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Quadriceps muscle weakness and wasting are common comorbidities in chronic obstructive pulmonary disease (COPD). Micro-RNA expression upregulation may favor muscle mass growth and differentiation. We hypothesized that the profile of muscle-enriched micro-RNAs in cultured myotubes differs between patients with COPD of a wide range of body composition and healthy controls and that expression levels of those micro-RNAs from patients with COPD and controls differ between in vivo and in vitro conditions. Twenty-nine patients with COPD [ n = 15 with muscle wasting and fat-free mass index (FFMI) 15 kg/m2 and n = 14 with normal body composition and FFMI 18 kg/m2] and 10 healthy controls (FFMI 19 kg/m2) were consecutively recruited. Biopsies from the vastus lateralis muscle were obtained in all study subjects. A fragment of each biopsy was used to obtain primary cultures, in which muscle cells were first proliferated to be then differentiated into actual myotubes. In both sets of experiments (in vivo biopsies and in vitro myotubes) the following muscle-enriched micro-RNAs from all the study subjects were analyzed using quantitative real-time PCR amplification: micro-RNA (miR)-1, miR-133a, miR-206, miR-486, miR-29b, miR-27a, and miR-181a. Whereas the expression of miR-1, miR-206, miR-486, and miR-29b was upregulated in the muscle biopsies of patients with COPD compared with those of healthy controls, levels of none of the studied micro-RNAs in the myotubes (primary cultured cells) significantly differed between patients with COPD and the controls. We conclude from these findings that environmental factors (blood flow, muscle metabolism, and inflammation) taking place in vivo (biopsies) in muscles may account for the differences observed in micro-RNA expression between patients with COPD and controls. In the myotubes, however, the expression of the same micro-RNAs did not differ between the study subjects as such environmental factors were not present. These findings suggest that therapeutic strategies should rather target environmental factors in COPD muscle wasting as the profile of micro-RNA expression in myotubes was similar in patients to that observed in the healthy controls. NEW & NOTEWORTHY Environmental factors taking place in vivo (biopsies) in the muscles may explain differences observed in micro-RNA expression between patients with chronic obstructive pulmonary disease (COPD) and controls. In the myotubes, however, the expression of the same micro-RNAs did not differ between the study subjects as such environmental factors were not present. These findings suggest that therapeutic strategies should rather target environmental factors in COPD muscle wasting and cachexia as micro-RNA expression profile in myotubes was similar between patients and controls. This study was supported by Centro de Investigación en Red de Enfermedades Respiratorias, Fondo de Investigación Sanitaria (FIS) 14/00713 [Fondo Europeo de Desarrollo Regional (FEDER)], FIS 18/00075 (FEDER), Grant SAF2014-54371, Sociedad Española de Neumología y Cirugía Torácica (SEPAR) 2016, SEPAR 2018, Fundació Catalana de Pneumologia 2016, and an unrestricted grant from Menarini SA 2018 (Spain). E. Barreiro was a recipient of the European Respiratory Society COPD Research Award 2008.

Chronic obstructive pulmonary disease (COPD) is an entity with a heterogeneous presentation. For this reason, attempts have been made to characterize different phenotypes and endotypes to enable a more individualized approach. The aim of the Biomarkers in COPD (BIOMEPOC) project is to identify useful biomarkers in blood to improve the characterization of patients. Clinical data and blood samples from a group of patients and healthy controls will be analyzed. The project will consist of an exploration phase and a validation phase. Analytical parameters in blood will be determined using standard techniques and certain 'omics' (transcriptomics, proteomics, and metabolomics). The former will be hypothesis-driven, whereas the latter will be exploratory. Finally, a multilevel analysis will be conducted. Currently, 269 patients and 83 controls have been recruited, and sample processing is beginning. Our hope is to use the results to identify new biomarkers that, alone or combined, will allow a better characterization of patients.

There is a specific COPD phenotype known as ‘frequent exacerbator’. Many of the exacerbations suffered by these patients are severe and require hospitalization (SFECOPD). In a previous study we analyzed the transcriptomic characteristics found in the blood of SFECOPD patients recruited in different hospitals. However, results were very heterogeneous. Our present AIM was to analyze the genetic expression profile in the blood of SFECOPD patients from a single center, where hospitalization requires strict objective criteria. Methods: 20 COPD patients (half of them with SFECOPD, >3 hospitalizations in the previous year) from a referral center were included, and blood samples were obtained and processed for the transcriptomic analysis. Results: SFECOPD showed changes in the expression (FDR 1500 genes, 60 of them showing a full change >50%. Those genes encoding transmembrane proteins TMEM176 A & B and KIR2DS4 (detection-destruction of ‘abnormal cells’, Δ -370%, -300% & +260%, respectively), cell adhesion molecule CEACAM6 (Δ+160%, increase in susceptibility to bacterial infections), as well as proteases CTSG & ELANE, defensines DEFA 3 & 4, and RNASE3 (Δ+130 to +140%, all of them with antibacterial activity) stood out among them. Moreover, GO analysis also revealed that pathways most significantly involved in changes were immune responses against bacteria and fungi (5 genes each; OR 89 and 67, respectively), and those mediated by antimicrobial peptides (6 genes, OR 48). Conclusion: Blood of SFECOPD exhibits a differentiated expression profile, which is mainly characterized by changes in genes related with the immune response against infections. Funded SAF2014-54371, Menarini, SEPAR 15 & 16

BackgroundThe respiratory microbiome is altered in COPD patients but its relationship with core components of the disease, such as the severity of airflow limitation, the frequency of exacerbations or the circulating levels of eosinophils, is unclear.MethodsCross-sectional study comprising 72 clinically stable COPD patients (mean age 68 [SD 7.9] years; FEV1 48.7 [SD 20.1]% of reference) who provided spontaneous sputum samples for 16S rRNA gene amplification and sequencing. The microbiome composition was analysed with QIIME.ResultsWe observed that: (1) more severe airflow limitation was associated with reduced relative abundance (RA) of Treponema and an increase in Pseudomonas; (2) patients with 2 exacerbations the previous year showed a significantly different bacterial community with respect to non-exacerbators (p=0.014), with changes in 13 genera, including an increase of Pseudomonas, and finally, (3) peripheral eosinophils levels 2% were associated with more diverse microbiome [Chao1 224.51 (74.88) vs 277.39 (78.92) p=0.006; Shannon 3.94 (1.05) vs 4.54 (1.06) p=0.020], and a significant increase in the RAs of 20 genera.ConclusionThe respiratory microbiome in clinically stable COPD patients varies significantly according to the severity of airflow limitation, previous history of exacerbations and circulating eosinophils levels., This study was funded by FIS PI15/00167 and PI15/02042 from Instituto de Salud Carlos III (ISCIII), SAF 2014-54371 (FEDER) from Ministerio de Economia y Competitividad, Sociedad Espanola de Neumologia y Cirugia Toracica (SEPAR), Laboratorios Menarini S. A, Fundacio Pla Armengol and Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBERES). CIBERES is an initiative of the ISCIII.; This work was also supported by grants from Agencia de Gestio d'Ajuts Universitaris i de Recerca-Generalitat de Catalunya (2017 GRC-716).

[ES] La enfermedad pulmonar obstructiva crónica (EPOC) es una entidad de presentación heterogénea. Por ello, se han intentado perfilar diferentes fenotipos y endotipos, que permitirían un manejo más diferenciado. El objetivo del proyecto Biomarcadores en la EPOC (BIOMEPOC) es identificar biomarcadores sanguíneos útiles para tipificar mejor a los enfermos. Se analizarán datos clínicos y muestras sanguíneas en un grupo de pacientes y controles sanos. El proyecto constará de fases de prospección y de validación. Se realizarán determinaciones analíticas sanguíneas con técnicas convencionales y de diversas ciencias «ómicas» (transcriptómica, proteómica y metabolómica). Las primeras se realizarán orientadas por hipótesis, mientras que con las segundas se realizará una exploración sin dicho condicionante. Finalmente se realizará un análisis multinivel. En el momento actual se han reclutado 269 pacientes y 83 controles, y se está iniciando el procesamiento de muestras. Con los resultados obtenidos se espera identificar nuevos biomarcadores que, en solitario o combinados, permitan una mejor tipificación de los pacientes., [EN] Chronic obstructive pulmonary disease (COPD) is an entity with a heterogeneous presentation. For this reason, attempts have been made to characterize different phenotypes and endotypes to enable a more individualized approach. The aim of the Biomarkers in COPD (BIOMEPOC) project is to identify useful biomarkers in blood to improve the characterization of patients. Clinical data and blood samples from a group of patients and healthy controls will be analyzed. The project will consist of an exploration phase and a validation phase. Analytical parameters in blood will be determined using standard techniques and certain ‘omics’ (transcriptomics, proteomics, and metabolomics). The former will be hypothesis-driven, whereas the latter will be exploratory. Finally, a multilevel analysis will be conducted. Currently, 269 patients and 83 controls have been recruited, and sample processing is beginning. Our hope is to use the results to identify new biomarkers that, alone or combined, will allow a better characterization of patients.

Objective: To evaluate the relationships between respiratory microbiome and main COPD phenotyes Methods: Sputum samples from COPD patients were obtained during stability and 16S rRNA gene was analyzed. Taxonomic and diversity information were correlated with frequent exacerbator (FE) and eosinophilic COPD (EC) phenotypes Results: 78 subjects were analyzed, mean age 67 (SD 9) years. Patients with ≥2 exacerbations the previous year were considered FE (n=23). Their bacterial community was significantly different (p=0.014) from patients with no exacerbations (n=31), with changes in RA of 3 phyla and 13 genera, which included an increase in Pseudomonas. Patients with 1 exacerbation (n=18) showed borderline differences in their bacterial community (p=0.081) compared to no exacerbations patients, with a loss of RA of 2 phyla and 13 genera. Patients with 1 or ≥2 exacerbations showed similar borderline differences (p=0.087) with changes in RA of 4 genera. EC patients, identified by a systemic eosinophil level ≥2%, showed a significantly more diverse respiratory microbiome [Chao1 224.5 (74.9) vs 280.5 (79.6) p=0.003; Shannon 3.9 (1.1) vs 4.6 (1.1) p= 0.010] and a different community structure (p=0.037), mediated by a significant increment of 4 phyla and 26 genera Conclusion: A specific bacterial composition was shown in stability samples of FE and EC phenotypes. The presence of 1 exacerbation the previous year produces significant changes in the RA of specific OTUs and in patients with ≥2 exacerbations these changes lead to significant differences in their bacterial community. The respiratory microbiome was more diverse in EC patients and with a specific bacterial community Funded by FIS 15/00167, SEPAR and BRN-Pla Armengol

Chronic obstructive pulmonary disease (COPD) is an entity with a heterogeneous presentation. For this reason, attempts have been made to characterize different phenotypes and endotypes to enable a more individualized approach. The aim of the Biomarkers in COPD (BIOMEPOC) project is to identify useful biomarkers in blood to improve the characterization of patients. Clinical data and blood samples from a group of patients and healthy controls will be analyzed. The project will consist of an exploration phase and a validation phase. Analytical parameters in blood will be determined using standard techniques and certain 'omics' (transcriptomics, proteomics, and metabolomics). The former will be hypothesis-driven, whereas the latter will be exploratory. Finally, a multilevel analysis will be conducted. Currently, 269 patients and 83 controls have been recruited, and sample processing is beginning. Our hope is to use the results to identify new biomarkers that, alone or combined, will allow a better characterization of patients.

High-intensity exercise induces oxidative stress and inflammatory events in muscles. Tumor necrosis factor (TNF)-α may alter muscle protein metabolism or promote muscle regeneration. We hypothesized that a program of noninvasive chronic inspiratory loading of different intensities induces a differential pattern of physiological, molecular, and cellular events within rat diaphragms. Antioxidants and TNF-α blockade may influence those events. In the diaphragm, gastrocnemius, and blood of rats exposed to high-intensity inspiratory threshold loads (2 hour every 24 hours for 14 days), with and without treatment with N-acetyl cysteine or infliximab (anti-TNF-α antibody), inflammatory cells and cytokines, superoxide anion production, myogenesis markers, and muscle structure were explored. In all animals, maximum inspiratory pressure (MIP) and body weight were determined. High-intensity inspiratory loading for 2 weeks caused a decline in MIP and body weight, and in the diaphragm induced a reduction in fast-twitch fiber proportions and sizes, whereas inflammatory cells and cytokine levels, including TNF-α immunohistochemical expression, superoxide anion, internal nuclei counts, and markers of myogenesis were increased. Blockade of TNF-α improved respiratory muscle function and structure, and animal weight, and, in the diaphragm, reduced inflammatory cell numbers and superoxide anion production drastically while inducing larger increases in protein and messenger RNA levels and immunohistochemical expression of TNF-α, internal nuclei, and markers of muscle regeneration. Blunting of TNF-α also induced a reduction in blood inflammatory cytokines and superoxide anion production. We conclude that TNF-α synthesized by inflammatory cells or myofibers could have differential effects on muscle structure and function in response to chronic, noninvasive, high-intensity inspiratory threshold loading.

Resumen Fundamento y objetivo El estres oxidativo y la inflamacion contribuyen al deterioro de la funcion contractil del diafragma observado en modelos animales de sepsis y endotoxemia. En los pacientes con sepsis, no se han evaluado nunca episodios moleculares en sus musculos respiratorios. En este estudio se evaluaron los niveles de estres oxidativo e inflamacion en un musculo respiratorio, el intercostal externo, y de las extremidades, el vasto lateral, de pacientes con sepsis. Pacientes y metodo En el intercostal externo y vasto lateral de pacientes con sepsis grave y/o shock septico se determinaron valores de proteinas oxidadas y nitradas, aductos proteicos de malondialdehido y de hidroxinonenal, enzimas antioxidantes catalasa y Mn-superoxido dismutasa, tumor necrosis factor (TNF, «factor de necrosis tumoral»)-α, receptores i y ii del TNF-α, interleucina (IL)-1 e IL-6, marcador panleucocitario CD18, y composicion fibrilar mediante Western blot, reaccion en cadena de la polimerasa, e inmunohistoquimica. Resultados Respecto de los controles, en los pacientes con sepsis, los niveles de proteinas oxidadas y nitradas estaban aumentados en el vasto lateral, pero no en el intercostal externo, los de enzimas antioxidantes no difirieron, los de citocinas inflamatorias fueron superiores en ambos musculos, y los porcentajes y tamanos de las fibras musculares no mostraron diferencias para ningun musculo entre ambos grupos. Conclusiones En pacientes con sepsis grave incipiente, la distinta actividad de los musculos respiratorios y de las extremidades puede explicar el patron diferencial de estres oxidativo e inflamacion observado en los mismos. Estos hallazgos podrian tener implicaciones importantes para el manejo clinico y terapeutico de estos pacientes.

Muscle dysfunction is frequently observed in chronic obstructive pulmonary disease (COPD) patients, contributing to their exercise limitation and a worsening prognosis. The main factor leading to limb muscle dysfunction is deconditioning, whereas respiratory muscle dysfunction is mostly the result of pulmonary hyperinflation. However, both limb and respiratory muscles are also influenced by other negative factors, including smoking, systemic inflammation, nutritional abnormalities, exacerbations and some drugs. Limb muscle weakness is generally diagnosed through voluntary isometric maneuvers such as handgrip or quadriceps muscle contraction (dynamometry); while respiratory muscle loss of strength is usually recognized through a decrease in maximal static pressures measured at the mouth. Both types of measurements have validated reference values. Respiratory muscle strength can also be evaluated determining esophageal, gastric and transdiaphragmatic maximal pressures although there is a lack of widely accepted reference equations. Non-volitional maneuvers, obtained through electrical or magnetic stimulation, can be employed in patients unable to cooperate. Muscle endurance can also be assessed, generally using repeated submaximal maneuvers until exhaustion, but no validated reference values are available yet. The treatment of muscle dysfunction is multidimensional and includes improvement in lifestyle habits (smoking abstinence, healthy diet and a good level of physical activity, preferably outside), nutritional measures (diet supplements and occasionally, anabolic drugs), and different modalities of general and muscle training.

Chronic obstructive pulmonary disease (COPD) is a complex disorder with extrapulmonary manifestations. Even though there is some knowledge regarding sex differences in the lung disease, little is known about extrapulmonary manifestations. Our aim was to analyze the specific profile of muscle dysfunction, structure, and biology in COPD women. Twenty-one women and 19 men with stable COPD as well as 15 controls were included. Nutritional status, physical activity, lung and muscle function, exercise capacity, and quality of life were assessed. In addition, blood, breath condensate, and quadriceps muscle samples were tested for inflammatory markers. Moreover, fiber phenotype, signs of damage–regeneration, and the expression of key genes linked to myogenesis and inflammation were assessed in the muscle. Inflammatory markers were increased in all body compartments but no correlation was found among them. Muscle dysfunction was present in both COPD groups but was more marked in women. The opposite occurred with the increase in the percentage of type II fibers that was lower in women despite a similar level of airway obstruction as in men. Female COPD also showed higher signs of muscle damage than COPD men who, in contrast, exhibited slightly higher signs of regeneration. We conclude that sex influences muscle phenotype and function in COPD.

Many respiratory diseases lead to impaired function of skeletal muscles, influencing quality of life and patient survival. Dysfunction of both respiratory and limb muscles in chronic obstructive pulmonary disease has been studied in depth, and seems to be caused by the complex interaction of general (inflammation, impaired gas exchange, malnutrition, comorbidity, drugs) and local factors (changes in respiratory mechanics and muscle activity, and molecular events). Some of these factors are also present in cystic fibrosis and asthma. In obstructive sleep apnea syndrome, repeated exposure to hypoxia and the absence of reparative rest are believed to be the main causes of muscle dysfunction. Deconditioning appears to be crucial for the functional impairment observed in scoliosis. Finally, cachexia seems to be the main mechanism of muscle dysfunction in advanced lung cancer. A multidimensional therapeutic approach is recommended, including pulmonary rehabilitation, an adequate level of physical activity, ventilatory support and nutritional interventions.

Resumen Introduccion Las acciones locales de las citocinas en los musculos de los pacientes con enfermedad pulmonar obstructiva cronica (EPOC) se hallan sometidas a debate. El objetivo del presente estudio ha sido analizar las relaciones entre su expresion y la activacion genetica de programas de reparacion muscular. Pacientes y metodos Se incluyo en el estudio a 25 pacientes con EPOC grave en situacion estable. Se les realizo una biopsia del musculo intercostal externo, donde se evaluaron los signos de lesion muscular (morfometria), la infiltracion de celulas inflamatorias (inmunohistoquimica) y la expresion de genes seleccionados (tecnica de reaccion en cadena de la polimerasa en tiempo real) correspondientes a las propias citocinas —factor de necrosis tumoral alfa (TNF-α) y sus receptores 1 y 2 (TNFR1 y TNFR2), e interleucinas-1β, 6 y 10—, un marcador panleucocitario (CD18) y moleculas clave en las vias de reparacion-miogenesis (Pax7, M-Caderina y Mio-D). Resultados La expresion de TNFR2 se relaciono directamente con la funcion muscular inspiratoria (representada por la presion inspiratoria maxima sostenible; r=0,496, p Conclusiones La expresion de los receptores del TNF-α guarda una estrecha relacion tanto con la activacion de los programas de miogenesis como con la propia funcion muscular inspiratoria. Este hecho refuerza nuestra hipotesis de que algunas citocinas locales participan en la reparacion de los musculos respiratorios en los pacientes con EPOC.

a b s t r a c t Objective: There is disagreement regarding the local action of cytokines in the respiratory muscles of patients with chronic obstructive pulmonary disease (COPD). The objective of this study was to analyze the relationships between cytokine expression and genetic activation of the mechanisms of muscle repair. Patients and Methods: Twenty-five patients with severe COPD and in stable condition were enrolled in the study. We performed a biopsy of the external intercostal muscle of the patients and analyzed the specimen for signs of muscle lesion (morphometry), infiltration of inflammatory cells (immunohistochemistry), and expression of selected genes (real-time polymerase chain reaction technique) corresponding to the cytokines (tumor necrosis factor α (TNF-α) and its type 1 and 2 receptors (TNFR1 and TNFR2), and interleukin (IL) 1β, IL-6, and IL-10), a pan-leukocyte marker (CD18), and key molecules in the repair- myogenesis pathways (Pax7, M-cadherin, and MyoD). Results: Expression of TNFR2 is directly related to inspiratory muscle function (represented by maximum sustainable inspiratory pressure; r=0.496; P

Objetivo Los musculos respiratorios de los pacientes con enfermedad pulmonar obstructiva cronica (EPOC) presentan lesiones estructurales, que coexisten con signos de adaptacion. Nuestra hipotesis es que esto solo puede explicarse si se produce simultaneamente la activacion de sus celulas satelite. El proposito del presente trabajo ha sido valorar el numero y la eventual activacion de dichas celulas, asi como la expresion de marcadores de microlesion estructural, ligados a la regeneracion. Pacientes y metodos Se incluyo en el estudio a 8 pacientes con EPOC grave —media ± desviacion estandar del volumen espiratorio forzado en el primer segundo: un 33 ± 9% del valor de referencia— y a 7 controles, a quienes se realizo una biopsia del musculo intercostal externo. La muestra se analizo mediante microscopia optica (fenotipo fibrilar), electronica (celulas satelite) y tecnica de reaccion en cadena de la polimerasa en tiempo real (marcadores de microlesion: factor de crecimiento similar a la insulina de tipo 1, factor de crecimiento mecanico e isoformas de cadenas pesadas de miosina [MyHC] embrionaria e isoformas de perinatal; de presencia y activacion de celulas satelite: Pax-7 y m-caderina, respectivamente; y condicionantes del fenotipo fibrilar: MyHC-I, IIa y IIx). Resultados Los pacientes tuvieron unas fibras mayores que los sujetos sanos (54 ± 6 frente a 42 ± 4 μm2; p Conclusiones Los musculos intercostales de pacientes con EPOC grave muestran signos indirectos de microlesion, acompanados de la activacion de sus celulas satelite. Esto apunta a la presencia de ciclos continuados de lesion y reparacion, lo que podria explicar parcialmente la conservacion de sus propiedades estructurales.

The respiratory muscles of patients with chronic obstructive pulmonary disease (COPD) display evidence of structural damage in parallel with signs of adaptation. We hypothesized that this can only be explained by the simultaneous activation of satellite cells. The aim of this study was to analyze the number and activation of those cells along with the expression of markers of microstructural damage that are frequently associated with regeneration.The study included 8 patients with severe COPD (mean [SD] forced expiratory volume in 1 second, 33% [9%] of predicted) and 7 control subjects in whom biopsies were performed of the external intercostal muscle. The samples were analyzed by light microscopy to assess muscle fiber phenotype, electron microscopy to identify satellite cells, and real-time polymerase chain reaction to analyze the expression of the following markers: insulin-like growth factor 1, mechano growth factor, and embryonic and perinatal myosin heavy chains (MHC) as markers of microstructural damage; Pax-7 and m-cadherin as markers of the presence and activation of satellite cells, respectively; and MHC-I, IIa, and IIx as determinants of muscle fiber phenotype.The patients had larger fibers than healthy subjects (54 [6] vs 42 [4] microm(2); P.01) with a similar or slightly increased proportion of satellite cells, as measured by ultrastructural analysis (4.3% [1%] vs 3.7% [3.5%]; P.05) or expression of Pax-7 (5.5 [4.1] vs 1.6 [0.8] arbitrary units [AU]; P.05). In addition, there was greater activation of satellite cells in the patients, as indicated by increased expression of m-cadherin (3.8 [2.1] vs 1.0 [1.2] AU; P=.05). This was associated with increased expression of markers of microstructural damage: insulin-like growth factor 1, 0.35 (0.34) vs 0.09 (0.08) AU (P.05); mechano growth factor, 0.45 (0.55) vs 0.13 (0.17) AU (P=.05).The intercostal muscles of patients with severe COPD show indirect signs of microstructural damage accompanied by satellite cell activation. This suggests the presence of ongoing cycles of lesion and repair that could partially explain the maintenance of the structural properties of the muscle.

Objetivo Los estudios estructurales y de expresion de diversas sustancias en musculos respiratorios de seres humanos se han servido de diversos modelos para la obtencion de las muestras. Entre ellos destacan la toma de tejidos en autopsias, la biopsia muscular en el curso de una toracotomia por lesion pulmonar localizada (TLL) y la biopsia ambulatoria en sujetos sin comorbilidad (TA). Los 2 primeros modelos adolecen de las posibles interferencias de factores relacionados, respectivamente, con la muerte o la enfermedad que motiva el acto quirurgico. La TA, aunque obvia los inconvenientes de los otros 2 modelos, no permite obtener muestras del diafragma, principal musculo respiratorio. El objetivo de este trabajo fue analizar la estructura fibrilar y expresion de la principal citocina inflamatoria -factor de necrosis tumoral alfa (TNF-α)- y del factor de crecimiento muscular insulina-like (IGF-1) en el musculo intercostal externo de pacientes con enfermedad pulmonar obstructiva cronica, comparando los resultados obtenidos con los modelos de TLL y TA Metodos Se tomaron prospectiva y consecutivamente muestras del musculo intercostal externo (quinto espacio, linea axilar anterior) en 15 pacientes con enfermedad pulmonar obstructiva cronica (66 ± 6 anos; volumen espiratorio forzado en el primer segundo del 49 ± 9% ref., presion arterial de oxigeno de 75 ± 9 mmHg). Las muestras se tomaron mediante TLL (8 pacientes, todos ellos con neoplasia pulmonar pero cuidadosamente seleccionados para descartar efectos tos sistemicos) o TA (7 pacientes), excluyendose en el segundo caso la presencia de comorbilidad importante. Las muestras se procesaron para analisis estructural fibrilar (inmunohistoquimica e histoquimica enzimatica) y de expresion genica de TNF-α e IGF-1 (reaccion en cadena de la polimerasa en tiempo real) Resultados El analisis estructural de las fibras no mostro diferencias entre ambos grupos. Tampoco se observaron diferencias en la expresion de TNF-α o IGF-1 Conclusiones Con criterios de seleccion rigurosos, el modelo de TLL parece adecuado para el estudio de las caracteristicas estructurales y de expresion de citocinas inflamatorias y factores de crecimiento en el musculo intercostal externo. Puede ademas inferirse que probablemente la TLL tambien sea util para esos objetivos en el caso del diafragma, para el que no existe una tecnica alternativa en la actualidad

Objective To analyse the DNA methylation status and the loss of heterozygosity (LOH) at the D17S5 locus (17p13.3) in urothelial cancer. Materials and methods DNA methylation was assayed and LOH analysed by Southern blotting in a series of 33 transitional cell carcinomas of the bladder and renal pelvis. Results DNA hypermethylation and LOH at the D17S5 locus were detected in six (18%) and 17 (52%) of the tumours, respectively. The six cases with DNA hypermethylation were of the papillary type, and four also had LOH at this locus. Conclusion In contrast to other epithelial tumours, DNA hypermethylation at the D17S5 locus is not a frequent event in human urothelial cancer.

Antecedentes El factor de necrosis tumoral alfa (TNF-a) se ha implicado en la perdida de masa muscular en la enfermedad pulmonar obstructiva cronica (EPOC) y otras enfermedades consumtivas. Su produccion estaria en relacion con la inflamacion derivada de la propia enfermedad pulmonar o el habito tabaquico, llegando al musculo por el torrente sanguineo. Sin embargo, tambien se ha sugerido que el TNF-α podria expresarse directamente en el tejido muscular. Sea cual fuere el lugar de produccion, la relacion del TNF-α con el dano muscular subsiguiente no es clara Objetivo Hemos estudiado la expresion del TNF-α y una interleucina inhibidora de su produccion (IL-10) en los musculos respiratorios principales y en un musculo periferico del perro Metodos Se han incluido nueve animales machos, adultos jovenes de raza Beagle. En todos ellos se tomaron biopsias del diafragma (Df), del intercostal externo (IExt) y de un musculo de las extremidades (vasto interno del cuadriceps o VInt). Se analizo la expresion de los genes de TNF-α e IL-10, a traves de la determinacion de sus ARNm corres-pondientes, utilizando la transcripcion reversa y reaccion en cadena de la polimerasa (RT-PCR). Tambien se evaluo el dano sarcolemico mediante deteccion de fibronectina intra-celular (inmunohistoquimica) Resultados Los valores de expresion de ambas citocinas evidenciaron una amplia dispersion interindividual. Por un lado, la expresion de TNF-α fue minima en Df y IExt (respectivamente, 0,02 ± 0,03 y 0,05 ± 0,06 u.a.), con valores relativamente elevados en VInt (0,14 ± 0,08 u.a.). A su vez, la IL-10 evidenciaba valores bajos en DF (0,6 ± 0,5 u.a.), que eran algo superiores en IExt (2,7 ± 1,9 u.a.; p Conclusiones a) TNF-α e IL-10 parecen expresarse constitutivamente en el musculo esqueletico canino; b) los valores basales de expresion del TNF-α son menores en los musculos repiratorios que en los de las extremidades mos-trando relacion en estos ultimos con el dano de membrana

We performed a study of loss of heterozygosity (LOH) at 3p by restriction fragment length polymorphism analysis in a series of 22 renal tumors. In 11 cases, molecular results could be compared with those of cytogenetic studies. The highest frequency of allelic losses at 3p was seen in clear cell non-papillary renal tumors, whereas none of the papillary renal cell carcinomas showed LOH at 3p. Allelic losses on 3p were found to be independent of tumor grade or stage or both. One case analyzed showed a discrepancy between cytogenetic and LOH studies. This tumor displayed rearrangements of chromosome 3 and no LOH at the c-RAF-1 (close to the Von Hippel Lindau gene) locus.

Oxidative stress and inflammation contribute to the diaphragm contractile dysfunction observed in animal models of sepsis and endotoxemia. In septic patients, molecular events have never been explored in their respiratory muscles. Levels of oxidative stress and inflammation were evaluated in a respiratory muscle, the external intercostal, and a limb muscle, the vastus lateralis, of patients with sepsis.Levels of oxidized and nitrated proteins, protein adducts of malondialdehyde and hydroxinonenal, antioxidant enzymes catalase and Mn-superoxide dismutase, tumor necrosis factor (TNF)-α, TNF-α receptors i and ii, interleukin (IL)-1 and IL-6, the panleukocyte marker CD18, and fiber type composition were explored using immunoblotting, real time-polymerase chain reaction, and immunohistochemistry in the external intercostal and vastus lateralis of patients with severe sepsis and/or septic shock.Compared to the controls, in septic patients, levels of oxidized and nitrated proteins were increased in the vastus lateralis, but not in the external intercostal, while those of the antioxidant enzymes did not differ, and the proportions and sizes of the muscle fibers were not significantly different in any muscle between patients and controls.Differences in activity between the respiratory and limb muscles may account for the differential pattern of oxidative stress and inflammation observed among patients with severe sepsis. These findings may have relevant implications for the clinical and therapeutic management of these patients.

Muscle dysfunction is a characteristic feature of chronic obstructive pulmonary disease (COPD). Recent studies suggest that cytokines may operate as local regulators of both muscle function and regeneration. The aim of the present study was to characterise the expression of different cytokines in the external intercostal muscle of COPD. Muscle biopsies were obtained from 25 stable COPD patients and eight healthy controls. Local tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, -6 and -10 expressions (real-time PCR and ELISA), sarcolemmal damage (immunohistochemistry), and the transcript levels of CD18 were assessed. Muscle TNF-alpha and IL-6 transcripts were significantly higher in COPD patients compared with controls, and IL-1beta and sarcolemmal damage showed a strong tendency in the same direction. Similar results were observed at protein level. The CD18 panleukocyte marker was similar in COPD and controls. Respiratory muscle function was impaired in COPD patients and it correlated to both the severity of lung function impairment and TNF-alpha muscle expression. Chronic obstructive pulmonary disease is associated with the upregulation of pro-inflammatory cytokines in the intercostal muscles. This phenomenon might be involved in respiratory muscle dysfunction.

Comparative genomic hybridization (CGH) and conventional cytogenetic karyotyping were used to screen for losses and gains of DNA sequences along chromosomes in ten renal tumors (RCC) of different histologic types (clear-cell RCC, papillary RCC, and one oncocytoma). Loss of 3p was the most common change in clear-cell RCC. All papillary tumors, either adenomas or carcinomas revealed gains of chromosomes 7 and 17q without limitation to size and grade. Homozygotic loss of the pseudoautosomal Xp or Yp region was detected in three RCC tumors. A dicentric (Y;14) was present as the sole chromosome abnormality in the oncocytoma. Both techniques showed concordant results in tumors with homogeneous karyotype. However, in tumors with several composite clones some discrepancies were observed, especially in cases of clear-cell RCC where chromosomal abnormalities present in a low number of metaphases could not be detected by CGH.

We used comparative genomic hybridization to analyze 10 primary tumor samples from patients with transitional cell carcinoma of the renal pelvis. The most frequent loss was located at 9q, that is, in 50% of the tumors. Gains of DNA sequences were most frequently observed in chromosome regions 1q21 approximately q23, 2p23 approximately p25, 8q21.1 approximately q22 and in the whole chromosome 20. High level amplifications at 1q21 approximately q25, 6p22 approximately p23, 8q21 approximately q22, 8q22 approximately q24.1, 11q13, and 12q14 approximately q21 were detected. Most of these regions have previously been reported to be involved in transitional cell carcinoma of the bladder, thus confirming the importance of an increasing number of chromosome imbalances in the development and progression of this type of tumors.

Forty-eight transitional cell carcinomas of the bladder and three transitional cell carcinomas of the renal pelvis were examined for loss of heterozygosity (LOH) on chromosomes 3p, 6q, and 17p. The most frequent allelic loss was seen on 17p (18/36, 50%) followed by 6q (6/22, 27%), and 3p (5/22, 23%). In cases with LOH at more than one locus, the same DNA sample often varied in degree of signal reduction for missing alleles. This observation indicates that LOH studies can serve to detect intratumor heterogeneity. No correlation was found between allelic losses at these chromosome arms and tumor grade and stage. Allelic losses on 6q were associated with tumors having a solid growth pattern; in this kind of tumors, allelic losses on 3p were associated with invasion.

We describe the first case of a familial renal cell carcinoma cytogenetically characterized as a papillary renal cell carcinoma. Cytogenetic and molecular studies were performed on primary renal cell carcinomas and normal kidney tissue from two members of the same family. Both patients showed a normal constitutional karyotype. The two tumors analyzed from the first patient showed the numerical chromosome alterations characteristic of papillary renal cell carcinomas. From the four tumors analyzed in the second patient, three of them presented the cytogenetic pattern of papillary renal cell tumors, and the fourth showed only structural chromosome abnormalities with the presence of a del(7)t(7;7) or dup(7) in all metaphases analyzed. Chromosome 3 was cytogenetically unaffected in all tumors from both patients, and restriction fragment length polymorphism analysis performed with probe pEFD145 (3p21.1–p23) did not detect any loss of heterozygosity.

Monosomy for chromosome 9, as well as loss of heterozygosity for markers on this chromosome, has been detected in a high percentage of transitional cell carcinomas (TCC) of the bladder. We report a case of a TCC of the bladder with an interstitial del(9)(q11q21.2) that could be indicative of the presence of a putative tumor-suppressor gene related to bladder tumor progression. To elucidate the role of chromosome 9 in bladder tumors, it would be interesting to study a possible loss of heterozygosity in this chromosome region.

Peripheral muscle weakness and mass loss are characteristic features in severe chronic obstructive pulmonary disease (COPD). We hypothesized that the phosphodiesterase (PDE)-4 inhibitor roflumilast-induced cAMP may ameliorate proteolysis and metabolism in skeletal muscles of COPD patients with severe muscle wasting. In myogenic precursor cells (isolated from muscle biopsies and cultured up to obtain differentiated myotubes) from 10 severe COPD patients and 10 healthy controls, which were treated with 1 μM roflumilast N-oxide (RNO) for three time cohorts (1, 6, and 24 h), genes of antioxidant defense and oxidative stress marker, myogenesis and muscle metabolism, proteolysis (tyrosine release assay) and ubiquitin-proteasome system markers, autophagy, and myosin isoforms were analyzed using RT-PCR and immunoblotting. In COPD patients at 6 h RNO treatment, myotube tyrosine release, total protein ubiquitination, and tripartite motif-containing protein 32 levels were significantly lower than healthy controls, whereas at 24 h RNO treatment, myotube myosin heavy chain ( MyHC) -I and MyHC-IIx expression levels were upregulated in both patients and controls. In the 6-h RNO cohort, in patients and controls, myotube expression of nuclear factor (erythroid-derived 2)-like 2 ( NRF2) and its downstream antioxidants sirtuin-1, FGF-inducible 14, and insulin-like growth factor-1 was upregulated, whereas that of myocyte-specific enhancer factor 2C, myogenic differentiation, myogenin, myostatin, atrogin-1, and muscle RING-finger protein-1 was downregulated. In myotubes of severe COPD patients with cachexia, roflumilast-induced cAMP signaling exerts beneficial effects by targeting muscle protein breakdown (tyrosine release), along with reduced expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In both patients and controls, roflumilast also favored antioxidant defense through upregulation of the NRF2 pathway and that of the histone deacetylase sirtuin-1, whereas it improved the expression of slow- and fast-twitch myosin isoforms. These findings show that muscle dysfunction and wasting may be targeted by roflumilast-induced cAMP signaling in COPD. These results have potential therapeutic implications, as this PDE-4 inhibitor is currently available for the treatment of systemic inflammation and exacerbations in patients with severe COPD. NEW & NOTEWORTHY In myotubes of cachectic chronic obstructive pulmonary disease (COPD) patients, cAMP signaling exerted beneficial effects by targeting muscle proteolysis and reducing gene expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In myotubes of patients and controls, roflumilast also favored antioxidant defense through upregulation of the nuclear factor (erythroid-derived 2)-like 2 pathway, of sirtuin-1, and of gene expression of slow- and fast-twitch isoforms. These findings have potential clinical implications for the treatment of muscle wasting in patients with COPD and cachexia. Support for this study was provided by CIBERES, FIS 14/00713 (FEDER), SAF-2014-54371-R, SEPAR 2016, FUCAP 2016 (Spain), and an unrestricted grant from Takeda (Japan). E. Barreiro was a recipient of the ERS COPD Research Award 2008.