Ola Rollman

Cutaneous gene transfer for skin and systemic diseases.

This article is partially based on the findings presented at a symposium on Cutaneous Gene Therapy, held in Uppsala, September 2001, and abstracted in Acta Derm Venereol 81: 227–239.

Treatment with LL-37 is safe and effective in enhancing healing of hard-to-heal venous leg ulcers : a randomized, placebo-controlled clinical trial

Venous leg ulcers (VLUs) are one of the most prevalent types of chronic wounds. The aim of this study was to determine the safety and dose–response efficacy of the human synthetic peptide LL‐37 in the treatment of hard‐to‐heal VLUs. This first‐in‐man trial included 34 participants with VLUs and comprised a 3‐week, open‐label, run‐in period on placebo, followed by a 4‐week randomized double‐blind treatment phase with twice weekly applications of LL‐37 (0.5, 1.6, or 3.2 mg/mL) or placebo, and a 4‐week follow‐up. The healing rate constants for 0.5 and 1.6 mg/mL of LL‐37 were approximately six‐ and threefold higher than for placebo (p = 0.003 for 0.5 mg/mL and p = 0.088 for 1.6 mg/mL). Square‐root transformed wound area data showed improved healing for the 0.5 and 1.6 mg/mL dose groups compared with pretreatment values (p < 0.001 and p = 0.011, respectively). Consistently, treatment with the two lower doses markedly decreased the mean ulcer area (68% for 0.5 mg/mL and 50% for 1.6 mg/mL groups). No difference in healing was observed between the groups receiving 3.2 mg/mL of LL‐37 and placebo. There were no safety concerns regarding local or systemic adverse events. In conclusion, topical treatment with LL‐37 for chronic leg ulcers was safe and well tolerated with the marked effect on healing predictors at the two lower doses warranting further investigations.

Treatment of Onychomycosis by Partial Nail Avulsion and Topical Miconazole

13 patients with distal subungual onychomycosis in a total of 48 dermatophyte-infected nails were treated with chemomechanical, partial nail avulsion followed by topical miconazole for 8 weeks. On examination, 6 months after cessation of therapy, 42% of the nails were cured by clinical and mycological criteria. The therapeutic response was related to the pretreatment extension of subungual hyperkeratosis. Periungual skin irritation was common during the initial avulsion period. Miconazole solution was well tolerated and this treatment modality proved to be a valuable alternative to other remedies for the treatment of onychomycosis limited to a few number of nails.

Fluorescence imaging of reepithelialization from skin explant cultures on acellular dermis

Reconstituted skin models are valuable tools in studies of cutaneous biology although they are not generally devised to visualize and quantify the time course of reepithelialization. We describe a skin explant culture technique coupled with vital microscopy allowing sequential imaging of epithelial outgrowth while maintaining the tissue in culture. Radial expansion of neo‐epidermis was initiated from a 2‐mm skin punch biopsy explanted onto acellular human dermis and maintained at the air–liquid interface in serum‐containing keratinocyte medium. Microscopic viewings and surface area measurements of newly formed epidermis were performed repeatedly using fluorescein‐based cell staining and subsequent image analysis. The labeling and visualization procedures did not interfere with neo‐epidermal growth or tissue architecture. In order to appraise the versatility of the model, we studied the effect of epidermal growth factor supplementation on the course of skin resurfacing as related to tissue morphology and proliferative activity over a 10‐day cultivation period. Exogenous epidermal growth factor at 10 ng/ml increased the radial outgrowth rate (mean, + 13.3 percent), papillomatosis index (+ 19.2 percent), epithelial thickness (+ 49.8 percent), and fraction of Ki‐67 positive basal cells (+ 18.4 percent) in neo‐epidermis produced from a biopsy of normal human skin. The contribution of this miniaturized and convenient format for concurrent studies of dynamic and qualitative features of neo‐epidermis should be a useful complement to existing assays of epidermalization.

The vitamin A metabolism and expression of retinoid-binding proteins differ in HaCaT cells and normal human keratinocytes

Abstract HaCaT keratinocytes differ from normal human epidermal keratinocytes (HEK) by constitutive expression of differentiation markers which are normally suppressed by vitamin A. In search of an explanation for this discrepancy we compared the vitamin A content, the expression of retinoid-binding proteins, and the vitamin A metabolism in the two cell types. The concentrations of retinol and 3,4-didehydroretinol in cultured HaCaT cells were less than one-fifth those in HEK, and the content of fatty acyl esters was even lower. Similarly, the concentrations of cellular retinol-binding protein and cellular retinoic acid-binding protein (CRBPI and CRABPII, respectively) were 10–30 times lower in HaCaT cells than in HEK corresponding to a reduced mRNA expression of these proteins. Unexpectedly, HaCaT cells expressed RARβ in addition to RARα, RAR>γ and RXRα, which are nuclear receptors normally found in HEK. Radioactive retinol added to the culture medium appeared only transiently in HaCaT cells, and pulse labeling confirmed a defective cellular retention of retinyl esters. After 24 h of incubation with [ 3 H]retinol, cell-associated radioactivity corresponding to retinol, 3,4-didehydroretinol, all- trans -retinoic acid and 3,4-didehydroretinoic acid was found in both HaCaT cells and HEK. [ 3 H]Retinoic acid showed a more rapid metabolism to 4-hydroxy/4-keto-retinoic acid in HaCaT cells than in HEK, which could be explained by a higher expression of cytochrome p450RAI in the former cells. In conclusion, the abnormal uptake of vitamin A and low levels of retinoid binding proteins in HaCaT cells, linked with an aberrant metabolism of retinol, may help to explain why these cells differentiate also in the presence of retinoids.

Clinical Pharmacology of 3 Generations of Retinoids

The bioavailability, plasma transport and tissue distribution of various retinoids are largely determined by their physicochemical properties; some are extremely lipidsoluble whereas others are relatively hydrophilic. Isotretinoin, a 1st generation retinoid, lacks the problematic affinity for fat. Etretinate, a 2nd generation aromatic retinoid, has been shown to accumulate in both fat tissues and in the adrenals. Etretin, the main free-acid metabolite of etretinate, is less lipophilic and is presently being tested as an alternative drug. Arotinoid ethyl ester, a 3rd generation aromatic retinoid which has as yet only undergone limited trials, is extremely potent making pharmacological evaluation difficult. The search for more potent retinoids has not so far resulted in a complete resolution of the efficacy and toxicity of the drugs.

Topical retinoic acid alters the expression of cellular retinoic acid-binding protein-I and cellular retinoic acid-binding protein-II in non-lesional but not lesional psoriatic skin.

Abstract: Therapeutic retinoids have profound effects on psoriatic skin pathology but their interactions with various retinoid‐binding proteins in lesional vs non‐lesional skin have not been investigated. Using quantitative real‐time PCR the mRNA expression of cellular retinol‐binding protein I (CRBPI) and retinoic acid‐binding protein I/II (CRABPI/CRABPII) was studied in psoriatic and healthy control (=normal) skin after 4 days of occlusive RA/vehicle treatment (n=6). Untreated psoriatic lesions showed a markedly elevated CRABPII/CRABPI ratio, while the CRBPI level was reduced in lesional and non‐lesional skin as compared to normal skin. In RA‐treated normal and non‐lesional skin, the mRNA expression of CRBPI was unaltered while that of CRABPI and CRABPII was reduced by ≈80% and increased ≈5‐fold, respectively, as compared to vehicle‐treated skin. In contrast, lesional skin exposed to RA showed an almost 90% increase in CRBPI transcripts but unaltered expression of CRABPI and CRABPII, yet, the mRNA expression of several inflammatory mediators, e.g. inducible nitric oxide synthase, interferon‐γ and interleukin‐1β, was clearly reduced. Immunohistochemistry localized CRABPII to suprabasal keratinocytes in normal skin and revealed markedly elevated levels in lesional skin. RA treatment induced CRABPII protein expression in normal and non‐lesional skin, to similar levels as in untreated lesions. The results indicate that the effects of RA differ in normal/non‐lesional psoriatic skin and lesional skin. Whether the high expression of CRABPII in psoriatic skin lesions is due to increased amounts of endogenous retinoids in lesional skin or reflects an abnormal regulation of the CRABPII gene in psoriasis remains to be studied.

Re-epithelialization from human skin explant cultures is promoted by ligand-activated HER3 receptor

BACKGROUND Ligand-stimulated epidermal growth factor receptor (EGFR/HER1) plays a fundamental role in skin biology as potent transducer of mitotic and anti-apoptotic stimuli in keratinocytes. In human epidermis, at least two additional EGFR family members--HER2 and HER3--are expressed but their biological functions in normal and diseased human skin remain obscure. OBJECTIVE Here, we studied the expression and biological impact of HER3 in regenerating human epidermis formed from skin explants adhered to acellular dermis. METHODS Neoepidermal HER3 expression was examined by quantitative real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot analysis. The dynamic effect of HER3 receptor stimulation by recombinant heregulin (HRG)-beta1 was assessed by fluorescence imaging of re-epithelialization. RESULTS In the neoepidermis, HER3 mRNA and protein were detected with activated receptors being immunolocalized at basal and low suprabasal levels. Exogenous HRG-beta1 at 10-20 ng/ml increased the outgrowth rate corresponding to approximately 30% the response of exogenous EGF. The growth-promoting effect of HRG-beta1 was associated with enhanced HER3 phosphorylation, keratinocyte proliferation and thickening of viable neoepidermis whereas blockade of ligand-binding to HER3 delayed the outgrowth process and inhibited both constitutive and ligand-induced HER3 phosphorylation. HER2 antagonism using an anti-dimerization antibody, pertuzumab, impeded the re-epithelialization rate. In addition, a selective HER2 kinase inhibitor, CP654577, downregulated phospho-HER3 expression suggesting that transactivation of kinase-deficient HER3 was accomplished through dimerization with HER2. CONCLUSION The study emphasizes the central role of EGFR in epidermal renewal and demonstrates that HRG-activated HER3 contributes to the outgrowth process of epidermis in vitro.

Differential uptake of chloroquine by human keratinocytes and melanocytes in culture

The antimalarial drug chloroquine has a high affinity for melanin and accumulates in melanin-rich compartments such as those of the eye. Chloroquine is also deposited in cutaneous tissue, but whether the drug distribution is restricted to melanin-producing cells of the skin is not known. In the present study, the uptake of chloroquine by normal human epidermal keratinocytes was compared with that by melanocytes. Selectively cultivated cells were incubated at drug concentrations ranging between 0 and 10 000 ng/ml for periods of up to 48 h. Chloroquine was quantified in cells and medium using high performance liquid chromatography and fluorometric detection. In both types of cells there was a rapid uptake of chloroquine within the first 2 h, followed by a slower uptake for 2–6 h until a steady-state condition was reached. Dose dependency was linear, with no sign of saturation, and approximately ten times higher drug concentrations were attained in melanocytes as compared with keratinocytes. No formation of desethylchloroquine, the major systemic metabolite, was detected in either cell type. The observed affinity of chloroquine for normal epidermal melanocytes in vitro suggests that the density and melanogenic activity of skin pigment cells may influence the cutaneous drug disposition of chloroquine.

Granzyme H Is a Novel Protease Expressed by Human Mast Cells

Background: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express granzyme H, the granzyme that may represent the functional counterpart to murine granzyme D. Methods: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. Results: Cord blood-derived mast cells and LAD2 cells were shown to express granzyme H and B mRNA, while granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of granzyme H. In contrast, granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that granzyme H is expressed by human skin mast cells in situ. Conclusions: The present findings implicate granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that granzymes B and H are reciprocally regulated.