Bernd Bonnekoh

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipfs law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.

Imiquimod, a Topical Immune Response Modifier, in the Treatment of Cutaneous Metastases of Malignant Melanoma

Background: Imiquimod 5% cream (Aldara®), a novel topical immune response modifier, has been approved for the topical treatment of anogenital HPV-induced warts. In addition, several studies have demonstrated antitumoral activity in solar keratoses, superficial basal cell carcinomas and Bowen’s disease. Aim: Given the convincing therapeutic results of imiquimod when used for treating selected types of epithelial skin cancer, we became interested to study imiquimod as an adjuvant for treating cutaneous metastases of malignant melanoma. Methods: Three patients with multiple, i.e. more than 15, cutaneous in-transit metastases of malignant melanoma in unilateral localization on the leg were treated topically with imiquimod 5% cream. Results: Twice daily application under occlusive conditions for a period of 21–28 weeks resulted in >90% regression of cutaneous metastases in 2 patients. The third patient showed marked response only when topical imiquimod was intermittently supplemented by intralesional interleukin (IL)-2 for 2 weeks. Unwanted side effects were mild in all patients. Conclusion: Overall, imiquimod as a single agent or in combination with intralesional IL-2 may be a promising immunomodulatory compound for the adjuvant topical treatment of patients with multiple cutaneous metastases of malignant melanoma.

Garlic-related dermatoses: Case report and review of the literature

BACKGROUND Garlic is widely appreciated as a spice and as a vegetable as well as an over-the-counter phytotherapeutic. From a dermato-allergological standpoint, several garlic-related adverse reactions have to be distinguished. OBJECTIVE The corresponding literature is reviewed briefly, with regard to our present observation of a cook, who contracted garlic-induced contact dermatitis being analyzed for its complex pathomechanism. METHODS The patient showed a positive type-IV patch test reaction for diallyl disulfide, a low molecular weight garlic ingredient; and strong, non-irritant reactions after 20 min and 24 hrs in the scratch chamber test with fresh total garlic. RESULTS Thus, in this case of an occupational dermatosis, protein contact dermatitis had to be considered, as well as allergic type-IV contact dermatitis as a co-existing pathomechanism. CONCLUSIONS The spectrum of garlic-related adverse reactions comprises irritant contact dermatitis, with the rare variant of zosteriform dermatitis; induction of pemphigus, allergic asthma and rhinitis; contact urticaria; protein contact dermatitis; allergic contact dermatitis, including the hematogenic variant; as well as combinations thereof, as evidenced by our present case observation.

Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity

SummaryThe application of a simple, rapid, and inexpensive colorimetric growth assay was tested for human epidermal cells subcultured in uncoated plastic dishes. Cell layers were incubated with a crystal violet (CV) solution (0.2% with ethanol 2% in 0.5 M Tris-Cl buffer, pH 7.8) for 10 min at room temperature. After rinsing with 0.5 M Tris-Cl (pH 7.8) the cell layer was dried and decolorized with a sodium-dodecylsulfate solution (0.5% with ethanol 50% in 0.5 M Tris-Cl, pH 7.8) for 60 min at 37°C. The extinction of the supernatant was read at the absorption maximum of 586 nm. The protein content of attached cells as classical parameter for quantifying cell growth was strongly related to CV extinction with a correlation coefficient of r=0.98. Furthermore, the subcellular protein binding qualities of CV were analyzed. The water-soluble protein fraction of cultured epidermal cells was separated by sodium-dodecylsulfate polyacrylamide gel electrophoresis and stained with CV. We found a staining pattern which was qualitatively very similar to that of Coomassie blue, however less intense. Keratin electrophoresis revealed an affinity of CV to the 48, 50, and 56 kD cytokeratins. In conslusion, this CV assay is a reliable and simple method for the monitoring of epidermal cell growth in cultures.

Antiproliferative and cytotoxic profiles of antipsoriatic fumaric acid derivatives in keratinocyte cultures.

Oral administration with complex mixtures of fumaric acid derivatives is known to have antipsoriatic efficacy. The present studies aimed to clarify the mode of action and toxicity of the individual compounds. Hyperproliferative HaCaT keratinocytes in monolayer cultures were exposed to fumaric acid, dimethylfumarate, zinc monoethylfumarate, calcium monoethylfumarate and magnesium monoethylfumarate at concentrations between 0.4 microM and 960 microM for 48 h. Cell proliferation was studied by [3H]thymidine incorporation. In addition 14C-labelled amino acid uptake and total protein content were measured. Direct cytotoxicity was determined by the release of cytoplasmic lactate dehydrogenase (LDH) into the culture medium. The corresponding 50% inhibition concentrations (IC50) were calculated for DNA/protein synthesis: 2.3/2.5 microM (dimethylfumarate), 133/145 microM (zinc monoethylfumarate), 215/230 microM (calcium monoethylfumarate), 275/270 microM (magnesium monoethylfumarate), > 960/> 960 microM (fumaric acid). The total protein content was less sensitive. Antiproliferative activity was found for dimethylfumarate and to a lesser degree for calcium monoethylfumarate already at the subtoxic concentrations of 1.3 and 4 microM, respectively. In the case of magnesium monoethylfumarate, zinc monoethylfumarate and fumaric acid there was no such dissociation between their cytotoxic and antiproliferative potential. These data indicate that most of the antipsoriatic potential of fumaric therapies is due to the dimethylfumarate compound.

Epidermolysis bullosa acquisita with ultraviolet radiationsensitivity

A 37‐year‐old male patient developed a bullous eruption and erythematous plaques mainly in exposed areas following prolonged sun exposure. In addition, blisters were noted on oral and nasal mucous membranes. Histopathological examination of a lesional skin biopsy revealed a subepidermal blister. Linear deposition of IgG and C3 at the epidermal basement membrane zone was revealed by direct immunofluorescence microscopy of a perilesional skin biopsy. Indirect immunofluorescence on 1 mol/L salt‐split skin showed binding of autoantibodies to the dermal side of the split. Immunoblot analysis of dermal extracts demonstrated that the patient’s serum contained IgG antibodies against type VII collagen, whereas no reaction was seen with epidermal extracts or by enzyme‐linked immunosorbent assay using a recombinant form of bullous pemphigoid 180. Standardized ultraviolet (UV) radiation provocation induced blistering with both UVA (13·5 J/cm2) and UVB (0·04 J/cm2) within 24 h clinically and histologically. External and systemic UV‐protective medication and nine cycles of high dosage immunoglobulins given intravenously (1·2 g/kg body weight over 2–3 days every 4 weeks) resulted in the cessation of blister formation. To the best of our knowledge, this is the first report of a case of epidermolysis bullosa acquisita with sensitivity to UV.

Arthus reaction to lepirudin, a new recombinant hirudin, and delayed-type hypersensitivity to several heparins and heparinoids, with tolerance to its intravenous administration.

The pathogenesis of allergic reactions to heparin is poorly understood. Clinically, this phenomenon is relevant because of its increasing incidence and the resulting therapeutic challenges due to various cross‐reactions between unfractionated and low‐molecular weight heparins as well as between heparins and heparinoids. A 44‐year‐old female patient had developed a delayed‐type hypersensitivity to certoparin‐sodium. Diagnostic allergy testing revealed various cross‐reactions between different heparins as well as an intolerance to heparinoids. After subcutaneous challenge with the recombinant hirudin lepirudin (Refludan®) the patient developed a local Arthus reaction at the injection site. In general, recombinant hirudins do not cross‐react with high‐ or low‐molecular weight heparins and heparinoids because of a different molecular structure and are therefore an alternative in case of adverse reactions to heparins and heparinoids. Whereas a local Arthus reaction has already been described twice for low‐molecular weight heparins, this is to the best of our knowledge the first observation of a superficial leukocytoclastic vasculitis due to s.c. applied lepirudin. Intravenous administration of heparins and heparinoids in case of hypersensitivity to these drugs following topical application risks a generalized eczematous reaction in patients with delayed‐type allergy to both groups of substances. In our patient with delayed‐type hypersensitivity to heparins and heparinoids and superficial vasculitis due to lepirudin, the intravenous challenge with heparin and a heparinoid was justified as an ultima ratio measure and proved to be the useful therapeutical alternative.

Persistent granulomatous contact dermatitis due to palladium body‐piercing ornaments

There are few reports of granuloma induction by metals and their salts. Beryllium-induced granulomas are an occupational disease in mining and fluorescent tube production (1, 2). Metal-induced granulomatous reactions have been reported for aluminium, used as an adjuvant in vaccines and hyposensitization extracts, as well as in antiperspirants and in tattoos (3–7), zirconium (antiperspirants) (7, 8), mercury, chrome, cobalt (pigments in tattoos and orofacial granulomatosis) (8–11), nickel (sternotomy wires) (12) and gold (jewelry) (13).

Lactate dehydrogenase release as an indicator of dithranol-induced membrane injury in cultured human keratinocytes

SummaryHaCaT cells, a rapidly multiplying human keratinocyte line, were tested for their sensitivity to antipsoriatic dithranol with regard to classical proliferation parameters and for the drugs action on the plasma membrane integrity by the dose- and time-dependent release of cytosolic lactate dehydrogenase (LDH). In the case of 3H thymidine as well as 14C amino acid incorporation the 50% inhibition concentration (IC50) was 0.2 ΜM dithranol 24 h after initial exposure to the drug. For protein content of attached cells the IC50 proved to be>3.0 ΜM. Using 0.3, 1.0 and 3.0 ΜM dithranol, significant (p<0.05) dose dependent LDH release of 0.866±0.387, 1.842±1.127 and 2.938±1.635 mU per hour and cm2 confluent culture area was measured between the 5th and the 24th hour, compared to an acetone control of 0.504±0.299 mU/ h×cm2. Between the 2nd and the 4th hour as well as from the 25th to the 48th hour and the 49th to the 72nd hour the LDH release after dithranol treatment did not exceed the control value. In accordance with these findings dose-dependent morphological signs of cell injury were detected by phase contrast microscopy beyond the 4th hour. The data reveal that: HaCaT cells are a very sensitive target for the antiproliferative action of dithranol; the drug causes considerable plasma membrane damage even at concentrations as low as 0.3 ΜM; and this membrane damage becomes evident after a latency of at least 4 h and for a limited period of up to 24 h.

Interferon-γ-Dependent in vitro Model for the Putative Keratin 17 Autoimmune Loop in Psoriasis: Exploration of Pharmaco- and Gene-Therapeutic Effects

In 1999, A.S. Gudmundsdottir et al. have envisaged an epitope on keratin 17 (K17) as a putative psoriasis major autoantigen recognized by T cells. In a HaCaT keratinocyte model, we now demonstrate that IFN-γ and to a less extent also TNF-α and TGF-α are able to induce K17 protein expression, in contrast to IL-1α, IL-1β, IL-6, IL-8 and IL-18. This supports our hypothesis of an existing proinflammatory cytokine/K17 autoimmune loop as a presumptive positive feedback mechanism driving psoriasis etiopathogenesis. K17 overexpression was now found to also coincide with suppression of keratinocyte proliferation, e.g. induced by NF-kappa B inhibitors (Bay 11-7082 and Bay 11-7085), and thereby correlated hyperapoptosis to be encountered in psoriatic epidermis. Acitretin as an established antipsoriatic drug and the tyrosine kinase inhibitor imatinib decreased, whereas hydrocortisone as well as dexamethasone increased the IFN-γ-induced K17 overexpression. The latter might be another mechanism explaining the well-known rebound phenomena after abrupt withdrawal of corticosteroids in psoriasis treatment. Finally, we defined a K17-directed and effective antisense oligodesoxynucleotide which may hold promise for future gene-therapeutic approaches in psoriasis.