Differential expression of inflammatory cytokines and chemokines in lipopolysaccharide-stimulated melanocytes from lightly and darkly pigmented skin.

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA-binding activity of NF-κB p50 and p65. We found substantial differences in the LPS-stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL-16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL-6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.

Evaluation of melanogenesis in A-375 melanoma cells treated with 5,7-dimethoxycoumarin and valproic acid.

Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10-150 µM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.

Evaluation of melanogenesis in A-375 cells in the presence of DMSO and analysis of pyrolytic profile of isolated melanin.

The increase of a skin malignant melanoma (melanoma malignum) incidence in the world has been observed in recent years. The tumour, especially in advanced stadium with metastases, is highly resistant to conventional treatment. One of the strategies is to modulate melanogenesis using chemical compounds. In this study, the processes of differentiation and melanogenesis induced by dimethylsulfoxide (DMSO) in human melanoma cells (A-375) were investigated. Natural melanin isolated from A-375 melanoma cell line treated with 0.3% DMSO was analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) method. The products derived from pheomelanin have not been stated in the pyrolytic profile of analyzed melanin. Within all products derived from eumelanins, 1,2-benzenediol has been predominated. It has been shown that in the melanoma cells stimulated with 0.3% and 1% DMSO, the increase of transcriptional activity of the tyrosinase gene took place. It was accompanied by the rise of tyrosinase activity and an accumulation of melanin in the cells. The better knowledge about the structure of melanins can contribute to establish the uniform criteria of malignant melanoma morbidity risk.

Detection and quantitation of a pheomelanin component in melanin pigments using pyrolysis-gas chromatography/tandem mass spectrometry system with multiple reaction monitoring mode.

Here, we describe the reliable method for the detection and quantitation of a pheomelanin component in melanin pigments. Synthetic melanins with various contents of pheomelanin-type structural units were thermally degraded, and the multiple reaction monitoring mode was applied to detect the pheomelanin markers in the pyrolysates by GC/MS/MS. The method allowed the specific detection and quantitation of a pheomelanin component in melanin with the incorporation of pheomelanin-type units as low as 0.05%. Considering highly universal character of the pheomelanin markers, the method could be applied for structural studies of natural melanin pigments being mixtures of eumelanin and pheomelanin.

Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide.

A large body of evidence suggests that epidermal melanocytes are an integral part of the skin immune system and can be considered immunocompetent cells. Recently, it has been reported that human melanocytes constitutively express Toll-like receptors and may be involved in the induction of several inflammatory cytokines. In the study the secretion of IL-1ß, IL-6 and TNF-α by cultured normal melanocytes was investigated after stimulation with lipopolysaccharide. LPS increased the secretion of IL-1ß in a dose-dependent manner. IL-1ß stimulated release of IL-6 and TNF-α by melanocytes, whereas LPS activated production of TNF-α, but not of IL-6. These observations indicate that LPS can participate in the regulation of cytokine activity in normal human melanocytes and suggest that cytokines released by melanocytes could affect melanocytes themselves or/and other cells of the epidermis.

Lack of effect of the CD14 promoter gene C-159T polymorphism on nutritional status parameters in hemodialysis patients.

BACKGROUND: CD14 is a membrane glycoprotein that acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS). Mutual interaction between CD14 and LPS plays an important role in the innate immune system. Increased serum soluble CD14 levels have been described in hemodialysis (HD) patients, and linked to increased mortality risk, inflammation and protein-energy wasting. The expression of CD14 may be influenced by CD14 promoter gene C-159T polymorphism. This study aimed to clarify the possible association between CD14 promoter gene C-159T polymorphism and nutritional status in hemodialysis patients. MATERIAL/METHODS: The study population consisted of 185 (104 males; 81 females) long-term HD patients treated in 5 dialysis centers. The control group consisted of 112 apparently healthy volunteers (32 males and 80 females). Nutritional status was assessed using a modified SGA scale, and anthropometric methods (BMI, WHR, waist, hip and mid-arm circumferences, biceps, triceps, subocular and subscapular skinfolds). Biochemical parameters evaluated included: CRP, albumin, creatinine, urea, cholesterol, triglycerides and TIBC. CD14 promoter gene C-159T polymorphism was determined by restriction fragment length polymorphism, after digestion of the PCR product with Hae III restriction endonuclease. RESULTS: Genotype and allele frequencies were similar to controls and compliant with Hardy-Weinberg equilibrium. No between-group differences were detected in measured variables with the exception of lower triglyceride levels in carriers of C allele in comparison to TT genotype. CONCLUSIONS: CD14 promoter gene C-159T polymorphism does not seem to be associated with nutritional status parameters in HD patients. It does seem, however, to influence triglyceride blood levels.

Occupational exposure to phenolic compounds at coke plants--urinary excretion of methoxyphenols as an indicator of exposure to methoxyphenols.

OBJECTIVES: This study describes the exposure of coke plant workers to methoxyphenols. The relationship between exposure to methoxyphenols and urinary excretion of metabolites was examined. METHODS: We determined concentrations of 2-methoxyphenol, 2-methoxy-4-methylphenol and 1-(4-hydroxy-3-methoxyphenyl)ethanone in the breathing-zone air and in the urine of workers, collected after the workshift. Urine metabolites were extracted after enzymatic hydrolysis by solid-phase extraction. Concentrations of methoxyphenols in air and urine were determined by gas chromatography with flame-ionization. RESULTS: The time-weighted average concentrations (median) of methoxyphenols in the breathing zone air were as follows: 9.9 ng/m(3), 15.4 ng/m(3) and 92.5 ng/m(3) for 2-methoxyphenol, 2-methoxy-4-methylphenol and 1-(4-hydroxy-3-methoxyphenyl)ethanone, respectively. The median values of urinary concentrations were as follows: 582.5, 190.1, 235.0 and 21.8 µmol/mol creatinine for 2-methoxyphenol, 2-methoxy-4 methylphenol, 1-(4-hydroxy-3-methoxyphenyl)ethanone and 2,6-dimethoxyphenol, respectively. A statistically significant correlation between the exposure level and the urinary level was found for 2-methoxyphenol (r=0.573, p<0.01). CONCLUSION: We found that the presence of 2-methoxyphenol in urine can be used as a biomarker for 2-methoxyphenol exposure. The analysis performed at the coke plant showed that the workers were exposed to relatively low concentrations of methoxyphenols.

[The role of melanocytes in protection against photooxidative stress Krystyna Stepien]. / Udzial melanocytów w ochronie przed stresem fotooksydacyjnym.

Excessive exposure to solar ultraviolet radiation is an essential etiological factor for skin cancer. UV radiation, directly or indirectly through the generation of reactive oxygen species (ROS), causes damage to DNA, proteins and lipids, and induces inflammation and immunosuppression. Cutaneous pigmentation afforded by melanocytes is the main photoprotective mechanism in human skin. In response to UV, melanocytes produce melanin pigments and transfer them to adjacent keratinocytes. This review describes: (i) the photoprotective action of melanin; (ii) the regulation of UV-induced melanogenesis and the role of p53 in this process; (iii) the relation between melanogenic and antioxidant activities in melanocytes. The possible involvement of UV-induced ROS in the stimulation of melanin synthesis is also discussed.

Serum concentrations of formation (PINP) and resorption (Ctx) bone turnover markers in rheumatoid arthritis.

Joint inflammation in rheumatoid arthritis (RA) induces local periarticular osteoporosis. Generalised bone mineral density (BMD) decrease concerns approximately 50% of rheumatic patients. Both types of bone mass depletion can issue from cytokine-induced (TNF-alpha, IL-1, IL-6) osteoclasts' activation, osteoprotegerin and its ligand's (RANKL) function disorders, patients' immobilisation and glucocorticosteroid (GCS) intake, as well as from hormonal alterations in postmenopausal women, predominate among RA individuals. The aim of the study was to compare serum concentrations of marker of bone formation--serum aminoterminal propeptide of type I collagen (PINP), and bone resorption, carboxy (C) terminal telopeptide (Ctx), bone turnover markers in RA and osteoarthritis (OA) patients and in RA groups of different disease activity, different degree of joint damage and the history of GCS intake. A total of 50 RA female patients and 50 women with knee OA were included in the study. Blood for morphology and biochemistry laboratory tests was taken. Joint X-rays to establish OA and RA diagnosis and the degree of RA progression, as well as DEXA BMD measurements were performed. PINP and Ctx concentrations were assessed. In RA patients the number of swollen and painful joints, the duration of morning stiffness, visual analogue scale values and Waaler-Rose's test activity were recorded. The Disease Activity Index (DAS 28) was counted from the appropriate formula. No differences in bone turnover markers' concentrations were noted neither between RA and OA patients nor between the RA group when compared to the one without the history of GCS use. Bone turnover markers' concentrations in RA were proportional to the number of swollen and painful joints. However, no correlation was found between the markers' concentrations and RA activity assessed by DAS 28 or by laboratory means. Ctx concentrations were higher in patients at II degree joint damage according to Larsen and Dale's than at more advanced stages. Ctx concentrations decreased with the disease duration. Serum morphogenesis and resorption markers' concentrations change in course of RA indicating the decrease in bone metabolic activity with the disease duration and progression. High RA activity and severity correlate with increased markers' levels-the resorption one. The influence of GCS on bone metabolism in RA requires further study.

Melanin from epidermal human melanocytes: study by pyrolytic GC/MS.

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.

Thermochemolysis as the useful method to assess the purity of melanin isolated from the human melanoma malignum.

Melanin formation in pigmented melanoma cells is considered as a target for the tumor therapy. The evaluation of potential correlation between melanin structure and the tumor type could be also of diagnostic and prognostic importance. One of the major problems in structural investigations of natural melanins is the lack of appropriate methods, which allow isolation of pure intact pigment. In this study the thermochemolysis technique was used to assess the purification grade of melanin isolated from the human melanoma malignum cells by two different enzymatic methods. Melanin samples were thermally degraded in the presence of tetramethylammonium hydroxide and the thermochemolysis products were analyzed by gas chromatography/mass spectrometry (GC/MS). Compounds of lipid origin, especially fatty acid methyl esters and aliphatic and cyclic hydrocarbons, were predominant among pyrolysis products of melanin isolated from the tumor cells by method I. In contrast, during thermochemolysis of the pigment sample isolated by the method II, mainly eumelanin markers (pyrrole and its methyl derivatives, toluene, styrene, phenol, benzyl nitrile and indole) were formed. The comparison of pyrolysis profiles of the analyzed samples indicate that method II is more efficient for melanoma pigment purification.

Diagnostic imaging approach to gastro-entero-pancreatic carcinomas of neuroendocrine origin - single NET center experience in Poland.

AIM: The aim of the study was to review the current diagnostic approach, based on the experience of one center performed during a 4-year period, according to WHO criteria of GEP - NET. MATERIAL AND METHODS: The study group comprised case records of 134 patients with confirmed GEP-NET carcinomas (WHO groups 2-4). All patients were subjected to clinical, biochemical and imaging examinations performed as routine clinical work-up. The imaging techniques consisted of anatomical (CT, EUS) and functional approaches (SRS, mIBG and FDG PET). RESULTS: The clinical classification considered the primary origin of the tumor as follows: 49% - foregut tumors, 44% - midgut, and 7% of tumors of unknown origin. Group of patients with WHO 2 consisted of 98 (73%) subjects. Considering those with foregut tumors EUS followed by CT and SRS were used in each case. SRS and CT imaging was used to assess the extent of the tumor. Patients with midgut tumors had CT and SRS as routine diagnostic imaging examinations. Considering the above-mentioned patients, CT and SRS were used to localize the primary tumor, and assess tumor extent. Overall sensitivity of CT considering the active disease amounted to 96%, while specificity - 75%. Sensitivity of SRS was 97%, while specificity- 85%. WHO 3-29 patients, 17 foregut and 9 midgut tumors, and 3 of unknown origin. Diagnostic imaging examinations consisted of CT. Standard SRS (sst2) was negative in most cases. The aggressive behaviour of this type of tumors was detected by means of FDG-PET. Sensitivity of CT amounted to 100%, and that of SRS - 44%. Specificity of CT amounted to 67% and that of SRS - 100%. WHO 4-7 patients, including 4 foregut and 3 midgut neoplasms. The imaging approach consisted of CT/MR and in 5 cases FDG-PET. Sensitivity of CT amounted to 100%. Only one patient presented with a SRS positive study. FDG-PET sensitivity amounted to 100%. CONCLUSION: Diagnostic imaging of GEP-NET, consider anatomical and functional techniques, which should be read together. The diagnostic value of CT and SRS were similar in case of WHO 2, while in case of WHO 3 CT had a higher diagnostic accuracy. FDG-PET seems to be a very attractive imaging functional modality in case of patients with WHO 3 and WHO 4.

Serum leptin in rheumatoid arthritis.

Leptin is a peptide hormone that has an essential role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. The role of leptin in the modulation of the immune response and inflammation has been regarded as important. In rheumatoid arthritis (RA) patients it was reported that fasting leads to an improvement of clinical and biological measures of disease activity, which was associated with a marked decrease in serum leptin. These features suggest that leptin may also influence the inflammatory mechanisms of arthritis in humans. In this study we assessed serum leptin levels in RA and osteoarthritis (OA) patients and found a correlation between serum leptin level and other markers as well as bone mass density changes, activity of disease, disease duration and the age of the patients. The blood was collected from 30 RA and 30 OA patients who constituted the control group. Serum leptin level was determined using the DRG Leptin ELISA Kit-a solid phase enzyme-linked immunosorbent assay based on the sandwich principle. The serum level of leptin in RA patients ranged from 1.8 to 81.1 ng/ml and median value was 11.2. There was a positive correlation between body mass index (BMI) of RA patients and serum level of leptin (correlation coefficients Spearman's r = 0.81). According to correlation coefficients, serum leptin level is independent of age of RA patients, stage of disease, number of painful and swollen joints, duration of morning stiffness, disease duration as well as value of titre of the Waaler-Rose, disease activity score (DAS 28) value and presence of rheumatoid nodules. There was a negative correlation between serum leptin level and glomerular filtration rate (GFR). No correlation between the serum leptin level and T-score was found. An influence of steroid treatment on the serum leptin level was not shown. The median serum leptin level in OA patients was 9.2 ng/ml. There was a positive correlation between body mass index of OA patients and serum level of leptin (correlation coefficients Spearman's r = 0.57). No correlation was found between serum leptin level and patient's age, duration of disease and value of laboratory data. There were no correlations between serum leptin level and visual analogue pain scale (VAS) for the lower-limb afflicted patients as well as stage of disease according to Kellgren and Lawrence's score in OA patients. There was a negative correlation between serum leptin level and T-score value in OA patients (r = -0.58, P < 0.05). No statistically significant differences were found between serum leptin levels for RA and OA patients.

[The role of neuromelanin in Parkinson's disease--new concepts]. / Rola neuromelaniny w chorobie Parkinsona--nowe koncepcje.

Progressive degeneration of dopaminergic neurons of the substantia nigra and the resulting dopamine deficiency in the striatum are neuropathological basis of the movement disorders in Parkinson's disease (PD). Neuromelanin-containing neurons are particularly susceptible to degeneration and their depigmentation is the hallmark of the advanced disease. The proposed mechanisms underlaying the pathogenesis and progression of neurodegeneration in the substantia nigra include iron-catalyzed oxidative stress, mitochondrial dysfunctions, inflammation and disturbances of protein metabolism. This review presents some new concepts concerning important but ambiguous role of neuromelanin in the above mentioned processes. It seems that the imbalance between cytoprotective and cytotoxic action of the pigment may cause neuronal death via mitochondrial oxidative stress, inhibition of ubiquitine-proteasome system and alpha-synuclein accumulation. Extraneuronal melanin may contribute to chronic inflammation by excessive secretion of cytokines and nitric oxide due to prolonged microglia activation. Recent reports suggest a possible role of the lipid component of neuromelanin in pigment-dependent cytotoxicity.

[The orthopedic philosophy of Professor Donat Tylman in the development of research on the pathomechanics, diagnostics, and treatment of scoliosis]. / Mysl ortopedyczna Profesora Donata Tylmana w rozwoju badan nad patomechanika, diagnostyka i leczeniem skolioz.

This article describes the innovative elements in the research activity of Prof. Donat Tylman, in terms of research philosophy, staging, and management in the etiology, pathomechanics and treatment of lateral curvature of the spine. Prof. Tylman's achievements are presented chronologically, following the course of numerous significant publications, which provide the basis for further work.

[Hypersensitivity to poppy seeds]. / Nadwrazliwosc na nasiona maku.

UNLABELLED: Hypersensitivity to poppy seeds is rare and it may develop due to immune (allergy) and non-immune reactions of fulminant course. Two cases of hypersensitivity to poppy seeds are presented: in a 21-year-old woman and 32-year-old man in whom life-threatening symptoms and signs of anaphylaxis (SSA) developed after consumption of poppy seeds in various situations, e.g in the street after consumption of a roll with poppy seeds or a cake prepared on a moulding board on which poppy seeds were previously squeezed. In the case of the woman, history data pointed to familial and individual occurrence of atopy, and positive cutaneous prick tests with allergens of grass/crop and weed pollen and tree pollen (birch, hazel) as well as hazelnuts. In the man the anamnesis and skin prick tests were negative. Serum concentrations of allergen-specific IgE (asIgE) for poppy seeds and, additionally, for food allergens, which, according to patients, also could be the cause of SSA: celery, pineapple, mixture of spices and nuts were determined. In the woman elevated concentrations of asIgE for celery (class 2) and nut mixture (class 2) with negative result for poppy were noted, whereas in the man elevated concentrations of asIgE for poppy (class 2) and nut mixture (class 2) were found. Additionally, in the male patient skin tests were performed with topical anesthetics and narcotic analgesics derived from poppy (morphine, codeine) finding positive reactions to xylocaine and codeine. CONCLUSIONS: The two presented cases illustrate that hypersensitivity to poppy seeds may occur in IgE-dependent or non-immune mechanisms and the presence of atopy is not decisive of its mechanism.

GC/MS analysis of thermally degraded neuromelanin from the human substantia nigra.

Neuromelanin (NM) is a complex polymer pigment found in catecholaminergic neurons of the human brain. The structure, formation pathway, and physiological function of NM have not yet been clarified, but interest in this polymer has been sparked by the suggestion that NM is involved in cell death in Parkinson's disease. In the current study, pyrolysis-gas chromatography/mass spectrometry analysis was applied for structural investigation of NM isolated from the human substantia nigra, using synthetic eumelanin and pheomelanin-type pigments as reference materials. None of the heterocyclic, sulfur-containing compounds being characteristic thermal degradation products of cysteinyldopamine-derived units of synthetic pheomelanin standard was detected in the pyrolysates of natural NM. The results suggest that nigral pigment isolated from normal brain tissue does not contain benzothiazine-type monomer units. Pyrolytic experiments in the presence of a derivatizing agent allowed identification of high levels of saturated and monounsaturated straight-chain C14-C18 fatty acids and led to the conclusion that a part of a lipid component is chemically bound to the NM macromolecule. The nigral pigment was also shown to be tightly associated with an isoprenoid-type compound.

Plasma homocysteine level and the course of ischemic stroke.

Increased level of homocysteine (Hcy) in blood seems to influence negatively the course of ischemic stroke (IS), the possible mechanism of this action could be acceleration of oxidative stress. The aim of this study is to assess the influence of Hcy level in patients with IS on the prognosis 3 months after the stroke onset. 75 patients aged 68.27 +/- 12.62 years, with the diagnosis of first ever IS were examined. Patients with the symptoms corresponding with TACS at the beginning of stroke and with diminished level of consciousness were not included. The level of Hcy over 15 micromol/l was assessed as mild hiperhomocysteinemia (MHcy). 74 (98.7%) patients were assessed 3 months after IS onset in the Rankin scale. Recovery was assessed, according to Rankin Scale: good recovery (GR) 0-2, bad recovery (BR) 3-5 and death. MHcy was seen in 9 (14.5%) with GR and in 8 (66.7%) with BR (P = 0.0005). MHcy increases the risk of BR 11.78 times (95% CI 2.93-47.42).