Photoprotective properties of new derivatives of kinetin.

The chronic exposure of skin to ultraviolet (UV) radiation causes adverse dermal reactions, such as erythema, sunburn, photoaging, and cancer, by altering several signalling pathways associated with oxidative stress, inflammation, and DNA damage. One of the possible UV light protection strategies is the use of dermal photoprotective preparations. The plant hormone kinetin (N6-furfuryladenine; KIN) exhibits antioxidant and anti-senescent effects in human cells. Topically applied KIN also reduced some of the clinical signs of photodamaged skin. To improve the biological activities of KIN, several derivatives have been recently prepared and their beneficial effects on cell viability of skin cells exposed to UVA and UVB light were screened. Two potent candidates, 6-(tetrahydrofuran-2-yl)methylamino-9-(tetrahydrofuran-2-yl)purine (HEO) and 6-(thiophen-2-yl)methylamino-9-(tetrahydrofuran-2-yl)purine (HEO6), were identified. Here the effects of KIN, its N9-substituted derivatives the tetrahydropyran-2-yl derivative of KIN (THP), tetrahydrofuran-2-yl KIN (THF), HEO and HEO6 (both THF derivatives) on oxidative stress, apoptosis and inflammation in UVA- or UVB-exposed skin cell was investigated. Human primary dermal fibroblasts and human keratinocytes HaCaT pre-treated with the tested compounds were then exposed to UVA/UVB light using a solar simulator. All compounds effectively prevented UVA-induced ROS generation and glutathione depletion in both cells. HEO6 was found to be the most potent. All compounds also reduced UVB-induced caspase-3 activity and interleukin-6 release. THP and THF exhibited the best UVB protection. In conclusion, our results demonstrated the UVA- and UVB-photoprotective potential of KIN and its derivatives. From this point of view, they seem to be useful agents for full UV spectrum protective dermatological preparations.

Effect of the flavonoids quercetin and taxifolin on UVA-induced damage to human primary skin keratinocytes and fibroblasts.

The ultraviolet (UV) part of solar radiation can permanently affect skin tissue. UVA photons represent the most abundant UV component and stimulate the formation of intracellular reactive oxygen species (ROS), leading to oxidative damage to various biomolecules. Several plant-derived polyphenols are known as effective photoprotective agents. This study evaluated the potential of quercetin (QE) and its structurally related flavonoid taxifolin (TA) to reduce UVA-caused damage to human primary dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK) obtained from identical donors. Cells pre-treated with QE or TA (1 h) were then exposed to UVA light using a solar simulator. Both flavonoids effectively prevented oxidative damage, such as ROS generation, glutathione depletion, single-strand breaks formation and caspase-3 activation in NHDF. These protective effects were accompanied by stimulation of Nrf2 nuclear translocation, found in non-irradiated and irradiated NHDF and NHEK, and expression of antioxidant proteins, such as heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and catalase. For most parameters, QE was more potent than TA. On the other hand, TA demonstrated protection within the whole concentration range, while QE lost its protective ability at the highest concentration tested (75 µM), suggesting its pro-oxidative potential. In summary, QE and TA demonstrated UVA-protective properties in NHEK and NHDF obtained from identical donors. However, due to the in vitro phototoxic potential of QE, published elsewhere and discussed herein, further studies are needed to evaluate QE safety in dermatological application for humans as well as to confirm our results on human skin ex vivo and in clinical trials.

Effect of UVA radiation on the Nrf2 signalling pathway in human skin cells.

The harmful effects of low energy UVA photons (315-400 nm) are associated with the massive production of reactive oxygen species resulting in oxidative stress. In response to oxidative damage, NF-E2-related factor 2 (Nrf2) is translocated to the nucleus and drives the expression of detoxication and antioxidant enzymes. UVA's effect on Nrf2 has been quite well characterised in dermal fibroblasts. However, there is a dearth of such information for keratinocytes. This study aimed to evaluate and compare the effect of UVA radiation on the Nrf2 pathway and oxidative stress related proteins in primary human dermal fibroblasts (NHDF), epidermal keratinocytes (NHEK) and human keratinocyte cell line HaCaT. NHDF were exposed to doses of 2.5-7.5 J/cm2, NHEK and HaCaT to 10-20 J/cm2 using a solar simulator. Effects on Nrf2 translocation were evaluated after 1, 3 and 6 h and Nrf2-controlled proteins (heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione reductase (GSR), glutathione-S-transferase (GST), interleukine-6 (IL-6), and matrix metalloproteinases (MMP-1, MMP-2)) after 3, 6 and 24 h. The results showed the fastest Nrf2 translocation was in UVA-irradiated HaCaT (1 h), persisting until the subsequent time interval (3 h), while in primary keratinocytes the effect of radiation was minimal. In NHDF, UVA-stimulated Nrf2 translocation was conspicuous 3 h after UVA treatment. In NHDF, most of the studied proteins (NQO1, HO-1, GSR, GSTM1 and MMP-1) showed the highest level 24 h after UVA exposure, except for MMP-2 and IL-6 which had their highest level at a shorter time incubation interval (3 h). In NHEK, NQO1, HO-1 and GST were increased 6 h after UVA exposure, GSR and MMP-2 level was slightly below or above the control level, and MMP-1 and IL-6 increased at shorter time intervals. When comparing NHEK and HaCaT, these cells displayed contrary responses in most of the Nrf2-controlled proteins. Thus, primary keratinocytes cannot be replaced with HaCaT when studying cell signalling such as the Nrf2 driven pathway and Nrf2-controlled proteins.

A pilot study of the UVA-photoprotective potential of dehydrosilybin, isosilybin, silychristin, and silydianin on human dermal fibroblasts.

The exposure of naked unprotected skin to solar radiation may result in numerous acute and chronic undesirable effects. Evidence suggests that silymarin, a standardized extract from Silybum marianum (L.) Gaertn. seeds, and its major component silybin suppress UVB-induced skin damage. Here, we aimed to investigate the UVA-protective effects of silymarin's less abundant flavonolignans, specifically isosilybin (ISB), silychristin (SC), silydianin (SD), and 2,3-dehydrosilybin (DHSB). Normal human dermal fibroblasts (NHDF) pre-treated for 1 h with flavonolignans were then exposed to UVA light using a solar simulator. Their effects on reactive oxygen species (ROS), carbonylated proteins and glutathione (GSH) level, caspase-3 activity, single-strand breaks' (SSBs) formation and protein level of matrix metalloproteinase-1 (MMP-1), heme oxygenase-1 (HO-1), and heat shock protein (HSP70) were evaluated. The most pronounced preventative potential was found for DHSB, a minor component of silymarin, and SC, the second most abundant flavonolignan in silymarin. They had significant effects on most of the studied parameters. Meanwhile, a photoprotective effect of SC was mostly found at double the concentration of DHSB. ISB and SD protected against GSH depletion, the generation of ROS, carbonylated proteins and SSBs, and caspase-3 activation, but had no significant effect on MMP-1, HO-1, or HSP70. In summary, DHSB and to a lesser extent other silymarin flavonolignans are potent UVA-protective compounds. However, due to the in vitro phototoxic potential of DHSB published elsewhere, further studies are needed to exclude phototoxicity for humans as well as to confirm our results on human skin ex vivo and in vivo.

Changes in antioxidant, inflammatory and metabolic markers during 1 week cultivation of human skin explants.

Human skin explant (HSE) seems to be a useful model for dermatological/cosmetic testing. HSE prepared from donor superfluous skin from plastic surgery operations is cheap and easily obtainable compared to reconstructed models. The HSE use, however, may be limited by the degeneration processes during cultivation. The aim was to monitor changes in metabolic activity and selected apoptotic, inflammatory and antioxidant parameters during 7 day cultivation. The significant changes were found in the superoxide dismutase-2 level from day 5, glutathione S-reductase level from day 6, metabolic activity and fibulin-5 level from day 4, cyclooxygenase-2, interleukin-6 and interleukin-10 from day 1 to 2. Other selected markers (lipid peroxidation products and glutathione level, glutathione S-transferase, catalase, superoxide dismutase and glutathione S-reductase activity, glutathione peroxidase and glutathione S-reductase levels) were not modified significantly due to high inter-individual variability of skin donors. The HSE microstructure as well as cytokeratin-10 and proliferation marker Ki67 expression was also only minimally affected during cultivation. Collectively, the results demonstrate that HSE represents a good model for short-term studies focused on the physical and chemical agent toxicity, protective potential of compounds or metabolic biotransformation. However, reduced metabolic activity, increased inflammation and the high inter-individual variability and sensitivity of donors have to be taken into consideration.

Dermal Delivery of Selected Polyphenols from Silybum marianum. Theoretical and Experimental Study.

Silymarin is a well-known standardized extract from the seeds of milk thistle (Silybum marianum L., Asteraceae) with a pleiotropic effect on human health, including skin anticancer potential. Detailed characterization of flavonolignans properties affecting interactions with human skin was of interest. The partition coefficients log Pow of main constitutive flavonolignans, taxifolin and their respective dehydro derivatives were determined by a High Performance Liquid Chromatography (HPLC) method and by mathematical (in silico) approaches in n-octanol/water and model lipid membranes. These parameters were compared with human skin intake ex vivo. The experimental log Pow values for individual diastereomers were estimated for the first time. The replacement of n-octanol with model lipid membranes in the theoretical lipophilicity estimation improved the prediction strength. During transdermal transport, all the studied compounds permeated the human skin ex vivo; none of them reached the acceptor liquid. Both experimental/theoretical tools allowed the studied polyphenols to be divided into two groups: low (taxifolin, silychristin, silydianin) vs. high (silybin, dehydrosilybin, isosilybin) lipophilicity and skin intake. In silico predictions can be usefully applied for estimating general lipophilicity trends, such as skin penetration or accumulation predictions. However, the theoretical models cannot yet provide the dermal delivery differences of compounds with very similar physico-chemical properties; e.g., between diastereomers.

UVA-photoprotective potential of silymarin and silybin.

Exposure to solar radiation is a major cause of environmental human skin damage. The main constituent of solar UV light is UVA radiation (320-400 nm); however, the need for protection against UVA has been marginalized for a long time. As a result, there is still a lack of useful agents for UVA protection. In this study, the effect of silymarin (SM) and its main constituent silybin (SB) pre-treatment on UVA-stimulated damage to primary human dermal fibroblasts were carried out. The cells were pre-treated for 1 h with SB or SM and then were exposed to UVA light, using a solar simulator. The effect of SB and SM on reactive oxygen species (ROS) and glutathione (GSH) level, caspase-3 activity, single-strand breaks (SSB) formation and protein level of matrix metalloproteinase-1 (MMP-1), heme oxygenase-1 (HO-1), and heat shock protein (HSP70) was evaluated. Treatment with both SM and SB resulted in a reduction in UVA-stimulated ROS generation and SSB production, as well as in the prevention of GSH depletion, a decrease in the activation of caspase-3 and protein level of MMP-1. They also moderately increased HO-1 level and reduced HSP70 level. Our data showed that both SM and SB are non-phototoxic and have UVA-photoprotective potential and could be useful agents for UV-protective dermatological preparations.

The Phototoxic Potential of the Flavonoids, Taxifolin and Quercetin.

Quercetin, one of the most abundant polyphenols in the plant kingdom has been shown to be photodegraded on exposure to UV light. Despite the fact, it is a component of several dermatological preparations. Its phototoxic potential has not been evaluated to date. The aim of this study was to assess whether photo-induced degradation of quercetin is linked to phototoxic effects on living cells. Its dihydro derivative, taxifolin, was included in the study. For evaluation, the 3T3 Neutral Red Uptake Phototoxicity Test according to OECD TG 432 was used. To better approximate human skin, HaCaT keratinocytes, normal human epidermal keratinocytes and dermal fibroblasts were used, apart from the Balb/c 3T3 cell line. Quercetin showed a dose-dependent photodegradation in aqueous and organic environments and a phototoxic effect on all used cells. Quercetin pretreatment and following UVA exposure resulted in increased reactive oxygen species production and intracellular glutathione level depletion in human dermal fibroblasts. Taxifolin was found completely nonphototoxic and photostable. As only in vitro methodology was used, further studies using 3D skin models and/or human volunteers are needed to confirm whether exposure to sunlight, tanning sunbeds and/or phototherapy in people using cosmetics containing quercetin is a health risk.

Reconstructive procedures in maxillofacial oncosurgery.

OBJECTIVE: Oropharyngeal cancers are a biologically heterogenous group of tumors with diverse risk factors including tobacco, alcohol, HPV, inherited disorders, the acquired immunodeficiency of Karposi's Sarcoma and non Hodgkin's lymphoma. In the Czech Republic, oropharyngeal cancers represent around 2% of all cancers. The treatment of these tumors is long and complex. Reconstructive procedures in maxillofacial oncosurgery demand good interdisciplinary collaboration and great professional preparedness of the surgical and nursing team. Patient age and stage of disease, including the presence of metastases are of key importance. A prerequisite for the success of surgical treatment is removal of the tumor with a sufficient safety margin. Reconstructive procedures then follow. AIM: To highlight the importance of radical tumor resection and describe reconstruction of the defect in a group of our patients. METHODS AND RESULTS: From 2008 to 2013, 23 patients with oropharyngeal carcinoma underwent radical surgical removal of tumor, followed by reconstruction of postoperative defects using distant and free flaps. The histopathology showed predominantly squamous cell carcinomas and one of Merkel cell carcinoma. 16 patients had malignant disease detected in III-IV. In only 7 cases was treatment initiated in the first and second stages of the disease. In these patients, the tumors were removed with a safety margin of healthy tissue and in none, did the basic cancer recur . The postoperative course in terms of flap engraftment and overall condition of the patient was uneventful. All of these patients still enjoy a good life quality with a current mean survival in range 5 - 76 months. Radical surgical removal of a malignant tumor in the early stages of the disease is associated with fewer postoperative complications and longer survival. CONCLUSION: To avoid the risk of local and/or systemic postoperative complications, appropriate patient selection is important. Overall, the traditional, classic reconstructive procedures with the use of prostheses, in many cases is still the best option in our experience.

Radiofrequency upper thoracic sympathectomy in the treatment of critical upper limb ischemia--a case series.

BACKGROUND: Patients with significant medical and social problems resulting from impaired perfusion of the upper limbs caused by micro- or macro-angiopathy are now frequent in clinical practice. Vasospastic disease of the upper limbs of combined origin is a difficult condition to treat by conservative methods and therapeutic strategies are usually multidisciplinary. In addition to standard pharmacotherapy, treatment may involve regional anaesthesia, thoracoscopic or radiofrequency sympathectomy and surgical treatment of defects, including plastic surgery. METHODS: This paper describes our successful work in the treatment of upper limb critical ischemia using radiofrequency upper thoracic sympathectomy. RESULTS: In three case reports we present the results of radiofrequency thermolysis applied to treat patients with chronic defects of the hand and fingers. These patients were diagnosed with upper limb critical ischemia of combined origin, standard conservative treatment methods failed and surgical intervention was originally not indicated, however, radiofrequency thermolysis proved to be beneficial. CONCLUSIONS: Radiofrequency thoracic sympathectomy could improve peripheral perfusion of the upper limbs and thereby contribute to healing of chronic defects, reduction of pain and improvement in the quality of life of the patients.

Pectoral muscle flap with V-Y skin paddle for covering sternal defects.

Deep sternal wound infection (DSWI) after a cardiac operation is a rare but serious complication associated with significant morbidity and mortality. It can lead to wound dehiscence with sternal osteomyelitis and both bony and soft tissue residual defects. When the infection is eradicated, reconstruction of the thoracic wall remains the main challenge. Tissue used for covering the defect must be well nourished and sutures must be tension free. We present our unique modification of the method using the pectoral muscle axial flap with a V-Y skin paddle.

Vacuum-assisted closure in the treatment of sternal wound infection after cardiac surgery.

AIM: Vacuum-assisted closure (VAC) was primarily designed for the treatment of pressure ulcers or chronic, debilitating wounds. Recently, VAC has become an encouraging treatment modality for sternal wound infection after cardiac surgery, providing superior results to conventional treatment strategies. METHODS: From November 2004 to September 2006, 34 patients, undergoing VAC therapy for sternal wound infection following cardiac surgery, were prospectively evaluated. Ten patients (29 %) were treated for superficial sternal wound infection and 24 (71 %) for deep sternal wound infection. The median age was 69.9 years (range 48 to 82) and the median BMI was 33.4 kg/m(2) (range 28 to 41). Twenty patients (59 %) were women and 19 patients (59 %) were diabetics. Owing to sternal wound infection complications, 16 patients (47 %) were readmitted to the department. VAC was used following the previous failure of the conventional treatment strategy in 7 patients (21 %). RESULTS: Thirty-three patients (97 %) were treated successfully. One patient (3 %) died of multiple organ failure. The overall length of hospitalization was 34.6 days (range 9 to 62). The median number of dressing changes was 4.6 (range 3 to 10). The median VAC treatment time until surgical closure was 9.2 days (range 6 to 21 days). VAC therapy was solely used as a bridge to definite wound closure. Three patients (9 %) with chronic fistula were re-admitted 1 to 6 months after VAC therapy. CONCLUSIONS: VAC therapy is a safe and reliable option in the treatment of sternal wound infection in cardiac surgery. VAC therapy should be considered an effective adjunct to conventional treatment modalities for the treatment of extensive and life-threatening wound infections following cardiac surgery, particularly in the presence of risk factors.

Possible restorations of the upper extremity motion in tetraplegic patients - 5-year clinical experience.

AIM: The aim of the work is to objectify the functional effectiveness of these operations and their influence on the quality of life of handicapped patients. METHOD: The authors evaluate the results of reconstructive surgery restoring hand grip in a group of 15 tetraplegic patients (3 women and 12 men) with complete spinal cord lesion of C5-C7 segments. The average age of patients in the group is 33 (22-50) years old. The reconstructions were performed using tendon transfer and tenodesis in the forearm and hand area. The effectiveness of the transfer was assessed objectively with regard to muscle strength by measuring the restored "thumb-index finger" grip and "into fist" grip. The range of motion achieved was also evaluated. Transfer effectiveness was evaluated on the basis of subjective patients' evaluation. An ADL (activities of daily living) questionnaire by Mohammed's (1992) took into account the effect of the surgery in a whole range of common daily activities. RESULTS: An extended range of daily activities was evident mainly in the fields of: communication, eating and drinking and operations associated with increase in general selfcare of the patient. There was no deterioration of condition in any of the activities. CONCLUSIONS: Up to 80 % of tetraplegic patients are suitable candidates for transfers and, to a certain extent, it is possible to improve the upper limb function. In a partial function restoration of the upper limbs there is immense potential for improvement in the quality of life of these patients.

Molecular changes in PDEGF and bFGF in malignant melanomas in relation to the stromal microenvironment.

BACKGROUND: Aberrant expression of either growth factors or growth factor-receptors by stromal cells can be an important factor promoting the growth of solid tumours. It may also affect differentiation of malignant cells and support tumour spread. The aim of the present study was to investigate the hypothesis that basic-fibroblast growth factor (bFGF) and platelet-derived growth factor (PDEGF) may be involved in tumour-stromal microenvironment interactions in primary malignant melanomas. MATERIALS AND METHODS: PDEGF and bFGF expression in malignant cells and surrounding stromal elements was assessed using indirect immunohistochemistry. RESULTS: It was confirmed that PDEGF can be involved in the reciprocal interactions between tumour cells and stroma, including aberrant angiogenesis. Interestingly, bFGF was present both in malignant melanoma lesions and benign nevi accompanied by different intracellular localisation of the protein, suggesting its implication in regulation of nevus cell proliferation and maturation. CONCLUSION: The present results suggest that bFGF and PDEGF participate in malignant melanoma progression.