Linda Milkova

Regenerative medicine in dermatology: biomaterials, tissue engineering, stem cells, gene transfer and beyond.

Please cite this paper as: Regenerative medicine in dermatology: biomaterials, tissue engineering, stem cells, gene transfer and beyond. Experimental Dermatology 2010; 19: 697–706.

ADAM10 is the constitutive functional sheddase of CD44 in human melanoma cells.

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

The NF-κB signalling pathway is involved in the LPS/IL-2-induced upregulation of FoxP3 expression in human CD4+CD25high regulatory T cells

Abstract:  Regulatory T cells (Treg) have been found to be central for host defense regulation against microbial antigens, the prevention of allergic and autoimmune diseases and the suppression of effective tumor immune responses. However, the influence of the microenvironment and the mechanisms leading to their activation in the periphery still remain unclear. In vitro infection models revealed that survival and suppressive function of Treg is improved when they are confronted with lipopolysaccharide (LPS). Because LPS initiates signalling via the receptor Toll‐like receptor 4 (TLR4) and the consequent activation of the transcription factor nuclear factor‐kappaB (NF‐κB), we investigated TLR4 expression and NF‐κB regulation in human Treg. We demonstrated that LPS in combination with IL‐2 induces human CD25+FoxP3+ T cells in vitro. FoxP3 expression of purified natural Treg increased and suppressive capacity was markedly improved compared with unstimulated Treg upon stimulation with LPS/IL‐2. Furthermore, blockade of the NF‐κB pathway by a selective inhibitor of IκB kinase (IKK)β abrogated the upregulation of FoxP3 expression. Taken together, our results suggest an important role of the NF‐κB signalling pathway for the induction and modulation of suppressive function of natural Treg, if they are confronted with TLR4‐stimulating agents such as Gram‐negative bacteria.

Improved method of differentiation, selection and amplification of human melanocytes from the hair follicle cell pool.

Hair root harbours a complex cell pool with an immense developmental potential. Several lineages, including skin, can be differentiated from the multipotent to pluripotent cells of outer root sheath (ORS) of hair follicle. Outer root sheath presents the most opulent non‐invasively gained adult stem cell source known. For the purposes of cultivating melanocytes designated for graft‐based treatments of depigmentation disorders, we have established an ex vivo/in vitro cultivation method by introducing several methodological improvements to the ORS explant method of Dieckmann. As a result, we gained a higher, purer yield of differentiated melanocytes in half the time (at least 106 of 95% pure cells in 4 weeks). This reliable cultivation procedure begins with the epilation of 60 hairs and yields high numbers of ORS melanocytes that could be used for grafting applications. The procedure not only utilises the developmental potential of hair root cell pool and favors differentiation into melanocytes, but also contributes to the general trend of minimal‐to‐non‐invasive strategies for regenerative medicine.

Human melanocytes can be isolated, propagated and expanded from plucked anagen hair follicles

Please cite this paper as: Human melanocytes can be isolated, propagated and expanded from plucked anagen hair follicles. Experimental Dermatology 2010; 19: 543–545.

Lupus erythematosus. Part II: Clinical picture, diagnosis and treatment

Lupus erythematosus (LE) is an important dermatologic autoimmune disease. In many respects, it may be regarded as a model autoimmune disease due to its spectrum of autoimmune antibodies and involvement of different organ systems, as well as response to immunosuppressive agents which target B cells and T cells and their cytokines. A recently published article in this Journal summarized the most important knowledge about epidemiology, genetics, and immunology of LE. Here, the different clinical manifestations, diagnostic procedures and current therapeutic approaches will be described. Special emphasis is placed on different cutaneous manifestations of LE. In regard to treatment, the classic treatment approaches such as corticosteroids, methotrexate, chloroquine and hydroxychloroquine will be described. Lastly, new therapeutic approaches with specific monoclonal antibodies which are currently used in systemic LE, such as belimumab (Benlysta®), will be addressed. The most recent developments in this area could have implications even for purely cutaneous forms of LE.

Facial Pyoderma Gangrenosum in Senescence

Clinically, pyoderma gangrenosum (PG) is characterized by a rapidly progressive, painful cutaneous ulcer with an irregular, violaceous and undermined border. PG occurs most frequently on the lower extremities and the trunk of middle-aged individuals. The face is only very rarely affected. We present an 89- and a 90-year-old patient, who developed a facial ulcer consistent with PG.

Asymptomatic dapsone-induced agranulocytosis in a patient with chronic spontaneous urticaria.

Dapsone-induced agranulocytosis is a rare, serious, and unpredictable intolerance to dapsone; it typically manifests after 4–12 weeks of treatment and is potentially highly fatal [1]. The clinical appearance consists of infections with high-fever, oral ulcers, pneumonia, and sepsis [1–4]. We describe a 74-year-old man who had severe urticaria for several months. The clinical presentation consisted of multiple annular wheals, which covered the entire surface of the skin. The patient also had palmar erythema. Laboratory tests were obtained for the following: total IgE (258 kU/l), tryptase (4.94 μg/l), and antinuclear antibodies (1 : 80). The remaining routine laboratory tests were normal. The results of a Helicobacter pylori breath test were positive. The bacteria were successfully eradicated, and dental restoration was performed. The patient was placed on a low-pseudoallergen diet and aspirin was discontinued. Yet, neither these measures, nor the eradication of infection, led to any significant improvement in his clinical appearance. Chronic spontaneous urticaria (CSU) was diagnosed and treatment initiated with oral antihistamines, which were administered up to four times daily, along with corticosteroidand polidocanol-based topical therapy, leading only to a slight improvement. As serum glucose-6-phosphate dehydrogenase (G6PD) and methemoglobin (MetHb) levels were Clinical Letter normal, a systemic therapy was initiated, starting with 50 mg diaminodiphenylsulfone (dapsone), which was given daily; over the course of treatment, the dosage was increased to 100 mg every other day. Seven weeks after initiating treatment, there was no significant improvement in clinical appearance. Routine laboratory tests revealed anemia (hemoglobin, 10.8 g/dl; erythrocyte count, 3.42 × 1012/l) and progressive leukocytopenia (white blood cell count below 1 000/μl and neutrophil count below detectable levels) (Figure 1). Flow cytometry of the lymphocyte population did not show any remarkable features related to the immunophenotype; the light-chain distribution of B cells was normal. MetHb was 1.1 % and remained within normal ranges during treatment. The patient was not taking any other substances that potentially form MetHb. The patient reported smoking 3–4 cigarettes per day. Due to suspected drug-associated agranulocytosis, dapsone was immediately discontinued and the patient hospitalized. He was given twice daily doses of 48 million units (total of 528 million units) of granulocyte colony stimulating factor (G-CSF). His neutrophil count gradually normalized, ultimately with excessive leukocyte release (leukocytes 43.6 × 109/l, neutrophilic granulocytes 36.5 × 109/l, Hb 10.7 g/dl, erythrocytes 3.4 × 1012/l) (Figure 1). With the exception of transient fatigue, the patient remained clinically asymptomatic, without any signs of infection. Subsequently, there was transient complete remission of CSU; over the course, episodes reappeared but were less frequent, with far fewer wheals, and treatment with desloratadine, administered 2 × daily, led to nearly complete relief of symptoms. The sulfone and aniline derivative diaminodiphenylsulfone (dapsone) has been used for 50 years as an antibiotic for treatment of leprosy; it is currently used only as co-medication, since more effective substances are now available [5, 6]. Due to its efficacy against protozoa, dapsone is also used as second-line

Agminated blue naevi in a patient with EMO syndrome.

© 2013 The Authors. doi: 10.2340/00015555-1366 Journal Compilation

Asymptomatische Dapson‐induzierte Agranulozytose bei einem Patienten mit chronisch spontaner Urtikaria

die Dapson-induzierte Agranulozytose ist eine seltene schwere und unvorhersehbare Unverträglichkeitsreaktion mit potenziell hoher Letalität, die sich typischerweise nach einer Therapiedauer von 4–12 Wochen manifestiert [1]. Klinisch bestehen üblicherweise hochfieberhafte Infektionen mit enoralen Ulzerationen, Pneumonien oder septische Zustände [1–4]. Wir berichten über einen 74-jährigen Patienten mit seit mehreren Monaten bestehender ausgeprägter Urtikaria. Klinisch bestanden am gesamten Integument multiple anuläre Quaddeln sowie palmare Erytheme. Im Labor zeigte sich: Gesamt-IgE 258 kU/l, Tryptase 4,94 μg/l, Antinukleäre Antikörper 1 : 80, die übrige Routinelabordiagnostik war unauffällig. Bei positivem Helicobacterpylori -Atemtest wurde eine erfolgreiche Eradikation vorgenommen, außerdem erfolgte eine Zahnsanierung. Eine pseudoallergenarme Diät und die Beendigung einer Komedikation mit Acetylsalicylsäure führten ebenso wie die Infektsanierung nicht zu einer entscheidenden Besserung des klinischen Bildes. Unter der Diagnose einer chronisch spontanen Urtikaria (CSU) erfolgte eine Therapie mit oralen Antihistaminika bis zu viermal täglich in Kombination mit Glukokortikoidund Polidocanol-haltigen Externa, was nur zu einer leichten Besserung führte. Bei normalen Werten für die Serum-Glucose-6-Phosphat-Dehydrogenase (G-6-P-DH) und das Methämoglobin (Met-Hb) wurde im Folgenden eine Systemtherapie zunächst mit 50 mg Diaminodiphenylsulfon (Dapson) täglich eingeleitet und im Verlauf umtägig auf 100 mg gesteigert. Sieben Wochen nach Therapiebeginn wurde ohne wesentliche Besserung des klinischen Befundes bei der routinemäßigen Laborkontrolle eine Anämie (Hämoglobin 10,8 g/dl, Erythrozyten 3,42 × 1012/l) und progrediente Leukozytopenie diagnostiziert (Leukozytenzahlen unter 1 000/μl und nicht mehr nachweisbare neutrophile Granulozyten) (Abbildung 1). Die durchflusszytometrische Analyse der Lymphozytenpopulationen zeigte keine Besonderheiten des Immunphänotyps einschließlich einer regelrechten Leichtkettenverteilung der B-Zellen. Das Met-Hb betrug 1,1 % und war im Verlauf stets im Normbereich, weitere potenzielle Met-Hb-Bildner wurden nicht eingenommen. Der Patient rauchte ca. 3–4 Zigaretten täglich. Bei Verdacht auf das Vorliegen einer medikamentenassoziierten Agranulozytose wurde Dapson umgehend abgesetzt und der Patient wurde stationär aufgenommen. Unter Therapie mit zweimal täglich 48 Mio. Einheiten (insgesamt 528 Mio. Einheiten) Granulozyten-Kolonie-stimulierendem Faktor (G-CSF) normalisierten sich allmählich die Neutrophilenzahlen mit schließlich überschießender Leukozytenfreisetzung (Leukozyten 43,6 × 109/l, Neutrophile Granulozyten 36,5 × 109/l, Hb 10,7 g/dl, Erythrozyten 3,4 × 1012/l) (Abbildung 1). Bis auf eine transiente vermehrte Müdigkeit bestand durchgehend klinische Asymptomatik ohne jegliche Infektzeichen. Nachfolgend trat zunächst Clinical Letter