Peter Knuschke

Untersuchungen zur Verfestigung der Hornhaut am Kaninchen

Hintergrund: Eine mechanische Stabilisierung der Hornhaut beim Keratokonus könnte das Fortschreiten dieser Erkrankung verzögern. Die an der Hornhaut von enukleierten Schweineaugen optimierten Vernetzungsmethoden zur Erhöhung der Festigkeit wurden im Langzeitversuch am Kaninchenmodell auf Beständigkeit und biologische Verträglichkeit getestet. Methode: Von 28 Kaninchen wurde das rechte Auge behandelt, das linke Auge diente jeweils als Kontrolle. Nach der mechanischen Entfernung des Epithels wurden 19 Augen mit Riboflavin-Dextranlösung getropft und anschließend mit UV-Strahlung (365 nm, Bestrahlungsstärke 2 mW/cm2) für 45 min exponiert. Auf 9 Augen wirkte ein Gemisch aus Methocel und 0,075% Glutaraldehyd für 20 min ein. Die Reaktion der Hornhaut wurde mit der Spaltlampenphotographie dokumentiert. Nach 1 Monat wurden 20 Tiere und nach 3 Monaten 8 Tiere getötet, die Augen enukleiert und von Streifen (Breite 5 mm, Länge 8 mm) der Hornhäute der Spannungs-Dehnungszusammenhang gemessen. Ergebnisse: Vier Wochen nach der Vernetzung konnte eine signifikant höhere Festigkeit in den mit Riboflavin+UV behandelten Hornhäuten nachgewiesen werden. Die vernetzten und unvernetzten Hornhäute unterschieden sich bei einer Dehnung von 6% in der Spannung um den Faktor 1,61±0,75 (p=0,041). Nach 12 Wochen lag diese Festigkeitszunahme noch bei 1,3±0,48 (p=0,07). Es kam zu keiner sichtbaren Entzündungsreaktion oder Beeinträchtigung der Transparenz. Die mit Glutaraldehyd behandelten Hornhäute wiesen zwar auch eine Zunahme der Festigkeit auf (Faktor 1,3±0,66), aber der Effekt war statistisch nicht signifikant (p=0,319), sodass ein Langzeittest dieser Methode nicht gerechtfertigt war. Schlussfolgerungen: Die Kombination von Riboflavin+UV-Strahlung ist geeignet zur Erzeugung von mindestens temporären Vernetzungen in der Hornhaut bei Kaninchen ohne klinische Zeichen für Nebenwirkungen wie Trübung oder Entzündung.Background: The mechanical stabilization of the cornea in keratoconus may delay progression of this disease. The cross-linking techniques optimized in corneas of enucleated porcine eyes were investigated under in vivo conditions in rabbits to estimate the biocompatibility and duration of the stiffening effect. Methods: Twenty-eight rabbits were treated monocularly, the fellow eye serving as control. The epithelium was mechanically removed and 19 eyes were treated with riboflavin plus ultraviolet irradiation (365 nm, 2 mW/cm2) for 45 min and 9 eyes with 0.075% glutaraldehyde for 20 min. After treatment, the eyelids were sutured for 3 days. The healing process was controlled by slit-lamp examination and photographically documented. After 1 month, 20 animals and after 3 months 8 animals were sacrificed, the eyes enucleated, and the stress-strain relation of the corneas measured and compared to the fellow eye. Results: The epithelium was closed after 4–5 days. The transparency of the corneas remained clear during follow-up, and there were no signs of inflammatory reaction. Stress for a strain of 6% was higher in the treated corneas by a factor of 1.3±0.66 (P=0.319) in the glutaraldehyde group and by a factor of 1.6±0.75 (P=0.0408) in the riboflavin group at 1 month, and by 1.3±0.48 (P=0.07) at 3 months after treatment. Conclusions: The cross-linking technique using riboflavin plus UV irradiation is suitable for at least temporarily stiffening the cornea in vivo and seems to be a promising method for conservative treatment of keratectasia.

UVB-induced Conversion of 7-Dehydrocholesterol to 1α,25-Dihydroxyvitamin D3 (Calcitriol) in the Human Keratinocyte Line HaCaT¶

Abstract We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D3 to the hormone calcitriol. The present study was initiated to test whether ultraviolet B (UVB)-induced photolysis of provitamin D3 (7-dehydrocholesterol, [7-DHC]) which results in the formation of vitamin D3 also leads to the generation of calcitriol in keratinocytes. Submerged monolayers of HaCaT keratinocytes were preincubated with 7-DHC (25 μM) at 37°C and irradiated with monochromatic UVB at different wavelengths (effective UV-doses: 7.5–60 mJ/cm2), or a narrow-band fluorescent lamp Philips TL-01 (UVB-doses: 125–1500 mJ/cm2). Irradiation with both sources of UVB resulted in the generation of different amounts of previtamin D3 in our in vitro model followed by time-dependent isomerization to vitamin D3 and consecutive formation of calcitriol in the picomolar range. Unirradiated cultures or cultures exposed to wavelengths >315 nm generated no or only trace amounts of calcitriol. The conversion of vitamin D3 generated after UVB irradiation to calcitriol is inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases in this chemical reaction. The generation of calcitriol was wavelength- and UVB dose dependent and reached approximately 18-fold higher levels after irradiation at 297 nm than at 310 nm (effective UVB dose: 30 mJ/cm2). Hence, keratinocytes may be a potential source of biologically active calcitriol within epidermis, when irradiated with therapeutical doses of UVB.

Evaluation of the atrophogenic potential of hydrocortisone 1% cream and pimecrolimus 1% cream in uninvolved forehead skin of patients with atopic dermatitis using optical coherence tomography

Topical corticosteroids are widely used to treat atopic dermatitis (AD), but their anti‐inflammatory mode of action can be accompanied by several unwanted side effects including skin atrophy and telangiectasia. In this 8‐week, investigator‐blinded, intraindividual right‐left comparison study with patients with mild‐to‐moderate AD, hydrocortisone 1% cream (HCT) was applied twice daily for 4 weeks on one side of forehead skin without clinical signs of AD and pimecrolimus 1% cream (PIM) on the other. Epidermal and dermal thickness were assessed by optical coherence tomography (OCT) and high‐frequency ultrasound, respectively. Skin atrophy and telangiectasia were assessed by contact dermatoscopic photography (Dermaphot®). Treatment with HCT leads to a significant decrease in epidermal thickness after only 2 weeks of treatment, while the decrease in PIM‐treated sites was less pronounced and was not statistically significant. By 4 weeks after the end of treatment, epidermal thickness returned to baseline values. No dermal thinning or development of telangiectasia could be observed by means of ultrasound or Dermaphot®, respectively. In summary, this study indicates that a 2‐week single course of topical treatment with a mildly potent steroid can cause transient epidermal thinning, an effect not seen in the PIM group. The slight decrease with PIM – although not significant – could be due to normalization of the increased skin thickness caused by a subclinical inflammation in AD. This study suggests that PIM may be safer for treatment of AD in sensitive skin areas like the face, especially when repeated application is required.

The UVB-induced synthesis of vitamin D3 and 1α,25-dihydroxyvitamin D3 (calcitriol) in organotypic cultures of keratinocytes: Effectiveness of the narrowband Philips TL-01 lamp (311 nm)

Both calcitriol and UVB radiation exert potent antipsoriatic effects. We hypothesize that the therapeutical effect of UVB radiation may be attributed at least in part to UVB-triggered cutaneous synthesis of calcitriol. The optimum wavelength for initiation of the vitamin D(3) pathway was found to be in the range of 300+/-5 nm in vitro and in vivo. The narrowband Philips TL-01 lamp which is commonly used as UVB source for phototherapy of psoriasis has maximum spectral irradiance at around 311 nm which is presumed to be, however, of lesser importance in photochemical activation of the vitamin D(3) pathway. The aim of this study was to compare the vitamin D(3) and calcitriol-inducing potential of UVB from the TL-01 lamp with that of monochromatic UVB at 300+/-2.5 nm and 310+/-2.5 nm in organotypic cultures of keratinocytes supplemented with 25 microM 7-DHC. We found that maximum calcitriol-generating capacity of the TL-01 lamp at 500 mJ/cm(2) and 16 h after irradiation still amounts up to 44% of that found after monochromatic irradiation at 300+/-2.5 nm and 30 mJ/cm(2). Thus, the antipsoriatic effect of UVB emitted from the TL-01 lamp may, at least in part, based on the antiproliferative and prodifferentiative action of newly synthesized calcitriol on epidermal keratinocytes.

Laboratory facilities and recommendations for the characterization of biological ultraviolet dosimeters.

A laboratory facility for characterizing biological dosimeters for the measurement of UV radiation has been built and tested. The facility is based on a solar simulator, stabilized by photofeedback, and monitored by a spectroradiometer, with a versatile filter arrangement. This enables the following characteristics of the dosimeters to be ascertained: spectral response, linearity, and reciprocity; angular acceptance and response; calibration in simulated sunlight. The system has been tested on a variety of dosimeters and has the potential to be used with other radiometers, subject currently to the size of their active surface.

Fluorescence spectroscopy of human skin

The advantages of optical spectroscopy in skin are discussed. The basis for fluorescence spectroscopy is briefly presented. The potential for in vitro and in vivo fluorescence spectroscopy of biotissues is significant, but not yet well developed. With the help of fluorescence spectroscopy and layer-by-layer skin surface strippings technique the important knowledge for autofluorescence spectra from different skin layers have been obtained. Resolution of autofluorescence spectra into discrete excitation-emission bands is presented, related to various skin fluorophores.

Knowledge of outdoor workers on the effects of natural UV radiation and methods of protection against exposure

The most important but influenceable risk factor in the development of skin cancer is the unprotected exposure to solar ultraviolet (UV) radiation. In order to assure adequate and effective protection against UV exposure, a level of knowledge about solar radiation and its effects is required. The objective of this study was to assess the knowledge of workers in outdoor professions on the effects of natural UV radiation and methods of protection against exposure. Forty outdoor workers were given a standardized questionnaire designed to ascertain their level of knowledge. The majority of participants knew exposure to solar radiation can be detrimental depending on exposure time. Eighty‐three percentage recognized that people working regularly in an outdoor environment may be at risk due to high exposure. Long‐sleeved clothing plus headgear and sunscreen containing sun‐protecting substances were deemed adequate methods of protection by 83% and 85% respectively. Seventy percentage of the outdoor workers were familiar with the definition of the sun protection factor (SPF), yet only 25% correctly identified the amount of sunscreen needed to achieve the SPF as indicated on the product. A mere 8% of participants knew that symptoms of a sunburn first became apparent 3 h after sun exposure and only 18% were able to accurately gauge the amount of time they could spend in the sun before developing one. Although 30% had heard of the ultraviolet index (UVI), only 13% understood that protecting your skin using additional measures is recommended as of UVI 3. Overall, 30% of the outdoor workers thought themselves sufficiently protected against the harmful effects of the sun. While the participants of this study had a basic fundamental understanding of the effects of solar radiation and methods of protection against exposure, there remains an urgent need for further clarification across all demographic groups.

A Novel Pathway for Hormonally Active Calcitriol

Calcitriol [1α,25(OH)2D3], the hormonally active form of vitamin D3 (D3) is produced in both renal and extrarenal tissues. Our findings demonstrate that physiological doses of UVB radiation at 300 nm induce the conversion of 7-dehydrocholesterol (7-DHC) via preD3 and D3 into calcitriol in the pmol range in epidermal keratinocytes. The hydroxylation of photosynthesized D3 to calcitriol is strongly suppressed by ketoconazole, a known inhibitor of cytochrome P450 mixed function oxidases. The UVB-induced formation of calcitriol in human skin is demonstrable in vivo by the microdialysis technique. These results suggest that human skin is an autonomous source of hormonally active calcitriol.

Common-path Fourier domain optical coherence tomography of irradiated human skin and ventilated isolated rabbit lungs

A compact common path Fourier domain optical coherence tomography (FD-OCT) system based on a broadband superluminescence diode is used for biomedical imaging. The epidermal thickening of human skin after exposure to ultraviolet radiation is measured to proof the feasibility of FD-OCT for future substitution of invasive biopsies in a long term study on natural UV skin protection. The FD-OCT system is also used for imaging lung parenchyma. FD-OCT images of a formalin fixated lung show the same alveolar structure as scanning electron microscopy images. In the ventilated and blood-free perfused isolated rabbit lung FD-OCT is used for real-time cross-sectional image capture of alveolar mechanics throughout tidal ventilation. The alveolar mechanics changing from alternating recruitment-derecruitment at zero positive end-expiratory pressure (PEEP) to persistent recruitment after applying a PEEP of 5 cm H2O is observed in the OCT images.

In vivo evaluation of sunscreens by spectroscopic methods.

Background/aims: The methods available for testing the efficacy of topical sunscreens have improved considerably in recent years. Nevertheless, so far no simple and rapid test has been proposed to measure in vivo transmission spectra of sunscreens in the UVA region.