RESUMEN
The number of forcibly displaced people has more than doubled over the past decade. Many people fleeing are left in limbo without a secure pathway to citizenship or residency. This mixed-methods systematic review reports the prevalence of mental disorders in migrants living in limbo, the association between limbo and mental illness, and the experiences of these migrants in high income countries. We searched electronic databases for quantitative and qualitative studies published after January 1, 2010, on mental illness in precarious migrants living in HICs and performed a meta-analysis of prevalence rates. Fifty-eight articles met inclusion criteria. The meta-analysis yielded prevalence rates of 43.0 % for anxiety disorders (95 % CI 29.0-57.0), 49.5 % for depression (40.9-58.0) and 40.8 % for posttraumatic stress disorder (30.7-50.9). Having an insecure status was associated with higher rates of mental illness in most studies comparing migrants in limbo to those with secure status. Six themes emerged from the qualitative synthesis: the threat of deportation, uncertainty, social exclusion, stigmatization, social connection and religion. Clinicians should take an ecosocial approach to care that attends to stressors and symptoms. Furthermore, policymakers can mitigate the development of mental disorders among migrants by adopting policies that ensure rapid pathways to protected status.
Asunto(s)
Migrantes , Humanos , Trastornos Mentales/epidemiología , Trastornos Mentales/psicología , Trastornos Mentales/etnología , Salud Mental , Prevalencia , Migrantes/psicología , Migrantes/estadística & datos numéricosRESUMEN
The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up-regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up-regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up-regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5-expressing leaves.
Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Humanos , Nicotiana/genética , Hemaglutininas , Respuesta de Proteína Desplegada/genética , Estrés del Retículo Endoplásmico/genéticaRESUMEN
The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co-expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin-related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.
Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Orthomyxoviridae , Humanos , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Oxilipinas/metabolismo , Agrobacterium tumefaciens/genética , Orthomyxoviridae/genética , Hojas de la Planta/genéticaRESUMEN
BACKGROUND: Inherited mutations in SERPINA1 coding for the alpha-1 antitrypsin (A1AT) protein is the only well established cause of hereditary emphysema. We aimed to identify the genetic ecause of early-onset emphysema in a five-generation French-Canadian family free of A1AT deficiency. METHODS: Between Dec 1, 2014, and April 1, 2017, we investigated 63 individuals from a single pedigree, including 55 with DNA available. Whole-exome sequencing was done in a convenience sample of 14 individuals (nine with unambiguous expression of the typical form of emphysema observed in this family). We filtered rare non-synonymous variants that were predicted to be damaging to identify a single mutation in a biologically relevant gene shared among all affected individuals. We assessed segregation with the disease in additional family members who were not evaluated by whole-exome sequencing. The effect of the candidate variant on protein function was evaluated in vitro. mRNA and protein expression of the candidate gene was assessed in lung samples from unrelated individuals (n=80) with and without emphysema who underwent surgery for lung cancer at our institution. FINDINGS: A rare in-silico-predicted damaging variant (Ala455Thr) was identified in the protein tyrosine phosphatase non-receptor type 6 (PTPN6) gene, also known as SHP-1, an important negative regulator of immune processes. 20 (95%) of 21 family members with computed tomography-confirmed emphysema were heterozygotes for the Ala455Thr mutation. No Thr455 homozygotes were identified. Emphysema or reduced diffusion capacity was observed in all heterozygotes with a history of smoking. Incomplete penetrance of the mutation and variable degrees of emphysema were observed in never smokers. The Ala455Thr mutation in SHP-1 caused a reduction in phosphatase activity in vitro, confirming the loss-of-function effect of the mutation. mRNA and protein expression of PTPN6 were upregulated in smokers, but were not associated with emphysema or severity of airflow limitation. INTERPRETATION: An inherited variant in the gene PTPN6 is responsible for early-onset emphysema in this family. To our knowledge, this is the second form of hereditary emphysema since the discovery of A1AT deficiency in the 1960s, representing a breakthrough in understanding the genetics and pathogenesis of emphysema. FUNDING: Fonds sur les maladies respiratoires J.-D. Bégin-P.-H. Lavoie de l'Université Laval, Fondation de l'Institut universitaire de cardiologie et de pneumologie de Québec, CIHR/GSK research Chair on COPD at Université Laval, and the Canadian Institutes of Health Research.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Mutación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Enfisema Pulmonar/genética , Adulto , Anciano , Anciano de 80 o más Años , Canadá , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Población BlancaRESUMEN
Smoking alters pulmonary reverse lipid transport and leads to intracellular lipid accumulation in alveolar macrophages. We investigated whether stimulating reverse lipid transport with an agonist of the liver X receptor (LXR) would help alveolar macrophages limit lipid accumulation and dampen lung inflammation in response to cigarette smoke. Mice were exposed to cigarette smoke and treated intraperitoneally with the LXR agonist T0901317. Expression of lipid capture and lipid export genes was assessed in lung tissue and alveolar macrophages. Pulmonary inflammation was assessed in the bronchoalveolar lavage (BAL). Finally, cholesterol efflux capacity and pulmonary surfactant levels were determined. In room air-exposed mice, T0901317 increased the expression of lipid export genes in macrophages and the whole lung and increased cholesterol efflux capacity without inducing inflammation or affecting the pulmonary surfactant. However, cigarette smoke-exposed mice treated with T0901317 showed a marked increase in BAL neutrophils, IL-1α, C-C motif chemokine ligand 2, and granulocyte-colony-stimulating factor levels. T0901317 treatment in cigarette smoke-exposed mice failed to increase the ability of alveolar macrophages to export cholesterol and markedly exacerbated IL-1α release. Finally, T0901317 led to pulmonary surfactant depletion only in cigarette smoke-exposed mice. This study shows that hyperactivation of LXR and the associated lipid capture/export mechanisms only have minor pulmonary effects on the normal lung. However, in the context of cigarette smoke exposure, where the pulmonary surfactant is constantly oxidized, hyperactivation of LXR has dramatic adverse effects, once again showing the central role of lipid homeostasis in the pulmonary response to cigarette smoke exposure.
Asunto(s)
Receptores X del Hígado/agonistas , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Nicotiana/toxicidad , Surfactantes Pulmonares/metabolismo , Humo/efectos adversos , Animales , Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/genética , Fumar Cigarrillos/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Homeostasis/efectos de los fármacos , Hidrocarburos Fluorados/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Cigarette smoke exposure can affect pulmonary lipid homeostasis and cause a progressive increase in pulmonary antibodies against oxidized low-density lipoproteins (OxLDL). Similarly, increased anti-OxLDL antibodies are observed in atherosclerosis, a pathology also tightly associated with smoking and lipid homeostasis disruption. Several immunization strategies against oxidized lipid species to help with their clearance have been shown to reduce the formation of atherosclerotic lesions. Since oxidized lipids are generated during cigarette smoke exposure, we investigated the impact of a prophylactic immunization protocol against OxLDL on the pulmonary effects of cigarette smoke exposure in mice. METHODS: Mice were immunized systemically with a mixture of human OxLDL (antigen source) and AddaVax (adjuvant) or PBS alone prior to the initiation of acute (2 week) or sub-chronic (8 weeks) cigarette smoke exposure protocols. Anti-OxLDL antibodies were measured in the bronchoalveolar lavage (BAL) fluid and serum by direct ELISA. Pulmonary impacts of cigarette smoke exposure and OxLDL immunization were assessed by measuring BAL inflammatory cells, lung functions, and changes in lung structure and gene levels of matrix/matrix-related genes. RESULTS: Immunization to OxLDL led to a marked increase in circulating and pulmonary antibodies against OxLDL that persisted during cigarette smoke exposure. OxLDL immunization did not exacerbate or reduce the inflammatory response following acute or sub-chronic exposure to cigarette smoke. OxLDL immunization alone had effects similar to cigarette smoke exposure on lung functions but OxLDL immunization and cigarette smoke exposure had no additive effects on these parameters. No obvious changes in lung histology, airspace or levels of matrix and matrix-related genes were caused by OxLDL immunization compared to vehicle treatment. CONCLUSIONS: Overall, this study shows for the first time that a prophylactic immunization protocol against OxLDL can potentially have detrimental effects lung functions, without having additive effects over cigarette smoke exposure. This work sheds light on a complex dynamic between anti-OxLDL antibodies and the pulmonary response to cigarette smoke exposure.
Asunto(s)
Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/inmunología , Lipoproteínas LDL/inmunología , Trastornos Respiratorios/inmunología , Trastornos Respiratorios/prevención & control , Humo/efectos adversos , Administración por Inhalación , Animales , Femenino , Humanos , Inmunización , Lipoproteínas LDL/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Trastornos Respiratorios/inducido químicamenteRESUMEN
E-cigarette use has exploded in the past years, especially among young adults and smokers desiring to quit. While concerns are mostly based on the presence of nicotine and flavors, pulmonary effects of propylene glycol and glycerol inhalation, the main solvents of e-liquid have not been thoroughly investigated. In this preclinical study, mice were exposed 2 h daily for up to 8 weeks to vapors of propylene glycol and/or glycerol generated by an e-cigarette. Lung transcriptome analysis revealed it affected the expression level of genes of the circadian molecular clock, despite causing no inflammatory response. Periodical sacrifices showed that the rhythmicity of these regulatory genes was indeed altered in the lungs, but also in the liver, kidney, skeletal muscle, and brain. E-cigarette exposure also altered the expression of rhythmic genes (i.e., hspa1a and hspa1b), suggesting that alterations to the 'clock genes' could translate into systemic biological alterations. This study reveals that the major solvents used in e-cigarettes propylene glycol and glycerol, not nicotine or flavors, have unsuspected effects on gene expression of the molecular clock that are to be taken seriously, especially considering the fundamental role of the circadian rhythm in health and disease.
Asunto(s)
Glicerol/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Pulmón/efectos de los fármacos , Propilenglicol/farmacología , Vapeo/efectos adversos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Femenino , Proteínas HSP70 de Choque Térmico/genética , Riñón/efectos de los fármacos , Riñón/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismoRESUMEN
Reverse lipid transport is critical to maintain homeostasis. Smoking causes lipid accumulation in macrophages, therefore suggesting suboptimal reverse lipid transport mechanisms. In this study, we investigated the interplay between smoking and reverse lipid transport and the consequences on smoking-induced lung and peripheral alterations.To investigate the relationship between smoking and reverse lipid transport, we used a clinical lung gene expression dataset and a mouse model of cigarette smoke exposure. We also used ApoA-1-/- mice, with reduced reverse lipid transport capacity, and a recombinant ApoA-1 Milano/phospholipid complex (MDCO-216) to boost reverse lipid transport. Cellular and functional analyses were performed on the lungs and impact on body composition was also assessed.Smoking affects pulmonary expression of abca1, abcg1, apoe and scarb1 in both mice and humans, key genes involved in reverse lipid transport. In mice, the capacity of bronchoalveolar lavage fluid and serum to stimulate cholesterol efflux in macrophages was increased after a single exposure to cigarette smoke. ApoA-1-/- mice showed increased lung neutrophilia, larger macrophages and greater loss in lean mass in response to smoking, whereas treatment with MDCO-216 reduced the size of macrophages and increased the lean mass of mice exposed to cigarette smoke.Altogether, this study shows a functional interaction between smoking and reverse lipid transport, and opens new avenues for better understanding the link between metabolic and pulmonary diseases related to smoking.
Asunto(s)
Apolipoproteína A-I/farmacología , Fumar Cigarrillos/efectos adversos , Metabolismo de los Lípidos , Pulmón/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Fosfatidilcolinas/farmacología , Animales , Apolipoproteína A-I/genética , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Combinación de Medicamentos , Femenino , Expresión Génica , Humanos , Pulmón/metabolismo , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
INTRODUCTION AND HYPOTHESIS: Obstetric fistula, caused by traumatic delivery and patient lack of access to obstetric care, is an important public health concern in developing countries, particularly in Sub-Saharan Africa. This research focuses on the experience of women living with obstetric fistula in Burkina Faso as well as their reintegration into community after surgery. METHODS: This project was funded by the Mères du Monde en Santé (MMS) Foundation and conducted in collaboration with the Boromo Hospital. A qualitative approach based on grounded theory and using the principles of participative action research (PAR) was used with semidirected interviews prior to surgery and follow-up interviews 1-2 years after surgery directly in the women's village of origin. Thirty-nine participants were recruited between 2012 and 2015. RESULTS: The results point to circumstances leading to obstetric fistula development: poverty, gender inequality in terms of decision making, healthcare-system deficiencies, and lack of services for referral and treatment of this condition. Our results reinforce the knowledge about the social and psychological repercussions of fistula by exploring the concepts of gossips, shame and self-exclusion as powerful mechanisms of exclusion, but they also show that social support was conserved for several women through their journey with this disease. There was complete social rehabilitation within the community after surgery; however, persistent barriers in term of anxiety regarding obstetric future and economic insecurity were present. CONCLUSIONS: Early recruitment for surgery and prevention are the main objectives when attempting to reduce the impact of obstetric fistula and facilitate patient reintegration. Improvements in local and governmental public health policies are required.
Asunto(s)
Fístula Vaginal/etnología , Fístula Vaginal/psicología , Adulto , Anciano , Burkina Faso , Parto Obstétrico/efectos adversos , Femenino , Humanos , Persona de Mediana Edad , Embarazo , Conducta Social , Fístula Vaginal/etiología , Fístula Vaginal/cirugía , Adulto JovenRESUMEN
Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/fisiopatología , Proteínas Quinasas/metabolismo , ARN Largo no Codificante/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Western Blotting , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Células HEK293 , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Activación Transcripcional , Regulación hacia ArribaRESUMEN
BACKGROUND: Impaired skeletal muscle regeneration could contribute to the progression of muscle atrophy in patients with chronic obstructive pulmonary disease (COPD). METHODS: Satellite cells and myogenesis-related proteins were compared between healthy subjects and patients with COPD, with or without muscle atrophy. Satellite cells were isolated and cultured to assess their proliferative and differentiation aptitudes. RESULTS: Although satellite cell numbers in muscle samples were similar between groups, the proportion of muscle fibers with central nuclei was increased in COPD. In muscle homogenates, increased expression of MyoD and decreased expression of myogenin and MRF4 were observed in COPD. In cultured satellite cells of patients with COPD, increased protein content was observed for Pax7, Myf5 (proliferation phase) and myogenin (differentiation phase) while myosin heavy chain protein content was significantly lower during differentiation. CONCLUSION: In COPD, the number of central nuclei was increased in muscle fibers suggesting a greater number of attempts to regenerate muscle tissue than in healthy subjects. Myogenesis signaling was also altered in muscle homogenates in patients with COPD and there was a profound reduction in the differentiation potential in this population as indicated by a reduced ability to incorporate myosin heavy chain into newly formed myotubes. Collectively, these results indicate that skeletal muscle regenerative capacity termination is impaired in COPD and could contribute to the progression of muscle atrophy progression in this population.
Asunto(s)
Atrofia Muscular/diagnóstico , Atrofia Muscular/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Músculo Cuádriceps/fisiopatología , Regeneración/fisiología , Anciano , Células Cultivadas , Humanos , Masculino , Persona de Mediana Edad , Músculo Cuádriceps/fisiologíaRESUMEN
RATIONALE: The maintenance of peripheral muscle mass may be compromised in chronic obstructive pulmonary disease (COPD) due to premature cellular senescence and exhaustion of the regenerative potential of the muscles. METHODS: Vastus lateralis biopsies were obtained from patients with COPD (n = 16) and healthy subjects (n = 7). Satellite cell number and the proportion of central nuclei, as a marker of muscle regenerative events, were assessed on cryosections. Telomere lengths, used as a marker of cellular senescence, were determined using Southern blot analyses. RESULTS: Central nuclei proportion was significantly higher in patients with COPD with a preserved muscle mass compared to controls and patients with COPD with muscle atrophy (p<0.001). In COPD, maximal telomere length was significantly decreased compared to controls (p<0.05). Similarly, minimal telomere length was significantly reduced in GOLD III-IV patients with muscle atrophy compared to controls (p<0.005). Minimal, mean and maximum telomere lengths correlated with mid-thigh muscle cross-sectional area (MTCSA) (R = 0.523, p = 0.005; R = 0.435, p = 0.019 and R = 0.491, p = 0.009, respectively). CONCLUSIONS: Evidence of increased regenerative events was seen in GOLD III-IV patients with preserved muscle mass. Shortening of telomeres in GOLD III-IV patients with muscle atrophy is consistent with an increased number of senescent satellite cells and an exhausted muscle regenerative capacity, compromising the maintenance of muscle mass in these individuals.
Asunto(s)
Músculo Esquelético/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Células Satélites Perineuronales/patología , Anciano , Southern Blotting , Técnica del Anticuerpo Fluorescente , Humanos , TelómeroRESUMEN
BACKGROUND: The mechanisms through which neuromuscular electrical stimulation (NMES) training may improve limb muscle function and exercise tolerance in COPD are poorly understood. We investigated the functional and muscular effects of NMES in advanced COPD. METHODS: Twenty of 22 patients with COPD were randomly assigned to NMES (n = 12) or sham (n = 8) training in a double-blind controlled study. NMES was performed on quadriceps and calf muscles, at home, 5 days per week for 6 weeks. Quadriceps and calf muscle cross-sectional area (CSA), quadriceps force and endurance, and the shuttle-walking distance with cardiorespiratory measurements were assessed before and after training. Quadriceps biopsy specimens were obtained to explore the insulin-like growth factor-1/AKT signaling pathway (70-kDa ribosomal S6 kinase [p70S6K] , atrogin-1). RESULTS: NMES training improved muscle CSA (P < .05), force, and endurance (P < .03) when compared with sham training. Phosphorylated p70S6K levels (anabolism) were increased after NMES as compared with sham (P = .03), whereas atrogin-1 levels (catabolism) were reduced (P = .01). Changes in quadriceps strength and ventilation during walking contributed independently to variations in walking distance after training (r = 0.77, P < .001). Gains in walking distance were related to the ability to tolerate increasing current intensities during training (r = 0.95, P < .001). CONCLUSIONS: In patients with severe COPD, NMES improved muscle CSA. This was associated with a more favorable muscle anabolic to catabolic balance. Improvement in walking distance after NMES training was associated with gains in muscle strength, reduced ventilation during walking, and the ability to tolerate higher stimulation intensity. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00874965; URL: www.clinicaltrials.gov.
Asunto(s)
Terapia por Estimulación Eléctrica , Fuerza Muscular/fisiología , Músculo Esquelético/fisiopatología , Unión Neuromuscular/fisiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/terapia , Índice de Severidad de la Enfermedad , Anciano , Biopsia , Método Doble Ciego , Tolerancia al Ejercicio/fisiología , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Resistencia Física/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal/fisiología , Caminata/fisiologíaRESUMEN
BACKGROUND: Factors involved in the regulation of muscle mass in chronic obstructive pulmonary disease (COPD) are still poorly understood. Comparing the signalisation involved in muscle mass regulation between two muscles with different levels of activation within the same subjects is an interesting strategy to tease out the impact of local (muscle activity) versus systemic factors in the regulation of muscle mass. A study was undertaken to measure and compare the protein levels of p-AKT, AKT, Atrogin-1, p-p70S6K, p-4E-BP1, p-GSK3ß as well as the mRNA expression of Atrogin-1, MuRF1 and FoxO-1 in the quadriceps and the diaphragm of 12 patients with COPD and 7 controls with normal lung function. METHODS: Diaphragm biopsies were obtained during thoracic surgery and quadriceps samples were obtained from needle biopsies. Protein content and mRNA expression were measured by western blot and quantitative PCR, respectively. RESULTS: Increased mRNA expressions of Atrogin-1, MuRF1 and FoxO-1 were found in the quadriceps compared with the diaphragm only in patients with COPD. The quadriceps/diaphragm ratio for MuRF1 was higher in COPD. The protein level of p-p70S6K was decreased in the quadriceps compared with the diaphragm in patients with COPD. The quadriceps/diaphragm ratios of p-p70S6K and p-GSK3ß were lower in patients with COPD than in controls. CONCLUSIONS: These results indicate a greater susceptibility to a catabolic/anabolic imbalance favouring muscle atrophy in the quadriceps compared with the diaphragm in patients with COPD. The balance between the atrophy and hypertrophy signalling is inhomogeneous between respiratory and lower limb muscles, suggesting that local factors are likely to be involved in the regulation of muscle mass in COPD.
Asunto(s)
Diafragma/patología , Atrofia Muscular/etiología , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Músculo Cuádriceps/patología , Anciano , Biopsia , Diafragma/metabolismo , Femenino , Volumen Espiratorio Forzado/fisiología , Regulación de la Expresión Génica , Humanos , Hipertrofia/etiología , Hipertrofia/patología , Masculino , Persona de Mediana Edad , Proteínas Musculares/biosíntesis , Proteínas Musculares/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Atrofia Muscular/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Músculo Cuádriceps/metabolismo , ARN Mensajero/genética , Capacidad Vital/fisiologíaRESUMEN
Since hypoxia might contribute to the development of muscle atrophy, we wished to provide direct evidence linking hypoxia to muscle atrophy. By evaluating protein degradation and synthesis in hypoxic myotubes we found a significant reduction in total protein content. Using functional assays we observed protein degradation elevation in the first 24 h while synthesis was maintained during this period and then significantly decrease at 48 h. These results demonstrate a temporal regulation of protein homeostasis, whereby elevated protein degradation is followed by a reduction in synthesis. These results are comparable to the cellular adaptation seen during development of muscle atrophy.
Asunto(s)
Hipoxia de la Célula , Homeostasis , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Cartilla de ADN , Hidrólisis , Contracción Muscular , Reacción en Cadena de la Polimerasa , RatasRESUMEN
BACKGROUND: Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. RESULTS: This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. CONCLUSION: These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.
Asunto(s)
Empalme Alternativo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , ADN sin Sentido/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Poli A/genética , Secuencias Repetidas Terminales/genética , Transcripción Genética , Proteínas Virales/genética , Leucina Zippers/genética , Proteínas de los RetroviridaeRESUMEN
Similar to several other viruses, human T cell leukemia virus type I (HTLV-I) induces the formation of multinucleated giant cells (also known as syncytium) when amplified in tissue culture. These syncytia result from the fusion of infected cells with uninfected cells. Due to the intrinsic difficulty of infecting cells with cell-free HTLV-I virions, syncytium formation has become an important tool in the study of HTLV-I infection and transmission. Since most HTLV-I-based cell fusion assays rely on the use of non-T cells, the aim of this study was to optimize a new HTLV-I-induced cell fusion assay in which HTLV-I-infected T cell lines are co-cultured with T cells that have been transfected with an HTLV-I long terminal repeat (LTR) luciferase reporter construct. We demonstrate that co-culture of various HTLV-I-infected T cells with different transfected T cell lines resulted in induction of luciferase activity. Cell-to-cell contact and expression of the viral gp46 envelope protein was crucial for this induction while other cell surface proteins (including HSC70) did not have a significant effect. This quantitative assay was shown to be very sensitive. In this assay, the cell fusion-mediated activation of NF-kappaB and the HTLV-I LTR occurred through previously described Tax-dependent signaling pathways. This assay also showed that cell fusion could activate Tax-inducible cellular promoters. These results thus demonstrate that this new quantitative HTLV-I-dependent cell fusion assay is versatile, highly sensitive, and can provide an important tool to investigate cellular promoter activation and intrinsic signaling cascades that modulate cellular gene expression.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Fusión Celular , Línea Celular , Técnicas de Cocultivo , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Células Jurkat , Mapeo Restrictivo , Sensibilidad y Especificidad , Virión/inmunologíaRESUMEN
Human T-cell leukemia virus type I (HTLV-I) transcription generally depends on the ability of the viral Tax protein to bind the CREB transcription factor and form an active complex by recruiting CBP/p300 coactivators to the long terminal repeat (LTR). Studies have demonstrated that T-cell activating agents that stimulate CREB are potent inducers of HTLV-I transcription. Herein, we demonstrate that bpV[pic], a protein tyrosine phosphatase (PTP) inhibitor activates the HTLV-I LTR in the presence and absence of Tax expression. Optimal activation occurred at 8 h and was synergistic with forskolin or PGE(2). Infected cell lines and cells transfected with HTLV-I proviral DNA were equally responsive to the synergistic effect of bpV and forskolin on HTLV-I gene expression. Activation of the LTR by bpV[pic] was T-cell receptor-independent, but required ZAP70, calcineurin activity and functional calcium entry. Inhibition of the SHP-1 PTP was suggested to be important. Transfection experiments with a CREB dominant-negative mutant and with isolated TRE1- or CREB-responsive reporter constructs and treatment with the MDL-12,330A adenylate cyclase inhibitor all supported the involvement of a CREB/ATF family member in this bpV-dependent activation of the HTLV-I LTR, although CREB itself did not seem to be involved. Analysis of HTLV-I reporter constructs containing mutated CREB-binding sites also implied the involvement of another element in this activation. These results demonstrate for the first time a powerful effect of PTP inhibitors on HTLV-I LTR activity and suggest participation of both CREB-dependent and -independent pathways in this activation.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus Linfotrópico T Tipo 1 Humano/genética , Proteínas Tirosina Fosfatasas/fisiología , Transcripción Genética/efectos de los fármacos , Vanadatos/farmacología , Línea Celular , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Dinoprostona/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen tax/fisiología , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Compuestos Organometálicos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencias Repetidas Terminales , Activación Viral , Proteína Tirosina Quinasa ZAP-70RESUMEN
The enhancer region in the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) is very important for viral transcription. This promoter sequence binds both nuclear factor-kappaB and NFAT, two important modulators of HIV-1 gene expression. Previous studies have indicated that the enhancer regions of the different HIV-1 clade LTRs differ in their number of NF-kappaB-binding sites. In this study, we have compared the activation potential of the different HIV-1 clade and HIV-2 LTRs and assessed their interaction with NFAT and NF-kappaB. In T-cell lines and primary CD4(+) T-cells, the results showed that the HIV-1 clade E LTR (with a single NF-kappaB-binding site) was the weakest LTR regardless of the tested activators, whereas the HIV-2 LTR was the most responsive LTR. The clade E enhancer region was also demonstrated to be the weakest enhancer region in transfection experiments with luciferase reporter-based vectors. Electrophoretic mobility shift assays with extracts from activated CD4(+) T-cells indicated that, although NF-kappaB and NFAT bound all enhancers, HIV-1 clade E and HIV-2 LTR enhancers were poor binding targets for these two factors. Weak NFAT binding to clade E enhancers was also confirmed using NFAT1-expressing 293T cells in competition experiments. We have also shown the absence of interaction of NF-kappaB or NFAT with the third NF-kappaB repeat present in clade C. However, the clade C enhancer bound NFAT more efficiently than all other enhancer regions tested. Our results hence demonstrate for the first time that differences in the binding of NF-kappaB and NFAT to the enhancer regions could be responsible for some of the observed variation in HIV-1 clade LTR activation, whereas HIV-2 LTR activation seems mostly independent of these interactions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , VIH-1/genética , Activación de Linfocitos , FN-kappa B/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Unión Competitiva , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Genes Reporteros , Vectores Genéticos , Humanos , Hibridomas/metabolismo , Células Jurkat , Luciferasas/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Secuencias Repetidas Terminales , Transcripción Genética , TransfecciónRESUMEN
Infection with human T-cell leukemia virus type 1 (HTLV-1) is characterized by long latency periods, indicating that viral gene expression is under tight control. There is presently little information available regarding the nature of extracellular stimuli that can transactivate the regulatory elements of HTLV-1 (i.e., long terminal repeat [LTR]). To gain insight into the biological importance of externally induced activation pathways in virus gene expression, primary and established T cells were transfected with HTLV-1-based reporter gene vectors and then were treated with agents that cross-linked the T-cell receptor (TCR) or the costimulatory CD28 molecule with prostaglandin E(2) (PGE(2)). We demonstrated that a potent induction of HTLV-1 LTR-driven reporter gene activity was seen only when the three agents were used in combination. Interestingly, similar observations were made when using C91/PL, a cell line that carries integrated HTLV-1 proviral DNA. This TCR-CD28-PGE(2)-mediated increase in virus transcription was dependent on protein kinase A activation and induction of the cAMP response element binding protein. Experiments with a mutated reporter construct further revealed the importance of the Tax-responsive elements in the HTLV-1 LTR in the observed up regulation of virus gene expression when TCR/CD28 engagement was combined with PGE(2) treatment. The protein tyrosine kinases p56(lck) and the transmembrane tyrosine phosphatase CD45 were all found to be involved in TCR-CD28-PGE(2)-directed increase in HTLV-1 LTR activity. This study presents new information on the possible mechanisms underlying reactivation of this retrovirus.