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Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin ß-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.
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Proteínas Filagrina , Complejo de la Endopetidasa Proteasomal , Humanos , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
BACKGROUND: Facial wrinkles are clear markers of the aging process, being chronological, photo-induced, or reflecting repetitive facial expressions. The aim of this study is to provide new insights into the biophysical and biological mechanisms involved in the formation, prevention, or elimination of the expression wrinkles. MATERIALS AND METHODS: We use a computational model to get a better understanding of the wrinkle mechanical behavior and evolution after skin softening and suggesting a possible antiaging mechanism. Then, we provide a clinical demonstration of the anti-wrinkle effect of a long-term application of a 20% glycerol in a moisturizer formula (GBM) versus its vehicle on crow's feet. Skin hydration, elasticity, and wrinkles visibility were evaluated by a combination of clinical and instrumental in vivo data, inverse finite element analysis, and proteomic data. RESULTS: The computational model shows a predominantly compressive stress beneath the wrinkle and its significant decrease by the softening of stratum corneum. The associated clinical study confirmed a significant increase of skin hydration and elasticity as well as a decrease of wrinkle visibility after 2 and 4 months as application for both formulas; this effect being stronger for GBM. A softening effect on stratum corneum and dermis was also observed for the GBM. Furthermore, proteomic data revealed an effect of upregulation of four proteins associated with desquamation, cell-glycan extracellular interactions, and protein glycation/oxidation, functions related to the tissue mechanics and adhesion. CONCLUSIONS: We provide an in vivo demonstration of the anti-ageing benefit of glycerol at high dose (20%) reflected by a cumulative skin surface softening effect. The use of high moisturizing potent formulations should bring additional performance to other conventional moisturizing formulations.
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Fármacos Dermatológicos , Glicerol , Envejecimiento de la Piel , Humanos , Envejecimiento , Glicerol/farmacología , Proteómica , Piel/efectos de los fármacos , Envejecimiento de la Piel/efectos de los fármacos , Cara , Expresión Facial , Simulación por Computador , Fármacos Dermatológicos/farmacologíaRESUMEN
We report a multi-modal study of the electrical, chemical and structural properties of a kesterite thin-film solar cell by combining the spatially-resolved X-ray beam induced current and fluorescence imaging techniques for the evaluation of a fully functional device on a cross-section. The data allowed the correlation of the chemical composition, defects at interfaces and inhomogeneous deposition of the layers with the local charge-collection efficiency of the device. We support our observations with Monte Carlo simulations of high-energy X-ray interactions with the semiconductor device, and finite-volume modeling of the charge-collection efficiency.
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FLG is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it colocalized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis using benzyloxycarbonyl-pro-prolinal induced the accumulation of FLG monomers. Downregulation of PREP expression in reconstructed human epidermis using RNA interference confirmed the impact of PREP on FLG metabolism and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, western blotting, and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification such as loricrin. Consequently, at the functional level, the transepidermal electric resistance was drastically reduced.
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In atopic dermatitis (AD), the skin barrier is disturbed, and the expression of calcium-dependent S100 proteins and the calcium gradient is also altered in the epidermis. The calmodulin-like skin protein (CLSP), which is expressed in the differentiated epidermis, is believed to modulate the function of calcium-dependent proteins involved in barrier formation and is significantly increased in the epidermis of psoriatic patients. We, therefore, investigated the CLSP level in skin biopsies taken from patients with acute exacerbated and non-exacerbated AD as well as from healthy control subjects. Immunohistochemical, Western blot and ELISA analyses showed significant increases (P < 0.03) in CLSP level in the epidermis from patients with acute exacerbated AD as compared to that from patients with non-exacerbated AD and from control subjects. Such increased expression of CLSP may help re-establish a functional epidermal barrier in acute AD.
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Proteínas de Unión al Calcio/metabolismo , Dermatitis Atópica/metabolismo , Epidermis/metabolismo , Regulación de la Expresión Génica , Biomarcadores/metabolismo , Biopsia , Calcio/metabolismo , Calmodulina/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Epidermis/patología , Humanos , Inflamación , Queratinocitos/citología , Psoriasis/metabolismo , Proteínas S100/metabolismo , Cicatrización de HeridasRESUMEN
Topically applied all-trans-retinoic acid (RA) is a gold-standard anti-aging molecule used in dermatology. As its cosmetic counterpart used in anti-aging, Retinol (ROL) is also a known metabolic precursor of RA. Despite this metabolic link, they haven't been compared exhaustively in vivo at a mechanistic level. Therefore, to highlight the effect of a topical application of both molecules on in vivo skin, we undertook a longitudinal 1-year study and performed an untargeted proteomic analysis to get a more holistic view on the underlying biological mechanisms of action. The generation of the temporal proteomics signatures of retinol and all-trans-retinoic acid reveals the impact of these molecules on biological functions related to the aging of skin. New biological functions impacted by retinoids were discovered: glycan metabolism and protein biosynthesis. In addition, the temporal analysis reveals highest modulations at early time points while the physical measures, such as epidermal thickening, was mostly observed at the latest time point, demonstrating a strong time lapse between molecular and morphological impacts. Finally, these global temporal signatures could be used to identify new cosmetic compounds of interest.
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Proteoma , Vitamina A , Humanos , Estudios Longitudinales , Proteómica , Tretinoina/farmacologíaRESUMEN
Introduction: Staphylococcus epidermidis is a commensal bacterium ubiquitously present on human skin. This species is considered as a key member of the healthy skin microbiota, involved in the defense against pathogens, modulating the immune system, and involved in wound repair. Simultaneously, S. epidermidis is the second cause of nosocomial infections and an overgrowth of S. epidermidis has been described in skin disorders such as atopic dermatitis. Diverse isolates of S. epidermidis co-exist on the skin. Elucidating the genetic and phenotypic specificities of these species in skin health and disease is key to better understand their role in various skin conditions. Additionally, the exact mechanisms by which commensals interact with host cells is partially understood. We hypothesized that S. epidermidis isolates identified from different skin origins could play distinct roles on skin differentiation and that these effects could be mediated by the aryl hydrocarbon receptor (AhR) pathway. Methods: For this purpose, a library of 12 strains originated from healthy skin (non-hyperseborrheic (NH) and hyperseborrheic (H) skin types) and disease skin (atopic (AD) skin type) was characterized at the genomic and phenotypic levels. Results and discussion: Here we showed that strains from atopic lesional skin alter the epidermis structure of a 3D reconstructed skin model whereas strains from NH healthy skin do not. All strains from NH healthy skin induced AhR/OVOL1 path and produced high quantities of indole metabolites in co-culture with NHEK; especially indole-3-aldehyde (IAld) and indole-3-lactic acid (ILA); while AD strains did not induce AhR/OVOL1 path but its inhibitor STAT6 and produced the lowest levels of indoles as compared to the other strains. As a consequence, strains from AD skin altered the differentiation markers FLG and DSG1. The results presented here, on a library of 12 strains, showed that S. epidermidis originated from NH healthy skin and atopic skin have opposite effects on the epidermal cohesion and structure and that these differences could be linked to their capacity to produce metabolites, which in turn could activate AHR pathway. Our results on a specific library of strains provide new insights into how S. epidermidis may interact with the skin to promote health or disease.
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Dermatitis Atópica , Staphylococcus epidermidis , Humanos , Promoción de la Salud , Receptores de Hidrocarburo de Aril , PielRESUMEN
A proteomic analysis of stratum corneum (SC) samples of normal healthy skin revealed the presence of more than 70 proteins by 2D electrophoresis. The majority of these proteins to our knowledge have not yet been described in normal SC. We analysed by Western blot the levels of 25 proteins in the SC taken from postmenopausal and dry skin compared with young and normal skin, respectively. In postmenopausal skin, there was a significantly increased amount of heat shock protein 27, plakoglobin and desmoglein 1, whereas transglutaminase 3, apolipoprotein D and acid ceramidase levels were significantly reduced compared with the SC of young skin. We confirmed corneodesmosin as a marker of dry skin. In addition, we showed for the first time that the levels of both phosphatidylethanolamine-binding protein 1 and annexin A2 were significantly increased in the SC of dry skin compared with the SC of normal skin. These results suggest that a proteomic analysis of the SC obtained using a non-invasive varnish stripping method is an attractive alternative to invasive methods to better characterize changes in the physiology of ageing and dry skin.
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Epidermis/química , Posmenopausia/metabolismo , Proteínas/análisis , Proteómica , Enfermedades de la Piel/metabolismo , Adulto , Envejecimiento/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Western Blotting , Epidermis/metabolismo , Femenino , Humanos , Persona de Mediana EdadRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Skin aspartic acid protease (SASPase) is believed to be a key enzyme involved in filaggrin processing during epidermal terminal differentiation. Since little is known about the regulation of SASPase function, the aim of this study was to identify involved protein partners in the process. Yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 binds to the N-terminal fragment of SASPase. This interaction was confirmed in reciprocal yeast two hybrid screens and by Surface Plasmon Resonance analyses. Immunohistochemical studies in human skin, using specific antibodies to SASPase and the N-terminal domain of filaggrin 2, showed that the two proteins partially co-localized to the stratum granulosum. In vitro enzymatic assays showed that the N-terminal domain of filaggrin 2 enhanced the autoactivation of SASPase to its 14 kDa active form. Taken together, the data suggest that the N-terminal domain of filaggrin 2 regulates the activation of SASPase that may be a key event upstream of filaggrin processing to natural moisturizing factors in the human epidermis.
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Ácido Aspártico Endopeptidasas/metabolismo , Proteínas S100/metabolismo , Piel/metabolismo , Ácido Aspártico Endopeptidasas/análisis , Activación Enzimática , Proteínas Filagrina , Humanos , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteínas S100/análisisRESUMEN
Clinical observations of both normal and pathological skin have shown that there is a heterogeneity based on the skin origin type. Beside external factors, intrinsic differences in skin cells could be a central element to determine skin types. This study aimed to understand the in vitro behaviour of epidermal cells of African and Caucasian skin types in the context of 3D reconstructed skin. Full-thickness skin models were constructed with site matched human keratinocytes and papillary fibroblasts to investigate potential skin type related differences. We report that reconstructed skin epidermis exhibited remarkable differences regarding stratification and differentiation according to skin types, as demonstrated by histological appearance, gene expression analysed by DNA microarray and quantitative proteomic analysis. Signalling pathways and processes related to terminal differentiation and lipid/ceramide metabolism were up-regulated in epidermis constructed with keratinocytes from Caucasian skin type when compared to that of keratinocytes from African skin type. Specifically, the expression of proteins involved in the processing of filaggrins was found different between skin models. Overall, we show unexpected differences in epidermal morphogenesis and differentiation between keratinocytes of Caucasian and African skin types in in vitro reconstructed skin containing papillary fibroblasts that could explain the differences in ethnic related skin behaviour.
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Epidermis/patología , Piel/metabolismo , Piel/patología , Población Negra/genética , Diferenciación Celular , Dermis/citología , Células Epidérmicas/metabolismo , Células Epidérmicas/patología , Fibroblastos/metabolismo , Humanos , Queratinocitos/citología , Modelos Biológicos , Morfogénesis , Proteómica/métodos , Población Blanca/genéticaRESUMEN
In tissue engineering, porous scaffolds are often used as three-dimensional (3D) supports for tissue growth. In scaffold design, it is imperative to be able to quantify the pore sizes and more importantly the interconnects between the pores. X-ray micro-computed tomography (microCT) has become a popular tool for obtaining 3D images of scaffold biomaterials, however images are only qualitative. In this work, methods were developed for obtaining pore size distributions for both the macropores and their interconnects. Scaffolds have been developed, by foaming sol-gel derived bioactive glasses, which have the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. MicroCT images were obtained from scaffolds with different pore structures. The images were thresholded and three algorithms were applied in 3D to identify pores and interconnects and to obtain pore size distributions. The results were validated against mercury intrusion porosimetry and manual 3D image analysis. The microCT data were then meshed such that predictions of permeability as a function of changes in the pore network could be made. Such predictions will be useful for optimising bioreactor conditions for tissue engineering applications. These techniques would be suitable for many other types of scaffolds.
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Imagenología Tridimensional , Ingeniería de Tejidos/estadística & datos numéricos , Materiales Biocompatibles , Huesos/anatomía & histología , Huesos/diagnóstico por imagen , Cerámica , Humanos , Ensayo de Materiales , Permeabilidad , Tomografía Computarizada por Rayos XRESUMEN
The study aimed at detecting differentially expressed proteins in the stratum corneum of dandruff versus non-dandruff scalps to better understand dandruff aetiology. iTRAQ-based quantitative proteomic analysis revealed a total of 68 differentially expressed biomarkers. A detailed analysis of their known physiological functions provided new insights into the affected metabolic pathways of a dandruff scalp. Dandruff scalp showed (1) profound changes in the expression and maturation of structural and epidermal differentiation related proteins, that are responsible for the integrity of the skin, (2) altered relevant factors that regulate skin hydration, and (3) an imbalanced physiological protease-protease inhibitor ratio. Stratum corneum proteins with antimicrobial activity, mainly those derived from sweat and sebaceous glands were also found modified. Comparing our data with those reported for atopic dermatitis revealed that about 50 % of the differentially expressed proteins in the superficial layers of the stratum corneum from dandruff and atopic dermatitis are identical.
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Dermatitis Atópica/metabolismo , Dermatitis Seborreica/etiología , Dermatitis Seborreica/metabolismo , Epidermis/metabolismo , Cuero Cabelludo/metabolismo , Adulto , Diferenciación Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteómica/métodos , Piel/metabolismo , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
BACKGROUND/AIMS: We investigated the relationship between postoperative liver failure and serum acute-phase protein level before and after liver resection. METHODOLOGY: Thirty-four patients who underwent liver resection were prospectively included. Serum concentrations of negative (albumin, prealbumin and retinol-binding protein) and positive (orosomucoid, haptoglobin and C-reactive protein) acute-phase proteins were assayed prior to surgery (baseline) and on postoperative day 3, 12 and 45. Postoperative liver failure was defined as serum bilirubin more than 50 micromol/L or prothrombin time less than 50% on postoperative day 7. Univariate analysis was performed to compare patients who did and did not present postoperative liver failure. RESULTS: Postoperative liver failure occurred in 8 cases and was correlated with: 1) higher negative and lower positive acute-phase protein levels (p<0.04) at baseline, 2) lower negative and lower positive acute-phase protein levels on postoperative day 3, 12 or 45 (p< or =0.05). CONCLUSIONS: Early onset of inflammatory serum protein profile was correlated with absence of postoperative liver failure. Serum acute-phase protein could be used as predictor as well as early postoperative diagnosis marker of postoperative liver failure. Relationship between preoperative inflammation and postoperative liver failure warrants further investigations because of potential therapeutic consequences.
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Proteínas de Fase Aguda/metabolismo , Hepatectomía , Fallo Hepático/diagnóstico , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Complicaciones Posoperatorias/diagnóstico , Adulto , Anciano , Femenino , Humanos , Fallo Hepático/sangre , Fallo Hepático/mortalidad , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/mortalidad , Pronóstico , Estadística como AsuntoRESUMEN
Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease-protease inhibitor balance.
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Desmosomas/enzimología , Epidermis/metabolismo , Glicoproteínas/metabolismo , Calicreínas/metabolismo , Serina Endopeptidasas/metabolismo , Especificidad de Anticuerpos , Cadherinas/inmunología , Cadherinas/metabolismo , Células Cultivadas , Desmocolinas , Desmogleína 1 , Expresión Génica , Glicoproteínas/inmunología , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular , Calicreínas/genética , Riñón/citología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Oligosacáridos/metabolismo , Oligosacáridos/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genéticaRESUMEN
TWEAK is a new TNF family member with proinflammatory and proliferative effects on different cell types, mediated by the recently identified Fn14 receptor. TWEAK expression was analyzed on mouse microglial cells and astrocytes. Both cell types express TWEAK mRNA. Astrocytes expressed Fn14 and proliferated in the presence of rTWEAK. TWEAK mRNA is expressed in normal CNS and its steady state level increases in spinal cord during EAE. Finally, EAE severity is enhanced in soluble TWEAK-overexpressing transgenic mice. These results support the contention that TWEAK is involved in CNS inflammation.
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Astrocitos/metabolismo , Proteínas Portadoras/genética , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Mediadores de Inflamación/farmacología , Microglía/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Astrocitos/inmunología , Proteínas Portadoras/farmacología , División Celular/efectos de los fármacos , División Celular/genética , División Celular/inmunología , Células Cultivadas , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/inmunología , Citocina TWEAK , Progresión de la Enfermedad , Susceptibilidad a Enfermedades/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Transgénicos , Microglía/inmunología , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Factores de Necrosis TumoralRESUMEN
High-affinity anti-GM1 antibodies are frequently described in several nervous system diseases, mainly in multifocal motor neuropathy (MMN) and some acute neuropathies. These antibodies are currently detected using enzyme-linked immunosorbent assay (ELISA) and immuno-thin-layer-chromatography (immuno-TLC) methods. We report in this article a new method based on the incorporation of exogenous GM1 in a selected cell line to detect anti-GM1 antibodies using flow cytometry (FC). This method is evaluated on 80 sera from normal blood donors (NBD) and patients suffering various nervous system diseases. It appears to be as sensitive as our method ELISA for the diagnosis of some motor neuron syndromes.
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Autoanticuerpos/análisis , Citometría de Flujo/métodos , Gangliósido G(M1)/inmunología , Autoanticuerpos/sangre , Glioblastoma , Humanos , Enfermedad de la Neurona Motora/diagnóstico , Enfermedad de la Neurona Motora/inmunología , Neuroblastoma , Sensibilidad y Especificidad , Células Tumorales CultivadasAsunto(s)
Enfermedad Celíaca/diagnóstico , Trastornos Mentales/etiología , Enfermedades del Sistema Nervioso/etiología , Anciano , Enfermedad Celíaca/complicaciones , Enfermedad Celíaca/terapia , Dietoterapia/métodos , Femenino , Glútenes , Humanos , Trastornos Mentales/diagnóstico , Trastornos Mentales/terapia , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/terapia , Vitamina B 12/uso terapéuticoRESUMEN
A multidisciplinary expert group had reviewed all scientific data available of post mastectomy pain syndrome. Seventy six publications were retained and thirty evidence based diagnosis, treatment and follow-up recommendations are listed. Few of theses recommendations are classed level A. Datas analysis make possible to propose a strategy based on systematic association of drugs, kinesitherapy and psychological support. Evaluation and closer follow-up are necessary. Several decisional trees are proposed.