Candida albicans strains 3153a, ATCC 48867, CBS 2730, DSM 70014, and Vir 13 were cultivated and sterile C. albicans filtrates were produced. The interaction of soluble Candida factors of these infiltrates with human HaCaT keratinocytes was assayed in vitro. The following parameters were analyzed: cell proliferation, protein synthesis, nuclear matrix protein (NMP) 41 release, cytokine release (IL-1beta, soluble IL-2 receptor, IL-6, and IL-8), and reactive oxygen species (ROS). Cell counts at 1, 12, and 24 h were significantly lower for C. albicans strains CBS 2730 and VIR 13 (P < 0.05). There was no significant change for the remaining strains. Neither the protein synthesis nor the NMP-41 release was significantly affected. IL-6 and IL-8 were stimulated by C. albicans filtrates to different amounts with higher levels in strains of low virulence. There was no effect on the other cytokines. The production of ROS by HaCaT keratinocytes was suppressed. The induction of an inflammatory keratinocyte response by soluble C. albicans factors may play a role among the host-yeast interactions.

An investigation of the antioxidative UV protective effect of melatonin was performed in an in vitro irradiation model with leukocytes. Leukocytes were isolated from EDTA-treated whole blood and taken up in phosphate-buffered saline (PBS). Five of 10 aliquots were incubated with 2 mmol/L melatonin and 5 with PBS as a control. The samples were irradiated by UV light (280-360 nm, max: 310 nm) at doses between 75 and 300 mJ/cm(2) or left unirradiated. Radical formation was measured using the chemiluminescence technique. Staining with trypan blue was performed to assess cell viability. Melatonin significantly suppressed radical formation in cell solutions irradiated from 75 to 300 mJ/cm(2) (P = 0.001). Controls showed an increase of reactive oxygen species (ROS) formation as a sign of oxidative stress when irradiated with increasing UV doses and a maximum ROS formation under 300 mJ/cm(2) UV light. The cytotoxicity of UV light was reduced by melatonin up to a UV dose of 1.5 J/cm(2). Leukocytes were suitable cells for the evaluation of the efficacy of melatonin as a radical scavenger under UV light. The results confirm that the clinically observed UV protective effects of melatonin may be at least partially based on its radical scavenging properties.

The epidermal growth factor (EGF) receptor plays a pivotal role in growth regulation of epidermal keratinocytes. Its expression and function can be markedly altered during malignant transformation in squamous cell carcinoma. The present study investigated the potential of growth inhibition by signal-transduction inhibitors in EGF-dependent epithelial cell lines in vitro. Benign HaCaT keratinocytes and malignant A431 cells were grown in vitro and exposed to various concentrations of a panel of eleven kinase and phosphodiesterase inhibitors. Cell growth was measured after 24 h and 48 h using fluorescence labeling with Hoechst 33342 and propidium iodide. Significant growth inhibition was achieved with all inhibitors when applied to HaCaT cells. The strongest growth inhibition was achieved with inhibitors H-7, A3 and diacylglycerol kinase inhibitors I and II. A431 cells were inhibited significantly by H-7, A3 and H-9. Selected signal-transduction inhibitors such as A3, H-7 and H-9 acting on intracellular kinases are capable of suppressing growth of EGF-dependent benign and malignant epithelial cell lines in vitro. They might be of future potential in the treatment of epithelial cancer but further studies are necessary.

To prove the relative potency of melatonin as a radical scavenger in UV-irradiated leukocytes, it was compared to other antioxidative substances such as trolox and vitamin C. Human leukocytes were isolated from EDTA whole blood and incubated with melatonin, trolox and vitamin C. The experiments were performed in a wide concentration range from 0.1 nM to 1 mM and in a small concentration range from 0.5 to 2 mM (mel), 5 mM (trolox) and 10 mM (vit. C). Irradiation was performed with UV-light (280-360 nm) at a dose of 750 mJ/cm(2). Radical formation was measured by the chemiluminescence technique. The maximum effect of radical suppression was seen at a concentration of 10 nM (p = 0.003) and 1 mM melatonin (p < 0.001) and vitamin C (p = 0.002; p < 0.001), respectively. ROS suppression by trolox was only significant at 1 mM (p < 0.001). In the small concentration range, a linear dose-response relationship was found and melatonin showed the strongest radical suppression (IC(50) = 0.21 mM) followed by vitamin C (IC(50) = 0.26 mM) and trolox (IC(50) = 1.03 mM).

Reactive oxygen species (ROS) are presumed to be involved in inflammatory UV reactions of the skin. This in vitro study was performed to investigate the suppressive effect of melatonin in interleukin-3 (IL-3) stimulated leukocytes. Neutrophilic granulocytes were isolated from EDTA-treated whole blood and placed in a phosphate-buffered saline (PBS) containing IL-3. Cell suspensions were either treated with PBS (control) or with increasing doses of melatonin (0.1, 0.5, 1, 2, 3, 5, 7.5, 10 mmol). One PBS solution was left unirradiated and the other nine solutions (PBS and melatonin) were irradiated with 750 mJ/cm2 UVB light (280-360 nm, max: 310 nm). Radical formation was measured by the chemiluminescence technique. UV-irradiated leukocytes showed a 5-fold higher radical formation than unirradiated leukocytes. Melatonin, in increasing doses in powers of ten, led to a maximum suppression of free radicals at 10 nmol (P= 0.01) and 1 mmol melatonin (P= 0.001), showing a biphasic, non-linear, dose response relationship. Melatonin, given in amounts of 0.1-10 mmol, led to a direct dose-dependent suppression of ROS. Radical formation was suppressed significantly in a range from 0.5 to 10 mmol (P= 0.001). Melatonin is known to function as a radical scavenger and antioxidant; some of these melatonin effects may be receptor independent, while others may be receptor dependent.

The treatment of progressive systemic sclerosis (PSS) is still unsatisfactory. We report on clinical, laboratory and immunological findings in 26 patients with PSS (6 males, 20 women) treated with extracorporeal photopheresis (ECP) for 8 cycles in a nonrandomized, uncontrolled study. ECP was performed on two consecutive days once a month. 8-methoxypsoralen concentrations in plasma and buffy coat were monitored by HPLC. We performed a standardized examination programme and determined parameters of inflammation and immune function. Global assessment revealed a partial remission in 18 patients, a stable disease in 8 patients and a slight progression in one patient. In the peripheral blood count a significant increase of CD3-positive NK cells was noted (p=0.03) although the leukocyte count decreased from 2,255 to 1,156 cells/microl. There was a non-significant decrease of elastase (102. 9 vs. 90.4 ng/ml), sulfidoleukotriens (2,255.4 vs. 1,688.9 pg/ml), ICAM-1 (301.9 vs. 276.6 ng/ml), soluble IL-2 receptor (609.0 vs. 422. 3 U/ml), and IL-10 (164.7 vs. 138.7 pg/ml). IL-6 and IL-8 did not show significant changes. The ECP treatment of patients with PSS shows immunomodulatory effects changing levels of pro-inflammatory and cytokine substances. Even after 8 cycles partial remission or stable disease is seen in patients as shown by global assessment and certain clinical symptoms. On the other hand, sufficient data on the long-term outcome are still missing.

M proteins are receptor proteins and one of the virulence factors of streptococci. M proteins seem to play a role in inflammatory skin disorders such as psoriasis. It is however unknown whether M proteins have a direct influence on proliferative activity of human keratinocytes. In the present study human HaCaT keratinocytes were exposed to M proteins (M1, M3, M5, M12) and the proliferative and proinflammatory response was analyzed. We found a dose-dependent inhibition of keratinocyte proliferation with crude extract of strain M3 4/55. Following affinity chromatography we found inhibitory activity for keratinocyte proliferation with a maximum of 80% at 10-8 M in the M protein. Additionally tested M1 protein preparation showed an inhibitory activity of 55% whereas other M preparations (5 and 12) did not show any effect. In supernatants from HaCaT cultures IL-1alpha, IL-1beta, IL-6, IL-8, TNFalpha and ICAM-1 were measured by ELISA. The levels of IL-8 were high and TNFalpha was upregulated, whereas ICAM-1 was decreased from around 20 ng/ml to almost zero. In contrast to the streptococcal-derived M3 protein preparation the recombinant M3 did not interfere with the proliferation of HaCaT cells. Because neither recombinant M3 protein nor M3 protein purified by ion exchange chromatography on a Q-resource column had any antiproliferative activity on keratinocytes we suggest, that a component different from M3 protein was responsible.

Comparative testing of seven wound dressings (WD) has been performed with human HaCaT keratinocyte and mouse 3T3 fibroblast cultures. To assess biocompatibility, morphologic examinations were combined with cell counting. Supernatants were subjected to measurements of tissue peptide antigen (TPS), soluble intercellular adhesion molecule 1 (ICAM-1), and interleukins (IL-1 alpha, -1 beta, -6). Furthermore, monoxygenation, the reduced glutathione/oxidized gluthathione (GSH/GSSG) ratio and lipid peroxides were determined. Initial morphologic events were noted within the first day of exposure to WD. After 72 h, inhibition of cell growth was observed in the presence of hydrocolloids and hydrogels. The cytochrome-P-450-dependent ethoxyresorufin 0-deethylation rate and the GSH/GSSG ratio were not altered by WD in HaCaT cells. Lipid peroxide generation, IL-1 and ICAM-1 were scarcely detectable. TPS and IL-6 release indicate the presence of an 'activated stage' of keratinocytes and fibroblasts exposed to WD. Peptide release in vivo may contribute to the beneficial effects of modern dressings in the treatment of superficial cutaneous wounds.