RESUMEN
BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin and mucous membranes. This disease typically affects the elderly and presents with itch and localized or, most frequently, generalized bullous lesions. A subset of patients only develops excoriations, prurigo-like lesions, and eczematous and/or urticarial erythematous lesions. The disease, which is significantly associated with neurological disorders, has high morbidity and severely impacts the quality of life. OBJECTIVES AND METHODOLOGY: The Autoimmune blistering diseases Task Force of the European Academy of Dermatology and Venereology sought to update the guidelines for the management of BP based on new clinical information, and new evidence on diagnostic tools and interventions. The recommendations are either evidence-based or rely on expert opinion. The degree of consent among all task force members was included. RESULTS: Treatment depends on the severity of BP and patients' comorbidities. High-potency topical corticosteroids are recommended as the mainstay of treatment whenever possible. Oral prednisone at a dose of 0.5 mg/kg/day is a recommended alternative. In case of contraindications or resistance to corticosteroids, immunosuppressive therapies, such as methotrexate, azathioprine, mycophenolate mofetil or mycophenolate acid, may be recommended. The use of doxycycline and dapsone is controversial. They may be recommended, in particular, in patients with contraindications to oral corticosteroids. B-cell-depleting therapy and intravenous immunoglobulins may be considered in treatment-resistant cases. Omalizumab and dupilumab have recently shown promising results. The final version of the guideline was consented to by several patient organizations. CONCLUSIONS: The guidelines for the management of BP were updated. They summarize evidence- and expert-based recommendations useful in clinical practice.
Asunto(s)
Dermatología , Penfigoide Ampolloso , Venereología , Corticoesteroides/uso terapéutico , Anciano , Vesícula/tratamiento farmacológico , Humanos , Penfigoide Ampolloso/diagnóstico , Penfigoide Ampolloso/tratamiento farmacológico , Calidad de VidaRESUMEN
BACKGROUND: The incidence of skin cancers has been increasing steadily over the last decades. Although there have been significant breakthroughs in the management of skin cancers with the introduction of novel diagnostic tools and innovative therapies, skin cancer mortality, morbidity and costs heavily burden the society. OBJECTIVE: Members of the European Association of Dermato-Oncology, European Academy of Dermatology and Venereology, International Dermoscopy Society, European Dermatology Forum, European Board of Dermatovenereology of the European Union of Medical Specialists and EORTC Cutaneous Lymphoma Task Force have joined this effort to emphasize the fundamental role that the specialist in Dermatology-Venereology has in the diagnosis and management of different types of skin cancer. We review the role of dermatologists in the prevention, diagnosis, treatment and follow-up of patients with melanoma, non-melanoma skin cancers and cutaneous lymphomas, and discuss approaches to optimize their involvement in effectively addressing the current needs and priorities of dermato-oncology. DISCUSSION: Dermatologists play a crucial role in virtually all aspects of skin cancer management including the implementation of primary and secondary prevention, the formation of standardized pathways of care for patients, the establishment of specialized skin cancer treatment centres, the coordination of an efficient multidisciplinary team and the setting up of specific follow-up plans for patients. CONCLUSION: Skin cancers represent an important health issue for modern societies. The role of dermatologists is central to improving patient care and outcomes. In view of the emerging diagnostic methods and treatments for early and advanced skin cancer, and considering the increasingly diverse skills, knowledge and expertise needed for managing this heterogeneous group of diseases, dermato-oncology should be considered as a specific subspecialty of Dermatology-Venereology.
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Dermatología , Melanoma , Enfermedades de la Piel , Neoplasias Cutáneas , Venereología , Dermatólogos , Humanos , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/terapiaRESUMEN
BACKGROUND: Pemphigus encompasses a group of life-threatening autoimmune bullous diseases characterized by blisters and erosions of the mucous membranes and skin. Before the era of immunosuppressive treatment, pemphigus was almost always fatal. Due to its rarity, only few randomized controlled therapeutic trials are available. Recently, rituximab has been approved as first-line treatment for moderate and severe pemphigus vulgaris in Europe and the United States. OBJECTIVES: The Autoimmune blistering diseases Task Force of the European Academy of Dermatology and Venereology (EADV) has initiated a throughout update of the guideline for the management of patients with pemphigus. RESULTS: The guidelines for the management of pemphigus were updated, and the degree of consent among all task force members was included. The final version of the guideline was consented by the European Dermatology Forum (EDF) and several patient organizations.
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Dermatología , Guías como Asunto , Pénfigo , Venereología , Academias e Institutos , Europa (Continente) , Humanos , Pénfigo/diagnóstico , Pénfigo/tratamiento farmacológicoRESUMEN
BACKGROUND: Survivin is detected in few adult normal cells and it is highly expressed in cancer. Nuclear survivin facilitates cell cycle entry, whereas the mitochondrial pool protects cells from apoptosis. Survivin is overexpressed in keratinocyte stem cells (KSCs) and protects them from apoptosis. METHODS: As KSCs are at the origin of squamous cell carcinoma (SCC), we evaluated survivin expression in normal and cancerous skin in vivo by immunohistochemistry and western blotting. HaCaT cells overexpressing survivin and wound-healing assay are used. Analysis of variance and Student's T-tests are used for statistical analysis. RESULTS: Survivin is localised in both the cytoplasm and nucleus of normal adult and young keratinocytes. Nuclear survivin is detected in one every 10 of 11 basal keratinocytes. When present in suprabasal cells, nuclear survivin is coexpressed with K10 but not with K15 or p75-neurotrophin receptor (p75NTR), a transit amplifying cell marker. Nuclear, but not cytoplasmic, survivin expression markedly increases in actinic keratosis and in SCC in situ, as compared with normal epidermis, and it is highest in poorly differentiated SCC. In SCC tumours, nuclear survivin-positive cells are mainly K10/p75NTR-negative and K15-positive. In poorly differentiated tumours, survivin mostly localises in the deep infiltrating areas. When overexpressed in keratinocytes, survivin increases cell migration. CONCLUSION: High survivin expression and the subcellular localisation of survivin correlate with keratinocyte differentiation and are associated with undifferentiated and more invasive SCC phenotype.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Neoplasias Cutáneas/metabolismo , Piel/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Linaje de la Célula , Núcleo Celular/metabolismo , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Queratinocitos/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Neoplasias Cutáneas/patología , Survivin , Adulto JovenRESUMEN
Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders.
Asunto(s)
Moléculas de Adhesión Celular/genética , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/terapia , Vectores Genéticos , Queratinocitos/metabolismo , Virus de la Leucemia Murina de Moloney/genética , Integración Viral , Animales , Moléculas de Adhesión Celular/metabolismo , Epidermólisis Ampollosa/metabolismo , Regulación de la Expresión Génica , Terapia Genética , VIH-1/genética , Células HeLa , Humanos , Ratones , Virus de la Leucemia Murina de Moloney/fisiología , Regiones Promotoras Genéticas , Células 3T3 Swiss , Transducción Genética , Transgenes , Inactivación de Virus , KalininaRESUMEN
Melanocytes and cells of the nervous system are of common ectodermal origin and neurotrophins (NT) have been shown to be released by human keratinocytes. We investigated the expression and function of NT [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/-5] and their receptors in human melanocytes. Human melanocytes produce all NT in different amounts, whereas they only release NT-4. NT-4 release is downregulated, whereas NT-3 is upregulated by ultraviolet (UVB) irradiation. Melanocytes treated with phorbol 12-myristate 13-acetate (PMA) express TrkA and TrkB, but not TrkC. NT fail to stimulate melanocyte proliferation, whereas they stimulate the synthesis of tyrosinase and tyrosinase-related protein-1 (TRP-1). Finally, NT-3, NT-4 and NGF increase melanin production. Taken together, these results demonstrate an intriguing interaction between melanocytes and the nervous system. We speculate that NT could be considered the target of therapy for disorders of skin pigmentation.
RESUMEN
Granulomatous tissue reactions appear in athymic mouse skin, indicating that initiation of granuloma formation may be T-cell independent. To further evaluate the relationships between granuloma formation and T-cell function, we treated euthymic BALB/c mice with cyclosporine (Cs), a potent immunosuppressive drug, injected intramuscularly (150 mg/kg/day) 5 times a week. Hepatic granulomas were isolated from mice with schistosomiasis and transplanted into the skin of mice treated with Cs for 2 weeks. Cyclosporine injection was continued for 3 additional weeks. Blood levels of the drug increased during treatment (489 ng/ml at 2 weeks and 822 ng/ml at 5 weeks). Morphologically identical granulomas developed in both treated and untreated mice. Examination for T-cell functions showed that by the end of 2 weeks treatment, concanavalin A, phytohemagglutinin responses, and IL-2 activity were markedly depressed, and IL-2 receptor expression was not detected in either lymph nodes or spleen of the Cs-treated mice; however, after hepatic granuloma graft, T-cell functions in regional lymph nodes, but not in spleen, as well as peripheral blood eosinophilia were stimulated in Cs-treated mice. These data strongly suggest that intact T-cell activity is not essential for the initiation of granuloma formation. In addition, granuloma grafts appear to stimulate Cs-resistant T-cell activation locally, which amplifies and organizes the granulomatous response.
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Ciclosporinas/farmacología , Granuloma/etiología , Enfermedades de la Piel/etiología , Animales , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Ultraviolet radiation is a potent inducer of apoptosis, whereas autocrine nerve growth factor protects human keratinocytes from programmed cell death. To evaluate the role of nerve growth factor in the mechanisms of ultraviolet B-induced apoptosis, cultured human keratinocytes were ultraviolet B irradiated following pretreatment with K252, a specific inhibitor of the tyrosine kinase high-affinity nerve growth factor receptor. Here we report that the addition of K252 significantly enhanced keratinocyte apoptosis. We then transfected normal human keratinocytes with pNUT-hNGF. Nerve growth factor overexpressing keratinocytes secreted the highest amounts of nerve growth factor in culture supernatants, were more viable, and had a higher rate of proliferation than mock-transfected cells. Whereas ultraviolet B radiation downregulated nerve growth factor mRNA and protein as well as the tyrosine kinase high-affinity nerve growth factor receptor in normal keratinocytes, it failed to do so in nerve growth factor-transfected cells. Moreover, nerve growth factor overexpressing keratinocytes were partially resistant to apoptosis induced by increasing doses of ultraviolet B at 24 and 48 h. These results indicate that downregulation of nerve growth factor function plays an important part in the mechanisms of ultraviolet B-induced apoptosis in human keratinocytes. In addition, ultraviolet B caused a decrease in BCL-2 and BCL-xL expression in mock-transfected keratinocytes, but not in nerve growth factor overexpressing cells. Finally, nerve growth factor prevented the cleavage of the enzyme poly(ADP-ribose) polymerase induced in human keratinocytes by ultraviolet B. These results are consistent with a model whereby the autocrine nerve growth factor protects human keratinocytes from ultraviolet B-induced apoptosis by maintaining constant levels of BCL-2 and BCL-xL, which in turn might block caspase activation.
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Apoptosis/efectos de la radiación , Queratinocitos/efectos de la radiación , Factor de Crecimiento Nervioso/fisiología , Rayos Ultravioleta , División Celular/efectos de la radiación , Humanos , Factor de Crecimiento Nervioso/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptor trkA/análisis , Transfección , Proteína bcl-XRESUMEN
Substance P has been detected in human skin and has been implicated in the pathogenesis of certain inflammatory cutaneous disorders. However, little is known about the number and distribution of substance P binding sites in the skin. Receptor autoradiography was employed to detect and quantitate substance P receptors in normal as well as psoriatic skin. Substance P binding sites were distributed in the epidermis and dermis both in normal and psoriatic skin. In the dermis, the highest densities of SP binding sites were found in the areas corresponding to the dermal papillae and the adnexal structures. Quantitative analysis revealed that saturable binding was obtained both in the epidermis and in the labeled dermal areas. Rosenthal plot values were consistent with a single population of binding sites. No difference in the binding measurements was observed between normal and psoriatic skin. The presence of substance P receptors in the epidermis and in the dermal papillae raises interesting issues on the possible targets of this peptide in human skin both under physiologic and pathologic conditions.
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Psoriasis/patología , Receptores de Neurotransmisores/análisis , Piel/ultraestructura , Autorradiografía , Sitios de Unión , Humanos , Radioisótopos de Yodo , Psoriasis/metabolismo , Receptores de Neuroquinina-1 , Receptores de Neurotransmisores/metabolismo , Piel/metabolismo , Sustancia P/metabolismoRESUMEN
An involvement of neurogenic components in the pathogenesis of psoriatic lesions has been suggested and neuropeptides are thought to play a modulatory role in cutaneous inflammation. In this study, we evaluated the immunoreactivity of the neuropeptides vasoactive intestinal polypeptide (VIP) and substance P (SP) in the skin of patients with chronic plaque psoriasis, by immunohistochemistry and radioimmunoassay. No differences were observed, by immunohistochemistry, in the expression and localization of VIP and SP between psoriatic and normal skin. Using the radioimmunologic technique on whole skin homogenates, VIP levels were significantly increased in psoriatic lesions as compared to normal skin. By contrast, SP levels were significantly lower in lesional and non-lesional psoriatic skin than in normal skin. In addition, we examined the effect of VIP and SP on the proliferation of cultured normal human keratinocytes. VIP (1-28) (1 nM-1 microM) as well as VIP fragments (10-28) (1 nM-1 microM) and (22-28) (1 nM-1 microM) stimulated the proliferation of keratinocytes in a dose-dependent manner, whereas the VIP fragment (1-12) (1 nM-1 microM) was ineffective. The VIP antagonist (N-Ac-Tyr1, D-Phe2)-GRF (1-29)-NH2 (0.1 microM) significantly inhibited the VIP effect on keratinocytes. On the other hand, SP (0.1 microM) not only failed to stimulate keratinocyte growth, but also blocked the VIP-induced stimulation of these cells. The imbalance of cutaneous VIP and SP and their disparate effects on the proliferation of normal human keratinocytes in culture would suggest that these peptides are involved in the pathogenesis of psoriasis and may exert different modulatory activities in the mechanisms underlying the psoriatic lesion.
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Queratinocitos/citología , Psoriasis/metabolismo , Sustancia P/análisis , Péptido Intestinal Vasoactivo/análisis , División Celular/efectos de los fármacos , Células Cultivadas , Humanos , Inmunohistoquímica , Queratinocitos/química , Psoriasis/patología , Radioinmunoensayo , Sustancia P/farmacología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Apoptosis is a cellular process of self-directed suicide that plays a key role during morphogenesis and in the maintenance of homeostasis in continuously renewing tissues. Currently, apoptosis is detected mainly by gel electrophoresis of fragmented DNA and by typical ultrastructural features such as cell shrinkage and chromatin condensation. Recently, an in situ technique was developed that allows the detection of the apoptotic process in cells and the quantitation of apoptosis in cell populations. We applied this technique to evaluate the apoptotic process in cultured normal human keratinocytes under basic conditions and after stimulation with factors and agents that are presumed but have never been proved to induce apoptosis in these cells. Apoptosis was analyzed after stimulation with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], transforming growth factor beta1 (TGFbeta1), calcium, UVB, or tumor necrosis factor alpha (TNFalpha). All these factors except TNFalpha induced apoptosis in human keratinocytes. Whereas UVB and calcium were good apoptogenic stimuli at 6 and 24 h, respectively, the vitamin D derivative and TGFbeta1 induced apoptosis after 5 and 6 d in culture. Apoptosis was also established by DNA fragmentation and electron microscopy. Finally, TUNEL technique showed that the number of apoptotic cells increases slightly (5-10%) from 24 to 144 h even in untreated keratinocytes. Our studies indicate that factors normally involved in the regulation of cell growth and differentiation can also control apoptosis.
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Calcitriol/farmacología , Calcio/farmacología , Queratinocitos/citología , Factor de Crecimiento Transformador beta/farmacología , Rayos Ultravioleta , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Humanos , Queratinocitos/ultraestructura , Microscopía ElectrónicaRESUMEN
Apoptosis plays a fundamental part in epidermal homeostasis, and apoptotic cells have been detected in normal and diseased skin. Little is known, however, on the inhibitory mechanisms of apoptosis at the skin level. In addition to bcl-2, a novel inhibitor of apoptosis designated survivin and structurally analogous to IAP apoptosis inhibitors has been recently identified. The expression of survivin in normal and pathologic skin was investigated. Immunohistochemical studies revealed that survivin is expressed in basal keratinocytes, but not in suprabasal epidermal layers, with a pattern similar to bcl-2. In western blots, the anti-survivin antibody recognized a single band of 16.5 kDa in protein extracts from normal human keratinocytes in culture, in agreement with the predicted size of survivin. In addition, survivin immunoreactivity was detected in benign and malignant melanocytic lesions, with strong expression in invasive lesions of melanomas. Whereas survivin staining was undetectable in benign epithelial tumors, such as seborrheic keratoses, it was observed in all epidermal layers in Bowen's disease. Interestingly, at variance with bcl-2, survivin was markedly expressed in squamous cell carcinoma, but virtually lacking in basal cell carcinoma, suggesting that these two apoptosis inhibitors may act through different anti-apoptotic pathways. Deregulation of survivin may influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer.
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Apoptosis/efectos de los fármacos , Proteínas Asociadas a Microtúbulos , Proteínas/análisis , Neoplasias Cutáneas/química , Piel/química , Adulto , Humanos , Immunoblotting , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis , Proteínas de Neoplasias , SurvivinRESUMEN
Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.
Asunto(s)
Queratinocitos/fisiología , Factores de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/genética , Apoptosis/fisiología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , División Celular/fisiología , Células Cultivadas , Expresión Génica/fisiología , Expresión Génica/efectos de la radiación , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , ARN Mensajero/análisis , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Rayos UltravioletaRESUMEN
Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti-low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.
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Queratinocitos/citología , Factores de Crecimiento Nervioso/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Carbazoles/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Alcaloides Indólicos , Queratinocitos/química , Queratinocitos/ultraestructura , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/análisis , Factores de Crecimiento Nervioso/farmacología , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores de Factor de Crecimiento Nervioso/genéticaRESUMEN
Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.
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Carbono/farmacología , Ácidos Carboxílicos/farmacología , Fulerenos , Queratinocitos/citología , Protectores contra Radiación/farmacología , Rayos Ultravioleta , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Desoxirribosa/farmacología , Citometría de Flujo , Humanos , Membranas Intracelulares/fisiología , Potenciales de la Membrana , Mitocondrias , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Normal human keratinocytes synthesize and release nerve growth factor (NGF) and express both the low- and the high-affinity NGF receptor. Because NGF has been shown to rescue certain cell types from programmed cell death, we investigated the role of endogenous NGF in preventing keratinocyte apoptosis. We report here that apoptosis is induced in normal human keratinocytes in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K252, a specific inhibitor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was assessed by DNA laddering, electron microscopy, and in situ nick end labeling technique. In anti-NGF-treated keratinocytes, the apoptotic process starts at 96 h, and is maximal at 120 h. After K252 treatment, apoptosis starts at 48 h and peaks at 120 h. Because the product of the bcl-2 proto-oncogene protects many cell types from apoptosis, we measured the levels of this protein in apoptotic keratinocytes. We found that both K252 and anti-NGF antibody strikingly downregulate bcl-2 expression, starting at 72 h. Furthermore, HaCat keratinocytes stably transfected with a plasmid containing bcl-2 cDNA fail to undergo apoptosis when treated with K252. These findings show that autocrine NGF acts as a survival factor for human keratinocytes in vitro through its high-affinity NGF receptor, possibly by maintaining constant levels of Bcl-2.
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Apoptosis/efectos de los fármacos , Queratinocitos/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Células Cultivadas , Fragmentación del ADN , Humanos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-bcl-2/análisisRESUMEN
Anoikia is a type of apoptotic cell death that occurs in cells that are substrate-restricted in their growth. Buckminsterfullerenes represent a new class of chemical compounds with wide potential pharmacological antioxidant activity. In this report we provide the first demonstration that a water-soluble fullerene derivative, C3-fullero-tris-methanodicarboxylic acid, synthesized in our laboratories, is capable of inducing anoikia resistance in epithelial cells by a mechanism involving a 'trophic' effect on cell spreading-associated cytoskeletal components, i.e. on actin microfilaments.
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Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Carbono/farmacología , Ácidos Dicarboxílicos/farmacología , Células Epiteliales/patología , Fulerenos , Apoptosis/efectos de la radiación , Carbono/química , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/efectos de la radiación , Humanos , Microscopía Electrónica de RastreoRESUMEN
Biologically active nerve growth factor (NGF) is synthesised and released by proliferating normal human keratinocytes. NGF up-regulates the expression of NGF mRNA in keratinocytes. Keratinocytes express both the low (p75)- and the high-affinity (TrkA) NGF-receptors, which are located in the basal layer of the epidermis. K252, a specific inhibitor of trk phosphorylation, blocks NGF-induced keratinocyte proliferation, in absence of exogenous NGF. Normal keratinocytes over-expressing TrkA proliferate better than control transfectants, while the NGF mimicking anti-Trk antibody induces an increased keratinocyte proliferation in Trk over-expressing cells as compared to mock transfected keratinocytes. In addition, NGF over-expressing keratinocytes proliferate better than mock transfected cells. K252, by blocking TrkA phosphorylation, induces apoptosis in normal keratinocytes, but not in keratinocytes over-expressing bcl-2. Furthermore, NGF transfected keratinocytes are protected from UV-B-induced keratinocyte apoptosis, by maintaining constant levels of Bcl-2 and Bcl-xL . Taken together these results support the concept of an autocrine survival system sustained by NGF and its high-affinity receptor in human keratinocytes. Because NGF and Trk levels are highly expressed in psoriasis. one could speculate that NGF autocrine system plays a role in the mechanisms associated with this and other hyperproliferative skin conditions, including cancer.
Asunto(s)
Queratinocitos/fisiología , Factor de Crecimiento Nervioso/fisiología , Apoptosis/fisiología , Comunicación Autocrina/fisiología , Humanos , Queratinocitos/patologíaRESUMEN
The immunohistochemical expression of neuron-specific enolase (gamma/gamma) (NSE) was studied comparatively with S100 protein in a group of Langerhans-cell-type ("X") (n = 8) and non-Langerhans-cell-type ("non X") (n = 24) cutaneous histiocytoses. NSE was expressed by the majority (70-90%) of histiocytic cells in all cases of Langerhans-cell histiocytoses, whereas it was absent from non-Langerhans-cell histiocytoses. S100 protein was expressed by the majority of Langerhans-cell histiocytosis cells but also by a small percentage (1-5%) of cells in non Langerhans-cell histiocytoses. These results show that NSE is almost as sensitive as, but more specific than, S100 protein in discriminating Langerhans-cell from non-Langerhans cell cutaneous histiocytoses, and that it consequently represents a useful adjunct in the immunohistochemical diagnosis of histiocytic skin diseases.
Asunto(s)
Histiocitoma Fibroso Benigno/patología , Histiocitosis de Células de Langerhans/patología , Fosfopiruvato Hidratasa/análisis , Proteínas S100/análisis , Enfermedades de la Piel/patología , Neoplasias Cutáneas/patología , Biomarcadores , Biomarcadores de Tumor/análisis , Histiocitoma Fibroso Benigno/enzimología , Histiocitosis de Células de Langerhans/enzimología , Humanos , Inmunohistoquímica , Enfermedades de la Piel/enzimología , Neoplasias Cutáneas/enzimologíaRESUMEN
Skin biopsy specimens from five patients with pityriasis lichenoides et varioliformis acuta and from six patients with pityriasis lichenoides chronica were studied by direct immunofluorescence and by an immunoperoxidase technique using a panel of monoclonal antibodies. The dermal inflammatory infiltrate was composed of T cells, macrophages, and a small proportion of CD1a+ cells, mostly perivascular. CD8+ cells (cytotoxic/suppressor phenotype) predominated in the epidermis according to the degree of epidermal necroses, whereas CD4+ cells (helper/inducer phenotype) were superior in number among dermal T cells. A few B cells and Leu7+ cells were detected in only a small proportion of lesions. The results obtained confirm that the two conditions are variants of a single disease process and suggest that cell-mediated immune mechanisms may be important in the pathogenesis of the epidermal and vascular damage. Endothelial cells (HLA-DR+ and HLA-DQ+) and CD1a+ cells (epidermal and possibly dermal) could be primarily involved, acting as antigen-presenting cells.