Optical constants and absorption properties of Te and TeO thin films in the 13-14†nm spectral range.

Undesired mask-induced effects caused by thick absorber layers in EUV photomasks reduce the quality of the projected patterns at the wafer stage in EUV photolithography scanners. New materials with better absorption properties than the state-of-the-art absorbers, TaN and TaBN, are required to mitigate these effects. In this work, we investigated the optical properties (δ and k) of Te and TeO films in the 13-14 nm range, and the absorption properties of these two materials at 13.5 nm. δ and k are obtained through fitting experimental values of reflectivity versus angle of incidence in the EUV range. We follow a methodology which combines different characterization techniques (X-ray reflectivity, EUV reflectivity, and X-ray photoemission spectroscopy) to reduce the number of free parameters in models and hence, increase the reliability of the optical constants obtained. At 13.5 nm, we obtain δ=0.03120, k = 0.07338 for Te, and δ=0.04099, k = 0.06555 for TeO. To experimentally verify the absorption properties of these materials, different thicknesses of Te and TeO films are cast on top of a state-of-the-art mask-quality EUV multilayer with 66.7% reflectivity at 13.5 nm. We found that a reflectivity of ∼0.7% can be attained with either 32.4 nm of Te, or 34.7 nm of TeO, greatly surpassing the absorption properties of TaN and TaBN. The morphology and surface roughness of the Te and TeO films deposited on the multilayer are also investigated.

Very efficient spin polarization analysis (VESPA): new exchange scattering-based setup for spin-resolved ARPES at APE-NFFA beamline at Elettra.

Complete photoemission experiments, enabling measurement of the full quantum set of the photoelectron final state, are in high demand for studying materials and nanostructures whose properties are determined by strong electron and spin correlations. Here the implementation of the new spin polarimeter VESPA (Very Efficient Spin Polarization Analysis) at the APE-NFFA beamline at Elettra is reported, which is based on the exchange coupling between the photoelectron spin and a ferromagnetic surface in a reflectometry setup. The system was designed to be integrated with a dedicated Scienta-Omicron DA30 electron energy analyzer allowing for two simultaneous reflectometry measurements, along perpendicular axes, that, after magnetization switching of the two targets, allow the three-dimensional vectorial reconstruction of the spin polarization to be performed while operating the DA30 in high-resolution mode. VESPA represents the very first installation for spin-resolved ARPES (SPARPES) at the Elettra synchrotron in Trieste, and is being heavily exploited by SPARPES users since autumn 2015.

Manipulating the Topological Interface by Molecular Adsorbates: Adsorption of Co-Phthalocyanine on Bi2Se3.

Topological insulators are a promising class of materials for applications in the field of spintronics. New perspectives in this field can arise from interfacing metal-organic molecules with the topological insulator spin-momentum locked surface states, which can be perturbed enhancing or suppressing spintronics-relevant properties such as spin coherence. Here we show results from an angle-resolved photemission spectroscopy (ARPES) and scanning tunnelling microscopy (STM) study of the prototypical cobalt phthalocyanine (CoPc)/Bi2Se3 interface. We demonstrate that that the hybrid interface can act on the topological protection of the surface and bury the Dirac cone below the first quintuple layer.

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

Immortalization of human melanocytes does not alter the de novo properties of nitric oxide to induce cell detachment from extracellular matrix components via cGMP.

Nitric oxide (NO) is an important mediator in many (patho)physiological processes including inflammation and skin cancer. A key transducer in NO signaling is the soluble guanylyl cyclase (sGC) that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP). The basic mechanism of NO-cGMP signaling in melanocytic cells is, however, not well elucidated. A setback for such studies is the limited availability of patient-derived melanocytes. Here, we report that immortalized human normal and vitiliginous cell lines generated via cell transfection with human papilloma virus 16 genes E6 and E7 express NO synthase and guanylyl cyclase isoforms and the multidrug resistance-associated proteins 4 and 5 as selective cGMP exporters. Donors of NO (e.g., the NONOate (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NO) and reactive nitrogen oxygen species (RNOS) like 3-morpholino-sydnonimine (SIN-1) as a donor of peroxynitrite as well as YC-1 as a NO-independent sGC stimulator increased intracellular cGMP levels in immortalized melanocytes (up to eightfold over controls), indicating the expression of functional sGC in these cells. PAPA-NO and SIN-1 also reduced the attachment of immortalized melanocytes to extracellular matrix (ECM) components like fibronectin which was dependent on cellular melanin content and cGMP. Such effects on melanoma cells were positively related to metastatic potential and were cGMP independent. Intriguingly, nonpigmented metastatic melanoma cells were more sensitive to exogenous sources of RNOS than of NO. Thus, immortalized melanocytes can be used as a tool for further research on differences in cell signaling between the different melanocytic lineages in particular towards impairment of cell-ECM adhesion by NO or RNOS, which may be important in metastasis and vitiligo pathogenesis.

Switching from Reactant to Substrate Engineering in the Selective Synthesis of Graphene Nanoribbons.

The challenge of synthesizing graphene nanoribbons (GNRs) with atomic precision is currently being pursued along a one-way road, based on the synthesis of adequate molecular precursors that react in predefined ways through self-assembly processes. The synthetic options for GNR generation would multiply by adding a new direction to this readily successful approach, especially if both of them can be combined. We show here how GNR synthesis can be guided by an adequately nanotemplated substrate instead of by the traditionally designed reactants. The structural atomic precision, unachievable to date through top-down methods, is preserved by the self-assembly process. This new strategy's proof-of-concept compares experiments using 4,4''-dibromo-para-terphenyl as a molecular precursor on flat Au(111) and stepped Au(322) substrates. As opposed to the former, the periodic steps of the latter drive the selective synthesis of 6 atom-wide armchair GNRs, whose electronic properties have been further characterized in detail by scanning tunneling spectroscopy, angle resolved photoemission, and density functional theory calculations.

Leprosy-specific B-cells within cellular infiltrates in active leprosy lesions.

Leprosy is a spectral disease with polar lepromatous and tuberculoid forms correlating with enhanced humoral and cell-mediated immunity, respectively, against Mycobacterium leprae and the borderline forms, borderline lepromatous, midborderline, and borderline tuberculoid showing in-between clinical and immunological characteristics. Histopathologically, the cellular infiltrates of leprosy lesions show predominantly the presence of interacting T-cells and antigen presenting cells like macrophages, whereas the presence of B-cells has only been sporadically reported. The present study demonstrates by immunohistochemical techniques the presence of B-cells, including plasma cells, in active lesions from lepromatous leprosy, skin smear negative borderline lepromatous, and paucibacillary borderline tuberculoid leprosy. Furthermore, the study demonstrates the in situ production of M leprae-specific antibodies from BT lesions using an organotypic skin explant culture model. Finally, analysis of the cytokine release profile in supernatants of lesional organotypic skin cultures showed a microenvironment conducive to the differentiation and maturation of B-cells. The results demonstrate the presence of different functionally active B-cell stages within lesions of patients with leprosy, including borderline tuberculoid patients, which could secrete anti-M leprae-specific antibodies. However, their role in leprosy pathology remains to be elucidated.

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

Therapeutic implications of autoimmune vitiligo T cells.

Vitiligo is an autoimmune disease presenting with progressive loss of skin pigmentation. The disease strikes 1% of the world population, generally during teenage years. The progressive loss of melanocytes from depigmenting vitiligo skin is accompanied by cellular infiltrates containing both CD4+ and CD8+ T lymphocytes. Infiltrating cytotoxic T cells with high affinity T cell receptors have likely escaped clonal deletion in the thymus, allowing such T cells to enter the circulation. Through the expression of CLA, these T cells home to the skin where they express type 1-cytokine profiles and mediate melanocyte apoptosis via the granzyme/perforin pathway. T cells found juxtapositionally apposed to remaining melanocytes can be isolated from the skin. Vitiligo T cells have demonstrated reactivity to antigens previously recognized as target antigens for T cells infiltrating melanoma tumors. In a comparison to existing melanoma-derived T cells, vitiligo T cells displayed superior reactivity towards melanoma cells. It is thought that genes encoding the TCRs expressed by vitiligo skin infiltrating T cells can be cloned and expressed in melanoma T cells, thereby generating a pool of circulating T cells with high affinity for their targets that can re-direct the immune response towards the tumor.

Accumulation of low-avidity anti-melanocortin receptor 1 (anti-MC1R) CD8+ T cells in the lesional skin of a patient with melanoma-related depigmentation.

Spontaneous or therapy-induced depigmentation in patients with melanoma has long been considered a favourable prognostic indicator. In this report, we isolated T cells infiltrating the depigmented skin of an HLA-A2+/DR4+ patient with melanoma, and detected a very high frequency of CD8+ T cells specific for melanocortin receptor 1 (MC1R), a hormone receptor involved in cutaneous pigmentation. In particular, tissue-infiltrating CD8+ T cells dominantly recognized the novel MC1R52-60 peptide epitope in an HLA-A2-restricted manner, and peptide-reactive CD8+ T cells were also detected in freshly isolated peripheral blood from this patient. Although type 1 CD4+ T-cell responses against MC1R were not detected in fresh tissue isolates, short-term in-vitro stimulation of peripheral blood lymphocytes resulted in the rapid expansion of CD4+ T cells reactive against novel HLA-DR4-presented epitopes derived from the MC1R protein (i.e. MC1R82-95, MC1R105-118 and MC1R149-161). MC1R peptide-specific CD8+ T-cell clones isolated from the depigmented skin of this patient were characterized by comparatively low functional avidity for specific major histocompatibility complex-peptide complexes and were poorly lytic; however, these effector cells were capable of secreting both interferon-gamma and granzyme B against relevant target cells in vitro, and may have played an important role in the induction of leucoderma in situ in this patient.

Factors Contributing to the Delay in Diagnosis and Continued Transmission of Leprosy in Brazil--An Explorative, Quantitative, Questionnaire Based Study.

BACKGROUND: Leprosy is a leading cause of preventable disability worldwide. Delay in diagnosis of patients augments the transmission of infection, and allows progression of disease and more severe disability. Delays in diagnosis greater than ten years have been reported in Brazil. To reduce this delay, it is important to identify factors that hinder patients from presenting to doctors, and those that delay doctors from diagnosing patients once they have presented. This study aimed to explore factors associated with the delayed diagnosis of leprosy in Brazil. METHODOLOGY/ PRINCIPAL FINDINGS: This is an exploratory study using a self-constructed questionnaire delivered to patients attending three leprosy referral clinics across three states in Brazil. Data were analysed to determine associations between variables and the time taken for participants to present to the health-service, and between variables and the time taken for doctors to diagnose participants once they had presented. Participants who suspected they had leprosy but feared community isolation were 10 times more likely to wait longer before consulting a doctor for their symptoms (OR 10.37, 95% CI 2.18-49.45, p = 0.003). Participants who thought their symptoms were not serious had a threefold greater chance of waiting longer before consulting than those who did (OR 3.114, 95% CI 1.235-7.856, p = 0.016). Forty-two point six per cent of participants reported initially receiving a diagnosis besides leprosy. These had a three times greater chance of receiving a later diagnosis of leprosy compared to those not misdiagnosed or not given a diagnosis (OR 2.867, 95% CI 1.288-6.384, p = 0.010). CONCLUSIONS/ SIGNIFICANCE: This study implies a need for patient education regarding leprosy symptoms and the reduction of stigma to encourage patients to present. The high rate of misdiagnosis reported suggests a need to increase clinician suspicion of leprosy. Further education regarding disease symptoms in medical school curriculums may be advisable.

UVB irradiation of normal human skin favors the development of type-2 T-cells in vivo and in primary dermal cell cultures.

To determine the effect of UVB exposure on the balance of type-1 or type-2 T-cells in skin, we examined the expression of key markers interferon (IFN)-gamma and interleukin (IL)-4 in cryostat sections. IFN-gamma mRNA was clearly detectable in nonirradiated control skin, and IFN-gamma protein was found in 2% of the dermal CD3pos T-cells, whereas IL-4 mRNA was hardly detectable, and no IL-4 protein was found. In contrast, IL-4 mRNA expression increased upon irradiation, and IL-4 was found in 2% of the T-cells at day 2 after UVB-exposure. Concomitantly, IFN-gamma mRNA expression decreased, and IFN-gamma protein became absent. We also analyzed T-cells present in primary dermal cell cultures, which were used as an in vitro equivalent of the in vivo situation. As compared with T-cells from control skin, T-cells in dermal cell cultures from UVB-exposed skin displayed an increased IL-4 and decreased IFN-gamma expression. No such skewing occurred when the T-cells from irradiated skin were cloned in the absence of a dermal microenvironment. Except for an occasional positive T-cell, type-1-associated cell-surface markers (CCR5, CXCR3) or type-2 markers (CCR3, CD30, CRTH2) were undetectable in situ. But these markers were expressed on cultured dermal T-cells from UVB-exposed and control skin at a comparable level, but did not correlate with the IFN-gamma and IL-4 production. Altogether, UVB-induced changes of the dermal microenvironment favor the development of type-2 T-cells.

Assessment of contact allergens by dissociation of irritant and sensitizing properties.

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose-response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay.

A two-centre evaluation of the human organotypic skin explant culture model for screening contact allergens.

Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin explant culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.

Conference scene: lessons from animal models of autoimmune diseases: from mechanisms to applications.

Autoimmune diseases are a group of maladies in which the patient's immune homeostasis becomes so deregulated that it mounts a destructive attack against the hosts tissues. Such diseases are characterized by an activation of autoreactive T and B cells and are associated, in some cases, with the production of pathogenic antibodies against self-molecules, culminating in inflammation and tissue damage. Target tissues can be from immune-vulnerable and immune-privileged sites. In view of the complex nature of autoimmune diseases, it is not surprising that they have long baffled immunologists, physicians and basic biomedical scientists who are struggling to combine known immunoinflammatory mechanisms into a unified general theory. The present seminar, organized by Euroscion, hosted a group of national and international scientists, affiliated to both academic and industrial research, to discuss state-of-the-art animal models for investigating pathomechanisms of autoimmune diseases, novel laboratory-based diagnostics and novel therapeutic prospects. The timely event on this important topic covered significant features of the basic pathomechanisms of autoimmune disease per se, the development of diagnostic and prognostic tests using functional biomarkers for monitoring patients and the development of targeted therapies. The absence of several prescheduled speakers allowed younger scientists, Stefan Kürten, Liliane Fossati-Jimack and Allan Holmes to shine. We are grateful for their participation. This meeting report describes key points and themes arising from this conference.

Antigenic profiles of individual-matched pairs of primary and melanoma metastases.

A unique collection of individual-matched pairs of primary and melanoma metastases were studied immunohistochemically with a panel of 6 monoclonal antibodies directed to gp-100, pigmentation-associated antigen, tyrosinase-related protein, human leukocyte antigen DR, MAA-1, and MAA-2 (high molecular weight melanoma-associated antigens). The antigenic profile of immunoreactive pigment cells was compared with the stage of tumor progression. Our data show consistent antigenic profiles of primary melanomas and their metastases within the same patient. Expression of tyrosinase-related protein and pigmentation-associated antigen was observed in the radial growth phase of primary melanomas but showed diminished or complete loss of expression in the vertical growth phase and in metastatic melanomas. HLA-DR was negative in the most primary lesions, but melanoma cells and a larger proportion of immunoreactive cells were observed at the metastatic site. The melanoma-associated antigens MAA-1 and MAA-2 were expressed throughout tumor progression. Although no clear distinction could be made between primary and secondary melanoma lesions for both melanoma-associated antigens, there was a profound variability in the topographical antigen distribution when compared with HLA-DR. The loss of expression of pigmentation-associated antigen and tyrosinase-related protein in the vertical growth phase of the primary lesions and metastatic melanomas did not reach statistical significance but still may be related to tumor progression. This indicates that primary melanomas can be distinguished from their metastases by evaluation of the antigenic profile and in this respect facilitate the recognition of tumor progression stages.

Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration.

Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant-like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical-induced migration of CD1a(+) LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a(+) LCs, CD83(+) mature dendritic cells (DCs) and CCR7(+) LN homing cells. After exposure to a toxic concentration of a non-immunogenic irritant, an increase of CD1a(+)CD83(+) LCs was found in the culture medium. A non-toxic concentration of an sensitizer induced an increase of CD1a(+) cells. About 50% of skin emigrating CD1a(+) LCs were CD83(-) (immature) but all were CCR7(+). Skin irritation by both non-allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation.

Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model: a novel method for identifying potential contact allergens.

The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83(+) cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model.

Non-lesional vitiliginous melanocytes are not characterized by an increased proneness to nitric oxide-induced apoptosis.

Nitric oxide (NO) is a reactive endogenous molecule with multiple functions including inflammation and immunity. NO stimulates melanogenesis by activating soluble guanylyl cyclase (sGC) resulting in increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP). In vitro experiments showed that NO could inhibit the de novo attachment of melanocytes to extracellular matrix (ECM) suggesting that NO-induced aberrant perturbation of melanocyte-ECM interaction could be a reason for melanocyte loss in vitiliginous lesions. Here, we examined whether there might be differences between normal melanocytes and vitiliginous melanocytes (VMs) with respect to NO-induced detachment from ECM and whether cGMP is involved. We used the direct NO donor (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and the peroxynitrite donor 3-morpholino-sydnonimine for the present studies. These donors induced detachment of both normal melanocytes and non-lesional VMs in a time- and concentration-dependent manner with comparable susceptibility and similar expression profile of sGC. Treatment of melanocytes with caspase inhibitors reduced cell detachment, indicating that a major part of the detachment is due to apoptosis. The NO-induced detachment but not apoptosis was partly inhibited in the presence of sGC and cGMP-dependent protein kinase inhibitors. In addition, the membrane-permeable cGMP analog 8-(4-chlorophenyethio/guanosine-3',5'-cyclic monophosphate (PCPT) cGMP was not able to induce apoptosis in melanocytes, suggesting that NO-induced detachment of melanocytes via apoptosis is cGMP-independent. The present results also indicate that there are no apparent differences between NO-induced detachment of non-lesional vitiliginous and normal melanocytes from ECM.

Gravity and cyclic GMP levels in melanocytic cells.

Guanosine 3',5'-cyclic monophosphate (cyclic GMP) is a major second messenger molecule, that is believed to play a role in various physiological and pathophysiological processes. Here we report that hypergravity induces differential effects on cyclic GMP turnover in melanocytic cells. Nonmetastatic melanoma cells responded to long-time exposure (24 h) of hypergravity (up to 5 x g) with decrease in intracellular cyclic GMP accumulation in the presence of an universal inhibitor of phosphodiesterases (IBMX), whereas the extracellular cyclic GMP increase. In contrast, there were no changes in cyclic GMP turnover in metastatic melanocytes. The expression of the guanylyl cyclases appeared to be not affected. These results suggest that cyclic GMP signaling may be involved in adaptation of human melanocytes to altered gravity conditions.