1-alpha-calcidol modulates major human monocyte antigens and toll-like receptors TLR 2 and TLR4 in vitro.

Since vitamin D derivatives are known to interfere with the cellular immune response, we analysed the possible effect of 1-alpha-calcidol (AC) on major monocyte antigens CD14 (an endotoxin receptor), HLA-DR, and toll-like recptors 2 and 4 (TLR2, TLR4). Peripheral blood monocytes were isolated from healthy donors and cultured by standard protocol followed by incubation with various concentrations of AC in unstimulated and LPS-activated cells. After 24, 48 and 72 hours cells were harvested and analysed for the expression of antigens by flowcytometry. Compared to the controls AC increased the expression of CD14 in a dose and time dependent manner (after 72 hours culture time p < 0.01). AC was capable of further stimulating CD14 expression in LPS activated monocytes (p < 0.05). Both LPS and AC downmodulated HLA-DR dramatically after 24 (p < 0.05), 48 (p < 0.01) and 72 hours (p < 0.0001). The expression of TLR2 but not of TLR4 was inhibited by 10-7M AC. The data reveal that AC significantly modulates the expression of CD14, HLA-DR as well as of TLR2, all involved as targets and effector molecules in antigen recognition and processing, relevant to overcome infections and organ lesions.

[Cultivated keratinocytes on micro-carriers: in vitro studies of a new carrier system]. / Kultivierte Keratinozyten auf Microcarriern: In-vitro-Untersuchungen zu einem neuartigen Trägersystem.

Epidermal grafts from confluently cultivated keratinocytes have been used since the early eighties for the treatment of severe burns, where the shortage of donor sites for split-thickness skin grafts did not allow for adequate wound coverage. The difficult handling of these grafts as well as the advanced differentiation of their epithelial cells into a multilayer sheet poses a problem for their clinical application. The aim of the study was to characterize cultivated keratinocytes, as well as to observe their migration and proliferation from the MC onto a surface. Keratinocytes were isolated from human foreskin and cultivated in serum-free and serum-containing medium according to a modified method by Rheinwald and Green. Collagen-coated Dextran beads were used as MC. The MC were colonized with keratinocytes using the Spinner culture technique. After seeding the colonized MC into culture flasks, their migration and proliferation was monitored regularly through immunohistochemical studies and measurement of the metabolic cell activity. Immunohistological staining proved that the cells isolated from human foreskin represent keratinocytes of the basal type. Keratinocytes, cultivated with serum-containing and serum free medium, both adhered to the surface of the MC, then migrated onto the surface of the flasks and proliferated to form a multilayer of epithelial cells. In the long-term, a flexible epithelial graft consisting of poorly differentiated keratinocytes should be available, which is simple to produce and easy to handle. This would be an alternative method for treating wounds, where the conventional multilayer epithelial graft (ET) is insufficient.

Time-resolved infrared ATR measurements of liposome transport kinetics in human keratinocyte cultures and skin reveals a dependence on liposome size and phase state.

A novel in vitro method for studying the permeation kinetics of superficially applied liposomes or vesicles through layers of human skin or keratinocytes on a solid support is presented, employing attenuated total reflection infrared spectroscopy. The method is applied to investigate transport kinetics of unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) through cultured human keratinocyte layers and through human skin. We find a strong resemblance of the qualitative features of the permeation kinetics of small unilamellar DMPC vesicles for skin and keratinocytes. Detailed studies of the vesicles transport through keratinocyte layers show that DMPC vesicles with an average diameter of 55 nm can readily permeate through the layer at 37 degrees C with a diffusion constant of D = (4.0 +/- 0.8) x 10(-15) m2/second, whereas larger vesicles of twice that diameter do not permeate at all. In contrast, liposomes containing a chemical permeation enhancer permeate through the layer significantly faster [D = (7.0 +/- 0.5) x 10(-15) m2/second] than the small DMPC vesicles despite their five-times-larger diameter. Moreover, the transport of the DMPC vesicles depends drastically on their phase state. No permeation was observed for small DMPC vesicles at a temperature of 10 degrees C when the lipid is in the crystalline phase state. Our results indicate that keratinocyte culture layers can pose a significant permeation barrier for vesicles. The permeation mechanism can be explained by diffusion of the vesicles through small pores and gaps in the layer, presumably driven by transdermal osmotic gradients.

[Use of keratinocyte cultures in treatment of severe burns--experiences up to now, outlook for further subsequent developments]. / Die Verwendung von Keratinozytenkulturen in der Schwerbrandverletztenbehandlung--bisherige Erfahrungen, Ausblicke zur weiteren Entwicklung.

There is a world-wide growing interest in cultured epithelium. It is commonly accepted that cultured epithelial auto- or allografts can stimulate wound healing and shorten re-epithelialization time. Sheets of cultured autologous epidermal cells have been used for more than 15 years as grafts to achieve permanent coverage of full-thickness burn wounds. Yet many surgeons who have used cultured epidermal grafts have reported a substantial variability in their outcome. The best results have been obtained by performing early excision, followed by temporary coverage with a cadaver homograft. Within 3 weeks the donor allodermis is incorporated and forms a neodermis. The epidermal parts of the donor skin are removed after about 3 weeks and cultured epidermal autografts are transplanted (composite graft technique). There is some hope that progress in the cultivation procedure and a modified transplantation technique will shorten the healing time. In our opinion, great progress was made when cryopreserved allogeneic epithelial grafts became available for the treatment of deep dermal burn wounds. We obtained a good re-epithelialization rate (56%) after 9.5 days in 56 cases. In the last 25 cases, the re-epithelialization time was 72% after 11.5 days. Especially burn wounds of the face have been treated successfully, avoiding over-grafting and achieving highly acceptable, aesthetic and functional results. Many laboratories are developing dermal equivalents, combining synthetic and biological materials in order to form a multilayer neodermis. Although it seems possible to cultivate adnexae of the skin, a neodermis with cultivated adnexae is not yet in sight.

[Keratinocyte culture and transplantation in burns]. / Keratinocytenzüchtung und -transplantation bei Verbrennungen.

The culture and transplantation of keratinocytes are considered an important progress in the treatment of severe burns. The keratinocyte grafts take best (50 to 90%) on remaining dermal structures after deep dermal (II b) burns. Since 1988 we culture also donor keratinocytes. They are cryopreserved in the 'skin bank' in large quantities. As vital wound cover they allow for a rapid and near scarless reepithelialization. For deep (III) burns we use composite grafts of cultured auto-keratinocytes on allo-dermis with increasing success (up to 75% take rate) without rejection.

Phospholipase A activity in Pseudomonas aeruginosa.

Our study describes the production, purification and properties of an enzyme from Pseudomonas aeruginosa displaying the properties of phospholipase A. Maximal amounts of enzyme could be detected in the culture supernatant when the bacterium was grown for 3 to 5 days at 37 degrees C in stirred flask cultures containing brain heart infusion. The enzyme was purified by polyethylenimine precipitation and ammonium sulfate precipitation followed by gel filtration. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme preparation exhibited two bands with molecular weights of 13.5 and 60 kD, respectively. Correspondingly, two peaks of the same molecular weight could be demonstrated by high performance size exclusion chromatography. The activity toward the sn-2 ester binding of phospholipids was characterized and found to be highest towards phosphatidylcholine. Enzymatic activity was not influenced by the addition of calcium or EDTA while magnesium and strontium caused a decrease of activity. The lyophilized enzyme was found to be stable when stored at -70 degrees C and most active at pH 8.0.

Composite grafts of autogenic cultured epidermis and glycerol-preserved allogeneic dermis for definitive coverage of full thickness burn wounds: case reports.

In patients with extensive deep burns and scarce donor sites autogenic cultured epithelial grafts (auto-CEG) have become a real alternative. In deep burns the 'take' rate of auto-CEG applied directly on subcutaneous fat, fascia or muscle is unreliable and frequently disappointing. The auto-CEG seems to need a dermal base. Improved results have been reported when auto-CEG were applied to the dermal base of a viable cryopreserved donor skin. We extended this principle by using the dermal layer of non-viable glycerol-preserved donor skin (GPDS). We report on two patients with deep burns of 55 and 80 per cent TBSA in whom we used the composite grafting of auto-CEG on non-viable allogeneic dermis from GPDS. The estimated 'take' rates were 70 and 77 per cent. The grafted areas remained stable for 4 and 8 months respectively. The two-layer skin substitute gave a permanent cover for full thickness burn wounds of higher quality and better 'take' rate than previous results, where the auto-CEG had been grafted directly onto the debrided wounds.

Discrimination of the irritancy potential of surfactants in vitro by two cytotoxicity assays using normal human keratinocytes, HaCaT cells and 3T3 mouse fibroblasts: correlation with in vivo data from a soap chamber assay.

Cell cultures have been proposed as a promising model for local tolerance testing. This study evaluated the cytotoxic effects of surfactants on early passage normal human keratinocytes, transformed human keratinocytes (HaCaT cells) and Swiss 3T3 embryonic mouse fibroblasts. Cell membrane integrity, as assessed by the release of the vital dye neutral red, and cell proliferation, as assessed by measurement of the total protein content, were both affected in a dose-dependent manner in response to surfactant exposure. There was a close correlation between the dose-response characteristics for the three cell types. Two surfactants exhibited differential effects on membrane integrity and proliferation, and thus no significant correlation was found between the two endpoints. The irritation potential of the surfactants to human forearm skin in vivo was assessed in a soap chamber test using transepidermal water loss and skin redness as quantitative endpoints. A comparison between the responses in vivo and in vitro yielded the highest correlation for the neutral red release test on normal keratinocytes. The total protein test did not significantly correlate with the soap chamber assay for keratinocytes and HaCaT cells. These results suggest that cultured normal human keratinocytes may be predictive for the irritancy of various surfactants in man. Definite judgement, however, has still to be based on confirmation in human volunteers of larger groups of compounds with diverse physico-chemical properties.

MTT-assay and neutral red release (NRR)-assay: relative role in the prediction of the irritancy potential of surfactants.

A comparative study on the in vitro and in vivo irritancy of anionic, amphoteric and non-ionic surfactants was performed. In vitro ED50 values of the surfactants were determined by two cytotoxicity assays, the dimethylthiazoldiphenyltetrazoliumbromide (MTT) assay and the neutral red release (NRR) assay on serum-free cultured human foreskin keratinocytes. In vivo human irritancy data were obtained by a 24 hour occlusive patch test in volunteers and the irritant skin response quantified by visual scoring, evaporimetry and colorimetry. A close relationship between the evaluation methods of the patch test was observed (r = 0.92 to r = 0.96), confirming that the 'bioengineering' methods, such as evaporimetry and colorimetry are suitable for measuring skin irritation. For six surfactants evaluated we found a good correlation (r = 0.91) between the ED50 values of the MTT assay and the in vivo irritancy data. The NRR assay yielded less satisfactory correlation coefficients with regard to MTT assay (r = 0.42) and in vivo irritancy data (r = 0.46). This can be mainly attributed to a misinterpretation of the amphoteric and non-ionic surfactants by the NRR assay. While the NRR assay may better evaluate the anionic surfactants, the MTT assay seems to be more suitable when testing a broader range of chemically diverse surfactants. Limitations of cell culture systems are noted, although the potential usefulness of cultured human skin cells for skin irritancy testing has been clearly demonstrated.

Evidence for the phagocytosis of intact oligolamellar liposomes by human keratinocytes in vitro and consecutive intracellular disintegration.

While there is evidence for the use of liposomes as drug carriers upon topical application to the skin, the underlying mechanisms are far from being clear. Therefore human keratinocytes grown in vitro were exposed to large oligolamellar liposomes. After attachment and invagination these particles can be found unchanged within the cytoplasm both inside and outside of lysosomes. If incorporated into lysosomes the structural lipids can be disintegrated and spread within the entire phagolysosome. Gold labeling adds further to the hypothesis of intact uptake of liposomes by the living cells of the human epidermis.

[Skin culture--keratinocytes]. / Hautzüchtung--Keratinozyten.

Our efforts to cultivate keratinocytes and to use cultivated epidermal grafts which are then transplanted onto deep second- and third-degree burns and donor sites date back in 1987. Our laboratory is now able to provide our intensive care unit with cultured epidermografts as a routine procedure. Furthermore, we have developed a simple method for cryopreservation of cultured human epidermal keratinocytes. So in 1980, a skin bank was set up which provides us with cryopreserved allogenic cultured epidermis. Indications, operative management, and results are presented and accompanied by typical clinical cases.

Absence of extracellular enzyme activity and cytotoxicity in cell-free culture filtrates of Haemophilus ducreyi.

Ten isolates of Haemophilus ducreyi, including seven clinical isolates and three laboratory reference strains, were assessed for their ability to produce extracellular enzymes which might contribute to their pathogenicity. Protease, elastase, lecithinase, lipase or collagenase enzyme activity were not detected in culture filtrates from any of the isolates tested using either plate or spectrophotometrical assays. Furthermore, cell-free culture filtrates did not exhibit cytotoxic activity in vitro when tested using either an established tissue culture cell line (Vero) or a primary cell culture of human keratinocytes. These results suggest that extracellular products do not play a role in host invasion or production of tissue damage by H. ducreyi.

Skin banking: a simple method for cryopreservation of split-thickness skin and cultured human epidermal keratinocytes.

A simple unit has been developed for the simultaneous passive cooling of small and large amounts of allograft or autograft split-thickness skin, as well as cultured human epidermis. An expanded polystyrene box of variable size, aluminum plates, and cellulose tissue are fused. This unit is cooled in a -70 degrees C constant-temperature mechanical refrigerator. Maximal cooling rates of -1.3 degrees C min-1 are obtained in a box with a constant wall thickness of cellulose tissue. The cooling rate can be varied by altering the number of cellulose layers. Exothermic temperature plateaus associated with skin cooled in this unit last for less than 0.3 minutes. The viability of the cryopreserved skin was determined by using up to four methods simultaneously: a dye-exclusion test with trypan blue; glucose consumption; production of lactate; and carbon-13 nuclear magnetic resonance spectroscopy. Using a cryoprotectant medium with 15% (vol/vol) glycerol for split-thickness skin 0.25 mm thick, a storage time of up to 509 days at -70 degrees C was observed, with only a small decrease in viability (trypan blue, 62.5%; glucose consumption, 71 to 90% compared with freshly harvested skin). Storage in liquid nitrogen did not significantly improve results (p greater than 0.05).

[Minimum inhibitory concentration and minimum bactericidal concentration of tetracycline and erythromycin in 35 recent Munich isolates of Chlamydia trachomatis]. / Minimale Hemmkonzentrationen (MHK) und minimale bakterizide Konzentrationen (MBK) von Tetrazyklin und Erythromycin bei 35 rezenten Münchener Chlamydia-trachomatis-Isolaten.

Thirty-five recent clinical isolates of Chlamydia trachomatis were subcultured and subjected to antimicrobial susceptibility testing with tetracycline and erythromycin. Detection of typical chlamydial inclusion bodies and elementary bodies was based on the use of fluorescence-labelled monoclonal antibodies. Minimum inhibitory concentration being defined as the lowest concentration suppressing all inclusion body formation and minimum bactericidal concentration as the lowest concentration preventing all detectable chlamydial growth, both these parameters were studied. With tetracycline the minimum inhibitory concentrations ranged from 0.03 to 0.08 microgram/ml, with erythromycin from 0.04 to 0.2 microgram/ml. The corresponding data for the minimum bactericidal concentrations were less than 0.2 to 1.0 and 0.2 to 2.0 respectively. Thus, at present, there still seems to be no major resistance problem with genital Chlamydia trachomatis isolates in the Federal Republic of Germany.

[In vitro effectiveness of ofloxacin and ciprofloxacin in genital Chlamydia trachomatis isolates measured by minimal inhibitory concentration and minimal bactericidal concentration]. / In-Vitro-Wirksamkeit von Ofloxacin und Ciprofloxacin bei genitalen Chlamydia trachomatis-Isolaten gemessen an minimaler Hemmkonzentration (MHK) und minimaler bakterizider Konzentration (MBK).

Although Chlamydia trachomatis is usually susceptible to the most frequently applied chemotherapeutic agents in non-gonococcal urethritis in men and its counterpart in women--i.e., tetracycline and erythromycin--the clinical results are less satisfying than those in comparable gonococcal infections. Therefore, potent therapeutical alternatives are urgently called for. From this point of view, we investigated the in vitro susceptibility of 35 recent isolates of genital Chlamydia trachomatis from Munich to the new quinolones ciprofloxacin and ofloxacin. With both chemotherapeutics, the highest minimum inhibitory concentration amounted to 2.0 micrograms/ml; 1.0 micrograms/ml of ofloxacin inhibited 97% of the strains, ciprofloxacin but 57%. The highest minimum bactericidal concentration was 6.0 micrograms/ml. 5.0 micrograms/ml of ciprofloxacin killed all infectious particles in 91% of the strains, the corresponding figure for ofloxacin read 74%. Thus both quinolones of the second generation proved more or less equally effective in vitro. In view of the limited clinical experience gained so far with regard to these chemotherapeutic agents, we suggest further clinical study in this matter.

[Genital chlamydia infections--clinical aspects, diagnosis and therapy]. / Genitale Chlamydieninfektionen--Klinik, Diagnostik und Therapie.

Non-gonococcal urethritis and its counterpart in women have become the most frequent genital infection worldwide. As Chlamydia trachomatis is the major causative agent interest has focused on this bacterium. While genital chlamydial infection in men often is manifest the opposite holds true for women. Major complications such as pelvic inflammatory disease can nevertheless turn up. Therefore efficient diagnostic tools are badly needed. If tissue culture procedures are not available direct specimen tests can be performed using fluorescence labelled monoclonal antibodies. For therapy tetracyclines and erythromycin are still the drugs of choice although the cure rates are not totally acceptable. Therefore evaluation of the new quinolones deserves interest.