The Evaluation of Vitiligous lesions Repigmentation after the Administration of Atorvastatin calcium salt and Simvastatin-acid sodium salt in patients with active vitiligo (EVRAAS), a pilot study: study protocol for a randomized controlled trial.

BACKGROUND: Vitiligo is a chronic skin disorder presenting with depigmentation, the pathogenesis of which is complex but the autoimmune theory is now preferred. Multiple immunologic processes, including stimulation of the T-helper (Th)1 response, formation of autoreactive melanocyte-specific CD8+ T lymphocytes, a decrease in the blood concentration of T regulatory (Treg) cells, and an increase in interleukin (IL)-17 and interferon (IFN) concentration, have been shown to contribute to vitiligo progression and maintenance. The aim of this study is to evaluate the influence of simvastatin and atorvastatin on vitiligous lesions in patients with nonsegmental vitiligo (NSV). According to available data, statins act through several immunological pathways, potentially reversing undesirable phenomena underlying autoimmune vitiligo pathogenesis. METHODS/DESIGN: A study has been designed as a single-center, randomized, double-blind, placebo-controlled pilot study with the enrollment of at least 24 active NSV patients presenting with vitiligous lesions on both upper and lower limbs. The clinical effects of ointments containing 1% simvastatin-acid sodium salt or 1% atorvastatin calcium salt applied on a preselected limb will be assessed in comparison with vehicle ointment applied on the opposite limb. All study participants will undergo clinical evaluation using body surface area (BSA) and Vitiligo Area Scoring Index (VASI) scales at baseline and at weeks 4, 8, and 12 time points. A precise assessment of skin lesions will be performed using photographic documentation obtained during each study visit and processed with NIS-Elements software. DISCUSSION: Currently available vitiligo topical therapeutic approaches including calcineurin inhibitors and corticosteroids remain poorly effective and are associated with either relatively high cost or potentially dangerous adverse effects. The clinical application of orally administrated statins, widely used as systemic cholesterol-lowering agents, in vitiligous patients has only been tested in two clinical trials; however, data on their potential usefulness is scarce. Moreover, due to a high risk of clinically significant toxicity, topical administration was recommended by researchers. This study is the first to evaluate safety and efficacy of the topical use of statins in patients presenting with NSV. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03247400 . Registered on 05 August 2017.

Anti-proliferative and cytotoxic activity of rosuvastatin against melanoma cells.

INTRODUCTION: Statins are considered potential candidate agents for melanoma chemoprevention. Statin-induced mevalonate pathway inhibition leads to reduction of cholesterol synthesis and also to decreased cellular levels of non-steroidal isoprenoids, geranylgeranyl pyrophosphate and farnesyl pyrophosphate. This results in the impairment of protein prenylation which affects carcinogenesis. AIM: To analyze anti-proliferative and cytotoxic activity of rosuvastatin against melanoma cells. MATERIAL AND METHODS: Melanoma cell lines (A375 and WM1552C) and normal fibroblasts (BJ) were used as the primary research material. Cells were treated with rosuvastatin at concentrations ranging from 0.01 µM to 10 µM. Cell viability was analyzed with the use of an MTT assay. Expression of proliferation marker Ki67 was assessed on the basis of immunofluorescence staining. RESULTS: Rosuvastatin reduced A375 and BJ cell viability in a time- and dose-dependent manner. After 72 h incubation, the IC50, half maximal inhibitory concentration, was 2.3 µM for melanoma cells and 7.4 µM for normal fibroblasts. In turn, rosuvastatin exhibited relatively lower activity against WM1552C cells. A significant reduction of Ki67 expression was also noted for BJ fibroblasts after prolonged incubation with the tested drug. CONCLUSIONS: The results indicate that the anti-melanoma properties of rosuvastatin are highly dependent on the tumor cell line assessed. However, the concentrations required to decrease melanoma cell viability in vitro exceed the plasma concentrations reached in patients treated with rosuvastatin at well-tolerated doses. What is more disturbing, reduction of proliferation and viability observed in BJ fibroblasts indicated that rosuvastatin at high doses may be toxic for normal cells.

Analysis of skin patch test results and metalloproteinase-2 levels in a patient with contact dermatitis.

INTRODUCTION: The complex course of skin reactions that contact eczema involves is due in part to abnormalities of the extracellular matrix function. Proteins that degrade extracellular matrix components include metalloproteinases (MMP), which are divided into subcategories depending on the chemical structure and substrate specificity. AIM: To analyse patch test results in contact dermatitis patients and to assess MMP-2 levels during skin lesion exacerbation and remission. MATERIAL AND METHODS: Fifty patients suffering from contact eczema were qualified to the study and 20 healthy volunteers as a control group. The study group patients had epidermal skin tests performed with the "European Standard" set. To assess the MMP-2 level in serum, venous blood was drawn, twice from study group patients - during contact dermatitis exacerbation and remission periods - and once from control group patients. Assessment of MMP-2 in serum was done with ELISA immunoassay. To verify the proposed hypotheses, parametric and nonparametric significance tests were used. RESULTS: Hands were the most frequent location of contact dermatitis. Nickel (II) sulphate was the most frequent sensitizing substance. Mean MMP-2 levels were statistically higher in the study group both in contact dermatitis exacerbation and remission periods than in the control group. There was no statistically significant difference between MMP-2 levels and skin patch test results. CONCLUSIONS: Nickel is one of the most allergenic contact allergens in patients with contact dermatitis. Metalloproteinase-2 is a good marker of contact dermatitis in various stages of the disease.

Lysosomal enzymes in serum and synovial fluid in patients with osteoarthritis.

OBJECTIVE: To assess the activity of arylsulfatase (AS), acid phosphatase (ACP), cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood serum and synovial fluid (SF) of patients with hip or knee osteoarthritis (OA). METHODS: The study included 43 subjects with OA (35 hip OA, 8 knee OA), submitted total joint replacement. The control group consisted of 58 subjects with no past history of musculoskeletal disorders. RESULTS: The OA blood serum samples showed a significantly higher level of lysosomal enzymes activity than in the control group (AS by 17.8%, AAT by 42.4%); only the CAT-D activity decreased by 50%). AS, ACP and CAT-D activities were about two-fold higher in SF when compared with blood of OA patients. The differences between the genders were visible in the SF: Total protein concentration, activity of ACP (both higher in OA men) and activity of CAT-D (higher in OA women). Between the involved hip and knee, there were no significant differences in all estimated parameters in the blood serum of the OA group. In regard to the SF, only ACP activity was significantly higher in patients with a hip involved. CONCLUSIONS: The osteoarthritic SF enzymatic profile differs from that in normal joints. The OA in joints is not reflected in the systemic response. Our preliminary results suggest further studies on role of lysosomal enzymes (ACP and AS) as biomarkers for the detection of osteoarthritis.

The activity of cathepsin D and alpha-1 antitrypsin in hip and knee osteoarthritis.

The progress of cartilage decay during joint degeneration is not well monitored with biochemical methods. The role of cathepsin D (CAT-D) in articular cartilage deterioration remains unclear. The aim of this study is to assess the activity of CAT-D and alpha-1 antitrypsin (AAT) in blood in patients with hip or knee osteoarthritis. The activity of CAT-D and AAT in blood serum of 40 women and 21 men with hip or knee osteoarthritis was determined before total joint replacement, on the tenth day after surgery, and once in 54 healthy patients. The preoperative activity of CAT-D in patients with osteoarthritis was lower by 53.6% (11.00 ± 4.54 10(-2) nM released tyrosine/mg protein/min, P < 0.001) and after surgery by 55.0% (10.67 ± 4.64 10(-2) nM released tyrosine/mg protein/min, P < 0.001) when compared to its activity in healthy patients. There was no significant statistical difference between CAT-D activity before the surgery and its activity on the tenth day after it in the analyzed group (P< 0.496). Simultaneously, the preoperative activity of AAT in the OA (osteoarthritis) patients was by 25.5% (0.93 ± 0.32 mg inhibited trypsin/ml blood serum, P < 0.001) and postoperative was by 44.9% higher (1.26 ± 0.36 mg inhibited trypsin/ml blood serum, P < 0.001) than in healthy patients. The low CAT-D activity in osteoarthritis of big joints is associated with a decrease of cartilage cells during the degenerative process. The higher activity of acute phase protein AAT in OA patients' blood serum confirms the inflammatory component in the osteoarthritis process.

The concentration of thiobarbituric acid reactive substances (TBARS) and paraoxonase activity in blood of patients with osteoarthrosis after endoprosthesis implantation.

BACKGROUND: The aim of this study was to evaluate the concentration of malondialdehyde (MDA) in erythrocytes and in blood plasma and the activity of blood paraoxonase (PON1) of patients with osteoarthrosis (OA) submitted to endoprosthesis implantation for evaluating oxidative stress. MATERIAL/METHODS: Study was conducted on 55 patients with OA and on 54 total movement-efficient volunteers. The material for the study was venous blood plasma, serum and erythrocytes. RESULTS: Increased concentration of MDAe before surgery was observed in the group of men and in patients with a degenerative process affecting hip joints. After an implantation of endoprosthesis, MDAe decreased to the level observed in the control groups. In the study group MDA concentration in plasma was slightly lower before surgery, and after an operation it reached the value of the parameter of the reference groups. Regardless of sex or age, paraoxonase activity was almost twice as high in almost all subgroups as in the reference group. A positive correlation between PON 1 activity and MDAe concentration was demonstrated both before and after surgery in the group of men. CONCLUSIONS: The increase of PON1 activity in patients' serum in relation to the control groups indicates a probable pathogenic role of the increased formation of reactive oxygen species in the course of OA and may suggest acute inflammation of the synovial joint. The high level of PON 1 activity after endoprosthesis implantation indicates that surgical treatment may additionally stimulate ROS generation. MDAe concentration indicate more intensive process of lipid peroxidation in the elderly.

Autologous cultured melanocytes in vitiligo treatment.

BACKGROUND: Surgical treatment of vitiligo is indicated when lesions are localized in poorly responding areas. OBJECTIVES: The objectives were: (1) to establish the melanocyte culture obtained from the epidermis of vitiligo patients for future treatment; (2) to estimate the influence of selected factors on the formation of suction blisters and the results of culture; and (3) to compare the results of treatment of vitiliginous macules localized in the dorsum of the hands and lower limbs by transplantation of cultured autologous melanocytes plus psoralen and ultraviolet A (PUVA) therapy (CMP), suction blister transplantation plus PUVA therapy (SBP), cryotherapy plus PUVA-therapy (CP), and only PUVA therapy (OP). METHODS: Forty patients were qualified for the study. The roofs of the suction blisters were used as a melanocyte source for culture establishment or were directly transplanted. RESULTS: The CMP procedure was successfully performed on only 10 of 20 patients because of the difficulties in cell culture establishment. The SBP method was carried out on all 20 patients. A total lack of effectiveness was found in CP and OP methods. CONCLUSIONS: The effectiveness of culture depends on time of suction blister forming, phototype, and previous PUVA therapy. This study demonstrated the advantage of the SBP over the CMP method.

Establishing melanocyte cultures in a serum-free system for transplantation in vitiligo patients.

BACKGROUND: The aim of the study was to establish melanocyte cultures obtained from the epidermis of vitiligo patients for future treatment and to estimate the influence of selected factors on the formation of suction blisters and the results of culture. MATERIAL/METHODS: Twenty patients with generalized vitiligo were qualified to the study. The melanocyte donor site was the arm or forearm where, using an electric vacuum suction machine and a plastic plate, suction blisters were formed. The removed roofs of the blisters were used as a melanocyte source for culture establishment. After an enzymatic bath, the cell suspension was transferred to culture flasks containing melanocyte growth medium (MGM M2). The melanocytes in culture were identified by immunocytochemistry. RESULTS: Suction blisters were formed in all patients. No melanocyte cultures were established from patients whose blisters appeared over a period 58 minutes. In addition, a suitable quantity of melanocytes was not obtained for eight other patients (with skin phototype II and/or without previous PUVA therapy). CONCLUSIONS: The epidermis of the arm and forearm is a good donor site for the establishment of melanocyte cultures. The effectiveness of culture depends mainly on the time of the formation of suction blisters, the phototype of the skin, and previous PUVA therapy.

Activity of superoxide dismutase and catalase and the level of lipid peroxidation products reactive with TBA in patients with psoriasis.

BACKGROUND: The etiology of psoriasis, one of the most common chronic dermatoses, has not been elucidated yet. Involvement of antioxidant enzymes is suspected. The study aimed to determine the concentrations of lipid peroxidation products reactive with thiobarbituric acid and antioxidant enzymes activity. MATERIAL/METHODS: 67 patients of the Chair and Clinic of Dermatology (22 females and 45 males) with psoriasis vulgaris of 3 to 34 years' duration were examined. All were treated topically with preparations for external use. Smooth skin was treated with salicyl ointment, cignoline and tar, while salicyl oil and betamethasone dipropionide with salicylic acid was used on the scalp. Lipid peroxidation products reactive with TBA, expressed as malondialdehyde (MDA) concentration, were determined in erythrocytes using Buege and Aust method. Activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were determined in erythrocytes according to Misra and Fridovich, and Beers and Sizer, respectively. RESULTS: In healthy subjects, SOD and CAT activity was 1049.2I261.9 U/gHb and 41.4I13.7 i 104 IU/gHb, respectively, and MDA concentration 35.8I10.5 nM/gHb. MDA concentrations in erythrocytes of psoriatic patients before treatment were 44% higher and SOD and CAT activities 20% and 27% lower, respectively, than those observed in the controls. Topical treatment improved the patients' clinical condition, resulting in slow increase of enzyme activity to values comparable with controls. CONCLUSIONS: The results confirm the involvement of antioxidant enzymes in the pathogenesis of psoriasis. It is, however, questionable whether the observed abnormalities are responsible for the onset of psoriasis, or resultant from ongoing pathologic process.