difference was found in the distribution of m2 alleles; the frequency was 3.13 and 3.06% of the alleles, respectively (odds ratio = 1.02, confidence limits 0.41–2.52, p = 0.96). In contrast, a trend to an overrepresentation of m1 alleles in acne patients was observed; allele frequency was 8.33 in the patients and 6.99% in the control subjects, respectively (odds ratio 1.21, 95% confidence limits 0.68–2.16, p = 0.52). As the m1 mutation might define a marker for alterations on regulatory sites, the biological efficacy of natural retinoids could be greatly impaired by their rapid metabolism to inactive compounds. The resulting deficit of active natural retinoids may lead to abnormal sebocyte differentiation and hyperkeratinization of the follicular canal implicating the development of acne in some patients.

Differentiation between Merkel Cell Carcinoma and Malignant Melanoma: An Immunohistochemical Study

Background: Although Merkel cell carcinoma (MCC) exhibits specific clinical and histologic features, differentiation from other cutaneous neoplasms, such as lymphoma, metastatic oat cell carcinoma and malignant melanoma (MM), may sometimes be difficult. Objective: The aim of our study was to immunohistochemically differentiate MCC from MM. Methods: Paraffin sections from 6 cases of primary MCC and 6 cases of primary MM were investigated. For immunostaining, the APAAP method was used. Results: Neuron-specific enolase was positive in all cases of MCC, as well as in 2 cases of MM. Marked positivity for cytokeratins 18, 20 and chromogranin A was observed in the MCC group, whereas a complete absence of expression of these three markers was noted in the MM group. Immunostaining with HMB45 and NKI/C3 was positive in all cases of MM and negative in all cases of MCC. S-100 protein was positive in all but 1 case of MM. In contrast, only 1 case of MCC reacted with S-100 protein. Conclusion: Our results underline the role of immunohistochemistry in the diagnosis and differential diagnosis of MCC. In particular, the combination of neuron-specific enolase, cytokeratins 18, 20 and chromogranin A positivity for MCC and HMB45, NKI/C3 and S-100 protein positivity for MM is of great value in the distinction between these two cutaneous neoplasms.

Twenty-eight cases of juvenile-onset Adamantiades-Behçet disease in Germany

Background and Objective: Adamantiades-Behçet disease is a rare entity at a juvenile age. We aimed to enlighten epidemiological and clinical characteristics of juvenile-onset disease in Germany. Methods: Data from the German Registry were used to compare clinical and epidemiological features of patients with juvenile-onset (≤16 years) and adult-onset (>16 years) disease diagnosed according to the criteria of the International Study Group. Results: Twenty-eight (17%) of 168 patients of the German Registry exhibited the onset of the disease and 8 (5%) of them the complete symptom complex at a juvenile age. Juvenile-onset disease was characterized by an increase in familial cases (25 vs. 8% in patients with adult-onset; p = 0.047). The frequency of diagnostic signs was similar between the two study groups. In juvenile-onset disease, delayed development of the complete symptom complex (median value 35 months vs. 12 months after onset; p = 0.014) and lower prevalence of severe complications (9 vs. 29%; p = 0.042) were detected. Conclusions: The major clinical features of juvenile-onset and adult-onset disease in Germany are comparable, but in juvenile-onset disease, the course is delayed and patients experience less severe complications. In addition, there is a higher rate of familial occurrence of the disease in patients with juvenile-onset.