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OBJECTIVES: Opioid use disorder is a public health problem worldwide with a treatment gap partially due to sociocultural representation and stigma. Taking the opportunity of an authorization to a subcutaneous (SC) injectable solution of buprenorphine, the first and only injectable treatment for opioid dependence available in France, we investigate potential obstacles to its implementation in France. METHODS: This study aimed to define the factors predicting the acceptance of a new SC form of opiate substitution treatment (OST) by comparing the social representations using an adapted version of the Explanatory Model Interview Catalogue (EMIC) and the internalized stigma of intravenous drug injection using the Internalized Stigma of Mental Illness Inventory (ISMI) between participants receiving OST likely to accept the SC form or not. We also observed whether the fear of an opiate withdrawal syndrome could influence this choice. RESULTS: Fifty OST patients were included, 54% of them accepted a new SC form of OST. Perceived causes of drug injection measured with EMIC were significantly lower among participants who would not accept the new SC form. No significant difference was found regarding the total score of the adapted ISMI or its items. The fear of opiate withdrawal syndrome did not seem to be statistically related to acceptance of a long-acting SC OST in either group. The most discriminating combination of factors in predicting patient acceptance of such treatment was related to the perceived causes of drug injection associated with a severe Diagnostic and Statistical Manual of Mental Disorders 5th version (DSM-5) diagnosis, and a lower alcohol consumption. CONCLUSIONS: We observed significant differences in social representations but not in internalized stigma between the two groups. Moreover, the predictive factors linked to the acceptance of a new SC form of OST suggest a multifactorial combination of elements that will have to be tested in a larger and prospective study delivering long-acting high-dose buprenorphine.
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According to the French recommendations, the elimination of the hepatitis C virus by 2025 could be a realistic public health goal. Screening policies are being intensified, and access to treatment is promoted for patients who escape the usual care pathway. The 'Scanvir' program is an original strategy based on dedicated screening days, as part of the 'test, treat and cure HCV' event in addiction care centers in a French region, during which innovative screening technologies (RDTs, FibroScan® and point-of-care HCV RNA testing) are brought on site and access to a multidisciplinary team is offered. A total of 392 patients attended the 67 regional Scanvir sessions: 31.6% were HCV Ab-positive and 66% of them were HCV RNA-positive. Treatment was initiated in 79.3% of the patients. RDTs were accepted by 62% of the PWIDs (including those who already knew their status) and FibroScan® by 99.5% of the patients. 80% of the viremic patients started their treatment on site and are now cured or still under treatment. Advanced fibrosis evaluated by FibroScan® (LSM > 8 KPa) was suspected in 13.4% and 14.1% of the global and the HCV population, respectively. Scanvir is an efficient strategy for HCV elimination based on dedicated days aimed at increasing cost-effectiveness and offering a multidisciplinary service while saving human care resources. It is an exportable strategy that also offers comprehensive screening of associated chronic liver diseases via the elastometry device and interviews.
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Consumidores de Drogas , Hepatitis C , Humanos , Hepacivirus/genética , Hepatitis C/epidemiología , Francia , ARN , Antivirales/uso terapéuticoRESUMEN
INTRODUCTION: Cancer has become a chronic disease thanks to therapeutic evolutions and justifies the early integration of supportive care in the management. The Advanced Practice Nurse (APN) was created to respond to the increase in the number of patients followed in the long term. The objective of this study is to identify the place and expected missions of an APN within a multidisciplinary supportive care team. MATERIAL AND METHOD: A qualitative study by semi-directed interview using a previously developed interview grid was carried out with 14 health professionals (doctors, nurses and health managers) working in a supportive care service in three Cancer Centres. RESULTS: The role expected by the participants is based on the optimisation of patients' follow-up, the integration of supportive care into the care pathway, the improvement of the relationship with the town, and the development of nursing leadership in the establishment. At the same time, the potential arrival of an APN as a change agent in a supportive care service is a source of fears. DISCUSSION: The APN seems to be a real link in the institutional organisations facilitating the link between the professionals of the institution and with the professionals of the territory. The identification of the origins of the fears expressed should enable work to be done to facilitate the integration of the APN into specific support care services, particularly for patients in palliative situations.
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Enfermería de Práctica Avanzada , Humanos , Cuidados Paliativos , Liderazgo , Investigación CualitativaRESUMEN
Requests for return to the country of origin of palliative patients are not exceptional and cause many difficulties for caregivers. This article provides a narrative review of the literature to examine the practical difficulties in dealing with these requests and to identify ways to overcome them. The return to the country of origin requires a medical assessment of both the conditions of the trip and the coordination with the teams that will take over the care of the patient. Transportation of patients is most often possible but requires preventive and therapeutic measures. The organization of the return journey may require collaboration between the social service, families, associations, or diplomatic representations in order to carry out the administrative procedures or to finance the return project. For patients with a language barrier, this process requires the use of professional interpreters. The return to the country of origin for patients in a palliative situation requires sufficient anticipation to take place under the best conditions. Early integration of palliative care and anticipation of the cessation of specific treatments and the end of life are ways of addressing these issues.
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Cuidados Paliativos , Migrantes , HumanosRESUMEN
BACKGROUND: Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystal-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. METHODS: Human calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1, and human tenocytes. The release of IL-1ß, IL-6, and IL-8 by cells was quantified by ELISA. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement were assessed in THP-1 cells using a NF-kB inhibitor and a caspase-1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. RESULTS: Human calcifications were able to induce a significant release of IL-1ß when incubated with monocytes, macrophages, and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL-1ß when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. The patient's crystals enhanced mRNA expression of pro-IL-1ß, as well as IL-18, NF-kB, and TGFß when IL-6 and TNFα expression were not. IL-1ß production was reduced by the inhibition of caspase-1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in the air pouch led to a significant increase in membrane thickness although significant overexpression of IL-1ß was only observed for synthetic hydroxyapatite. CONCLUSIONS: As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1ß after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1ß induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape, and protein content may explain this observation.
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Proteína con Dominio Pirina 3 de la Familia NLR , Tendinopatía , Apatitas , Caspasa 1 , Humanos , Inflamasomas , Interleucina-1beta , Tendinopatía/tratamiento farmacológicoRESUMEN
The PGC-1 (Peroxisome proliferator-activated receptor Gamma Coactivator-1) family of coactivators (PGC-1α, PGC-1ß, and PRC) plays a central role in the transcriptional control of mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) processes. These coactivators integrate mitochondrial energy production into cell metabolism using complementary pathways. The XTC.UC1 cell line is a mitochondria-rich model of thyroid tumors whose biogenesis is almost exclusively dependent on PRC. Here we aim to propose an integrative view of the cellular pathways regulated by PRC through integration of cDNA and miRNA microarray data and chromatin immunoprecipitation results obtained from XTC.UC1 cells invalidated for PRC. This study showes that PRC induces a complex network of cellular functions interacting with at least one to five of the studied transcription factors (Estrogen Related Receptor alpha, ERR1; Nuclear-Respiratory Factors, NRF1 and NRF2; cAMP Response Element Binding, CREB; and Ying Yang, YY1). Our data confirm that ERR1 is a key partner of PRC in the regulation of mitochondrial functions and suggest a potential role of this complex in RNA processing. PRC is also involved in transcriptional regulatory complexes targeting 12 miRNAs, five of which are involved in the control of the OXPHOS process. Our findings demonstrate that the PRC coactivator can act in complex with several transcription factors and regulate miRNA expression to control the fine regulation of main metabolic functions in the cell. Therefore, in PGC-1α/ß-associated pathologies, PRC, as a metabolic sensor, may ensure mitochondrial homeostasis.
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The collagen ColQ anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction (NMJ). It also binds MuSK and perlecan/dystroglycan, 2 signaling platforms of the postsynaptic domain. Mutations in ColQ cause a congenital myasthenic syndrome (CMS) with AChE deficiency. Because the absence of AChE does not fully explain the complexity of the syndrome and there is no curative treatment for the disease, we explored additional potential targets of ColQ by conducting a large genetic screening of ColQ-deficient mice, a model for CMS with AChE deficiency, and analyzed their NMJ and muscle phenotypes. We demonstrated that ColQ controls the development and the maturation of the postsynaptic domain by regulating synaptic gene expression. Notably, ColQ deficiency leads to an up-regulation of the 5 subunits of the nicotinic acetylcholine receptor (AChR), leading to mixed mature and immature AChRs at the NMJ of adult mice. ColQ also regulates the expression of extracellular matrix (ECM) components. However, whereas the ECM mRNAs were down-regulated in vitro, compensation seemed to occur in vivo to maintain normal levels of these mRNAs. Finally, ColQ deficiency leads to a general atrophic phenotype and hypoplasia that affect fast muscles. This study points to new specific hallmarks for this CMS.-Sigoillot, S. M., Bourgeois, F., Karmouch, J., Molgó, J., Dobbertin, A., Chevalier, C., Houlgatte, R., Léger, J., Legay, C. Neuromuscular junction immaturity and muscle atrophy are hallmarks of the ColQ-deficient mouse, a model of congenital myasthenic syndrome with acetylcholinesterase deficiency.
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Acetilcolinesterasa/deficiencia , Colágeno/metabolismo , Modelos Animales de Enfermedad , Proteínas Musculares/metabolismo , Atrofia Muscular/patología , Síndromes Miasténicos Congénitos/patología , Unión Neuromuscular/fisiología , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos , Colágeno/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Síndromes Miasténicos Congénitos/enzimología , Síndromes Miasténicos Congénitos/genética , TranscriptomaRESUMEN
SCOPE: Our study aims to investigate molecular events associated to methyl donor deficiency (MDD) by analyzing the transcriptome and the methylome of MDD rats in liver. METHODS AND RESULTS: Twenty-one-day-old rats born to mothers fed either with a standard diet or a MDD diet during gestation and lactation were compared. From a total of 44 000 probes for 26 456 genes, we found two gene clusters in MDD rats whose expression levels had significant differences compared with controls: 3269 overexpressed (p < 0.0009) and 2841 underexpressed (p < 0.0004) genes. Modifications of DNA methylation were found in the promoter regions of 1032 genes out of 14 981 genes. Ontological analyses revealed that these genes are mainly involved in glucose and lipid metabolism, nervous system, coagulation, ER stress, and mitochondrial function. CONCLUSION: Putative master genes exhibiting changes in both gene expression and DNA methylation are limited to 266 genes and are mainly involved in the renin-angiotensin system (n = 3), mitochondrion metabolism (n = 18), and phospholipid homeostasis (n = 3). Most of these master genes participate in nonalcoholic fatty liver disease. The adverse effects of MDD on the metabolic process indicate the beneficial impact of folate and vitamin B12, especially during the perinatal period.
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Metilación de ADN , Epigenómica , Hepatopatías/genética , Hígado/fisiología , Fenómenos Fisiologicos Nutricionales Maternos , Transcriptoma , Animales , Biología Computacional , Dieta/veterinaria , Femenino , Ácido Fólico/sangre , Ácido Fólico/farmacología , Expresión Génica , Lactancia , Metabolismo de los Lípidos , Atención Perinatal , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Vitamina B 12/sangre , Vitamina B 12/farmacologíaRESUMEN
BACKGROUND: Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence. METHODOLOGY/PRINCIPAL FINDINGS: 197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy. CONCLUSION/SIGNIFICANCE: RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.
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Células del Cúmulo/metabolismo , Regulación de la Expresión Génica/fisiología , Genoma Humano , Oocitos/metabolismo , Embarazo/metabolismo , Proteínas RGS/biosíntesis , Adulto , Biomarcadores/metabolismo , Comunicación Celular/fisiología , Células del Cúmulo/citología , Femenino , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/citología , Perilipina-2 , Ribonucleasa Pancreática/biosíntesis , Transferencia de un Solo Embrión , Inyecciones de Esperma IntracitoplasmáticasRESUMEN
Duchenne Muscular Dystrophy (DMD) is a complex process involving multiple pathways downstream of the primary genetic insult leading to fatal muscle degeneration. Aging muscle is a multifactorial neuromuscular process characterized by impaired muscle regeneration leading to progressive atrophy. We hypothesized that these chronic atrophying situations may share specific myogenic adaptative responses at transcriptional level according to tissue remodeling. Muscle biopsies from four young DMD and four AGED subjects were referred to a group of seven muscle biopsies from young subjects without any neuromuscular disorder and explored through a dedicated expression microarray. We identified 528 differentially expressed genes (out of 2,745 analyzed), of which 328 could be validated by an exhaustive meta-analysis of public microarray datasets referring to DMD and Aging in skeletal muscle. Among the 328 validated co-expressed genes, 50% had the same expression profile in both groups and corresponded to immune/fibrosis responses and mitochondrial metabolism. Generalizing these observed meta-signatures with large compendia of public datasets reinforced our results as they could be also identified in other pathological processes and in diverse physiological conditions. Focusing on the common gene signatures in these two atrophying conditions, we observed enrichment in motifs for candidate transcription factors that may coordinate either the immune/fibrosis responses (ETS1, IRF1, NF1) or the mitochondrial metabolism (ESRRA). Deregulation in their expression could be responsible, at least in part, for the same transcriptome changes initiating the chronic muscle atrophy. This study suggests that distinct pathophysiological processes may share common gene responses and pathways related to specific transcription factors.
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Inmunidad Adaptativa/fisiología , Mitocondrias/metabolismo , Músculo Esquelético/inmunología , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/inmunología , Distrofia Muscular de Duchenne/metabolismo , Inmunidad Adaptativa/genética , Adolescente , Envejecimiento/genética , Niño , Análisis por Conglomerados , Humanos , Técnicas In Vitro , Factor 1 Regulador del Interferón/genética , Masculino , Distrofia Muscular de Duchenne/genética , Neurofibromina 1/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Proto-Oncogénica c-ets-1/genética , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
PURPOSE: To evaluate the prognostic value of the cell of origin (COO) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBLC), prospectively treated by rituximab, dexamethasone, high-dose cytarabine, and cisplatin (R-DHAP) versus rituximab, ifosfamide, carboplatin, and etoposide and followed by intensive therapy plus autologous stem-cell transplantation on the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) trial. PATIENTS AND METHODS: Among the 396 patients included on the trial, histologic material was available for a total of 249 patients at diagnosis (n = 189 patients) and/or at relapse (n = 147 patients), which included 87 matched pairs. The patient data were analyzed by immunochemistry for CD10, BCL6, MUM1, FOXP1, and BCL2 expression and by fluorescent in situ hybridization for BCL2, BCL6 and c-MYC breakpoints. The correlation with survival data was performed by using the log-rank test and the Cox model. RESULTS: Characteristics of immunophenotype and chromosomal abnormalities were statistically highly concordant in the matched biopsies. In univariate analysis, the presence of c-MYC gene rearrangement was the only parameter to be significantly correlated with a worse progression-free survival (PFS; P = .02) and a worse overall survival (P = .04). When treatment interaction was tested, the germinal center B (GCB) -like DLBCL that was based on the algorithm by Hans was significantly associated with a better PFS in the R-DHAP arm. In multivariate analysis, independent prognostic relevance was found for the GCB/non-GCB the Hans phenotype interaction treatment (P = .04), prior rituximab exposure (P = .0052), secondary age-adjusted International Prognostic Index (P = .039), and FoxP1 expression (P = .047). Confirmation was obtained by gene expression profiling in a subset of 39 patients. CONCLUSION: COO remains a major and independent factor in relapsed/refractory DLBCL, with a better response to R-DHAP in GCB-like DLBCL. This needs confirmation by a prospective study.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Centro Germinal/patología , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Neoplasia Residual/tratamiento farmacológico , Terapia Recuperativa , Adulto , Anciano , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Antígenos CD20/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Biomarcadores de Tumor/genética , Carboplatino/administración & dosificación , Cisplatino/administración & dosificación , Citarabina/administración & dosificación , Dexametasona/administración & dosificación , Supervivencia sin Enfermedad , Etopósido/administración & dosificación , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ifosfamida/administración & dosificación , Inmunohistoquímica , Hibridación Fluorescente in Situ , Linfoma de Células B Grandes Difuso/química , Linfoma de Células B Grandes Difuso/inmunología , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Análisis Multivariante , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Ensayos Clínicos Controlados Aleatorios como Asunto , Recurrencia , Factores de Riesgo , Rituximab , Terapia Recuperativa/métodosRESUMEN
Risk stratification in advanced heart failure (HF) is crucial for the individualization of therapeutic strategy, in particular for heart transplantation and ventricular assist device implantation. We tested the hypothesis that cardiac gene expression profiling can distinguish between HF patients with different disease severity. We obtained tissue samples from both left (LV) and right (RV) ventricle of explanted hearts of 44 patients undergoing cardiac transplantation or ventricular assist device placement. Gene expression profiles were obtained using an in-house microarray containing 4217 muscular organ-relevant genes. Based on their clinical status, patients were classified into three HF-severity groups: deteriorating (n= 12), intermediate (n= 19) and stable (n= 13). Two-class statistical analysis of gene expression profiles of deteriorating and stable patients identified a 170-gene and a 129-gene predictor for LV and RV samples, respectively. The LV molecular predictor identified patients with stable and deteriorating status with a sensitivity of 88% and 92%, and a specificity of 100% and 96%, respectively. The RV molecular predictor identified patients with stable and deteriorating status with a sensitivity of 100% and 96%, and a specificity of 100% and 100%, respectively. The molecular prediction was reproducible across biological replicates in LV and RV samples. Gene expression profiling has the potential to reproducibly detect HF patients with highest HF severity with high sensitivity and specificity. In addition, not only LV but also RV samples could be used for molecular risk stratification with similar predictive power.
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Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Sesgo , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Medición de RiesgoRESUMEN
Conduction slowing of the electric impulse that drives the heartbeat may evoke lethal cardiac arrhythmias. Mutations in SCN5A, which encodes the pore-forming cardiac sodium channel alpha subunit, are associated with familial arrhythmia syndromes based on conduction slowing. However, disease severity among mutation carriers is highly variable. We hypothesized that genetic modifiers underlie the variability in conduction slowing and disease severity. With the aim of identifying such modifiers, we studied the Scn5a(1798insD/+) mutation in 2 distinct mouse strains, FVB/N and 129P2. In 129P2 mice, the mutation resulted in more severe conduction slowing particularly in the right ventricle (RV) compared to FVB/N. Pan-genomic mRNA expression profiling in the 2 mouse strains uncovered a drastic reduction in mRNA encoding the sodium channel auxiliary subunit beta4 (Scn4b) in 129P2 mice compared to FVB/N. This corresponded to low to undetectable beta4 protein levels in 129P2 ventricular tissue, whereas abundant beta4 protein was detected in FVB/N. Sodium current measurements in isolated myocytes from the 2 mouse strains indicated that sodium channel activation in myocytes from 129P2 mice occurred at more positive potentials compared to FVB/N. Using computer simulations, this difference in activation kinetics was predicted to explain the observed differences in conduction disease severity between the 2 strains. In conclusion, genetically determined differences in sodium current characteristics on the myocyte level modulate disease severity in cardiac sodium channelopathies. In particular, the sodium channel subunit beta4 (SCN4B) may constitute a potential genetic modifier of conduction and cardiac sodium channel disease.
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Canalopatías/genética , Sistema de Conducción Cardíaco/fisiopatología , Animales , Arritmias Cardíacas/fisiopatología , Canalopatías/fisiopatología , Elementos Transponibles de ADN , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Ratones , Ratones Endogámicos , Células Musculares/citología , Células Musculares/fisiología , Mutación , Canal de Sodio Activado por Voltaje NAV1.5 , ARN Mensajero/genética , Canales de Sodio/deficiencia , Canales de Sodio/genética , Canales de Sodio/fisiología , Subunidad beta-4 de Canal de Sodio Activado por VoltajeRESUMEN
BACKGROUND: Myostatin (MSTN), a member of the TGF-beta superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass. RESULTS: Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed. CONCLUSION: All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3epsilon protein, TCTP/GSK-3beta). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo.
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Apoptosis , Perfilación de la Expresión Génica , Miostatina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Músculo Cuádriceps/metabolismo , Animales , Biología Computacional , Expresión Génica , Ratones , Ratones Noqueados , Miostatina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
Recent large-scale cDNA cloning studies have shown that a significant proportion of the transcripts expressed from vertebrate genomes do not appear to encode protein. Moreover, it was reported in mammals (human and mice) that these non-coding transcripts are expressed and regulated by mechanisms similar to those involved in the control of protein-coding genes. We have produced a collection of cDNA sequences from immunologically active tissues with the aim of discovering chicken genes involved in immune mechanisms, and we decided to explore the non-coding component of these immune-related libraries. After finding known non-coding RNAs (miRNA, snRNA, snoRNA), we identified new putative mRNA-like non-coding RNAs. We characterised their expression profiles in immune-related samples. Some of them showed changes in expression following viral infections. As they exhibit patterns of expression that parallel the behaviour of protein-coding RNAs in immune tissues, our study suggests that they could play an active role in the immune response.
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Pollos/genética , ADN Complementario/genética , ARN no Traducido/genética , Animales , Infecciones por Birnaviridae/genética , Infecciones por Birnaviridae/inmunología , Pollos/inmunología , Etiquetas de Secuencia Expresada , Femenino , Perfilación de la Expresión Génica , Biblioteca de Genes , Activación de Linfocitos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Masculino , Enfermedad de Marek/genética , Enfermedad de Marek/inmunología , MicroARNs/genética , MicroARNs/aislamiento & purificación , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , ARN no Traducido/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Linfocitos T/inmunologíaRESUMEN
Survival of solid organ grafts depends on life-long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous "operational tolerance" can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross-validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis.
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Perfilación de la Expresión Génica , Trasplante de Riñón/inmunología , Adulto , Anciano , Análisis por Conglomerados , Femenino , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Humanos , Terapia de Inmunosupresión , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) are the key regulators of bone metabolism. Recent findings demonstrated a crucial role of RANK in several bone-associated tumors. Indeed, we have recently demonstrated functional RANK expression both in a mouse and several human osteosarcoma cell lines. However, RANKL effects on osteosarcoma cells remain to be determined. In this study, we determined RANKL effects on RANK-positive Saos-2 human osteosarcoma cells. cDNA microarray and quantitative RT-PCR analyses clearly demonstrated that RANK-positive osteosarcoma cells were the target of RANKL as well as osteoclasts/osteoclast precursors. Thus, we present for the first time that RANKL can directly and significantly modulate gene expression of RANK-expressing Saos-2 cells. RANKL-modulated genes included genes that were implicated in protein metabolism, nucleic acid metabolism, intracellular transport, cytoskeleton organization and biogenesis, apoptosis and signaling cascade. Our results strengthen the involvement of the RANK/RANKL/OPG axis in osteosarcoma biology and capability to identify novel therapeutic approaches targeting RANK-positive osteosarcomas.
Asunto(s)
Neoplasias Óseas/genética , Perfilación de la Expresión Génica , Osteosarcoma/genética , Ligando RANK/fisiología , Receptor Activador del Factor Nuclear kappa-B/genética , División Celular , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/fisiología , Osteoclastos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Heme oxygenase-1 (HO-1), a cytoprotective enzyme, can be induced in tumors in response to anti-cancer therapies. We investigated the role of HO-1 in B16(F10), S91, and Sk-mel188 melanoma cells. Overexpression of HO-1 after transduction with adenoviral vectors increased cell proliferation, resistance to oxidative stress generated by H2O2, and angiogenic potential as determined by induction of endothelial cell divisions. Likewise, cells stably transfected with HO-1 cDNA (B16-HO-1) showed higher proliferation, stress resistance, and angiogenic activity than the wild-type line (B16-WT). HO-1 overexpression in tumors significantly shortened survival of mice after subcutaneous injection of cancer cells (38 and 22 days for B16-WT and B16-HO-1, respectively; P=0.017). This also resulted in development of more packed tumors, with more melanoma cells, and reduced inflammatory edemas. Mice injected with B16-HO-1 had lower levels of tumor necrosis factor and higher serum concentrations of its soluble receptor tumor necrosis factor-RI, whereas tumors overexpressing HO-1 displayed augmented vascularization and stronger production of vascular endothelial growth factor. Finally, B16-HO-1 cells injected intravenously formed more metastases in lungs. Thus, HO-1 overexpression increased viability, proliferation, and angiogenic potential of melanoma cells, augmented metastasis, and decreased survival of tumor-bearing mice, suggesting that induction of HO-1 may be detrimental in anti-cancer therapy of melanoma.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Melanoma Experimental/patología , Neovascularización Patológica/etiología , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Citocinas/metabolismo , Humanos , Neoplasias Pulmonares/secundario , Melanoma Experimental/enzimología , Melanoma Experimental/mortalidad , Ratones , Metástasis de la Neoplasia , Neovascularización Patológica/metabolismo , Tasa de Supervivencia , Transfección , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Array-comparative genomic hybridization (CGH) has evolved as a useful technique for the detection and characterization of deletions, and, to a lesser extent, of duplications. The resolution of the technique is dictated by the genomic distance between targets spotted on the microarray, and by the targets' sizes. The use of region-specific, high-resolution microarrays is a specific goal when studying regions that are prone to rearrangements, such as those involved in deletion syndromes. The aim of the present study was to evaluate the best experimental conditions to be used for array-CGH analysis using low molecular weight (LMW) targets. The parameters tested were: the target concentration, the way LMW targets are prepared (either as linearized plasmids or as purified PCR products), and the way the targets are attached to the array-CGH slide (in a random fashion on amino-silane coated slides, or by one amino-modified end on epoxysilane-coated slides). As a test case, we constructed a microarray harboring LMW targets located in the CREBBP gene, mutations of which cause the Rubinstein-Taybi syndrome (RTS). From 10 to 15% of RTS patients have a CREBBP deletion. We showed that aminosilane- and epoxysilane-coated slides were equally efficient with targets above 1,000 bp in size. On the other hand, with the smallest targets, especially those below 500 bp, epoxysilane-coated slides were superior to aminosilane-coated slides, which did not allow deletion detection. Use of the high resolution array allowed us to map intragenic breakpoints with precision and to identify a very small deletion and a duplication that were not detected by the currently available techniques for finding CREBBP deletions.