Apocynin prevents cigarette smoking-induced loss of skeletal muscle mass and function in mice by preserving proteostatic signalling.

BACKGROUND AND PURPOSE: Skeletal muscle dysfunction is a major comorbidity of chronic obstructive pulmonary disease (COPD). This type of muscle dysfunction may be a direct consequence of oxidative insults evoked by cigarette smoke (CS) exposure. The present study examined the effects of a potent Nox inhibitor and reactive oxygen species (ROS) scavenger, apocynin, on CS-induced muscle dysfunction. EXPERIMENTAL APPROACH: Male BALB/c mice were exposed to either room air (sham) or CS generated from nine cigarettes per day, 5 days a week for 8 weeks, with or without the coadministration of apocynin (5 mg·kg-1 , i.p.). C2C12 myotubes exposed to either hydrogen peroxide (H2 O2 ) or water-soluble cigarette smoke extract (CSE) with or without apocynin (500 nM) were used as an experimental model in vitro. KEY RESULTS: Eight weeks of CS exposure caused muscle dysfunction in mice, reflected by 10% loss of muscle mass and 54% loss of strength of tibialis anterior which were prevented by apocynin administration. In C2C12 myotubes, direct exposure to H2 O2 or CSE caused myofibre wasting, accompanied by ~50% loss of muscle-derived insulin-like growth factor (IGF)-1 and two-fold induction of Cybb, independent of cellular inflammation. Expression of myostatin and MAFbx, negative regulators of muscle mass, were up-regulated under H2 O2 but not CSE conditions. Apocynin treatment abolished CSE-induced Cybb expression, preserving muscle-derived IGF-1 expression and signalling pathway downstream of mammalian target of rapamycin (mTOR), thereby preventing myofibre wasting. CONCLUSION AND IMPLICATIONS: Targeted pharmacological inhibition of Nox-derived ROS may alleviate the lung and systemic manifestations in smokers with COPD.

Cigarette Smoking Exacerbates Skeletal Muscle Injury without Compromising Its Regenerative Capacity.

Skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease negatively impacts quality of life and survival. Cigarette smoking (CS) is the major risk factor for chronic obstructive pulmonary disease and skeletal muscle dysfunction; however, how CS affects skeletal muscle function remains enigmatic. To examine the impact of CS on skeletal muscle inflammation and regeneration, male BALB/c mice were exposed to CS for 8 weeks before muscle injury was induced by barium chloride injection, and were maintained on the CS protocol for up to 21 days after injury. Barium chloride injection resulted in architectural damage to the tibialis anterior muscle, resulting in a decrease contractile function, which was worsened by CS exposure. CS exposure caused muscle atrophy (reduction in gross weight and myofiber cross-sectional area) and altered fiber type composition (31% reduction of oxidative fibers). Both contractile function and loss in myofiber cross-sectional area by CS exposure gradually recovered over time. Satellite cells are muscle stem cells that confer skeletal muscle the plasticity to adapt to changing demands. CS exposure blunted Pax7+ centralized nuclei within satellite cells and thus prevented the activation of these muscle stem cells. Finally, CS triggered muscle inflammation; in particular, there was an exacerbated recruitment of F4/80+ monocytic cells to the site of injury along with enhanced proinflammatory cytokine expression. In conclusion, CS exposure amplified the local inflammatory response at the site of skeletal muscle injury, and this was associated with impaired satellite cell activation, leading to a worsened muscle injury and contractile function without detectable impacts on the recovery outcomes.

Maraviroc improves hepatic triglyceride content but not inflammation in a murine nonalcoholic fatty liver disease model induced by a chronic exposure to high-fat diet.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the general population. Its severity ranges from simple steatosis to cirrhosis. C-C chemokine ligand type 5 or RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) plays an important role in the progression of hepatic inflammation and fibrosis. Our objective was to examine the preventive and therapeutic effects of maraviroc (MVC), a C-C chemokine receptor 5 antagonist, on liver pathology in an NAFLD mouse model. A total of 60 male C57BL/6 mice were randomly assigned to 1 of 4 groups: (1) high-fat diet (HFD) group or control group, (2) preventive group (HFD group plus MVC in drinking water since the beginning of the study), (3) early-therapeutic group (HFD group plus MVC in drinking starting at week 24 of the study), and (4) late-therapeutic group (HFD group plus MVC in drinking water starting at week 36 of the study). All mice were sacrificed at week 48. The hepatic triglyceride concentration in the HFD group was significantly higher than that in the groups treated with MVC at any time. Gene expression associated with lipogenesis (diacylglycerol acyltransferase 2 and proliferator-activated receptor-γ), insulin resistance (insulin receptor substrate-2), and ß-oxidation (carnitine palmitoyltransferase 1A and acyl-CoA oxidase) was significantly reduced in all the groups treated with MVC. In summary, the beneficial effect of MVC on hepatic steatosis is maintained throughout the study.

Evaluation of right heart function in a rat model using modified echocardiographic views.

Echocardiography plays a major role in assessing cardiac function in animal models. We investigated use of a modified parasternal mid right-ventricular (MRV) and right ventricle (RV) outflow (RVOT) view, in assessing RV size and function, and the suitability of advanced 2D-strain analysis. 15 WKY rats were examined using transthoracic echocardiography. The left heart was assessed using standard short and long axis views. For the right ventricle a MRV and RVOT view were used to measure RV chamber and free wall area. 2D-strain analysis was applied to both ventricles using off-line analysis. RV chamber volume was determined by injection of 2% agarose gel, and RV free wall dissected and weighed. Echocardiography measurement was correlated with necropsy findings. The RV mid-ventricular dimension (R1) was 0.42±0.07cm and the right ventricular outflow tract dimension (R2) was 0.34±0.06cm, chamber end-diastolic area measurements were 0.38±0.09cm2 and 0.29±0.08cm2 for MRV and RVOT views respectively. RVOT and MRV chamber area correlated with gel mass. Doppler RV stroke volume was 0.32±0.08ml, cardiac output (CO) 110±27 ml.min-1 and RV free wall contractility assessed using 2D-strain analysis was demonstrated. We have shown that modified MRV and RVOT views can provide detailed assessment of the RV in rodents, with 2D-strain analysis of the RV free wall potentially feasible.

Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods.

BACKGROUND: The discovery of mutations in the epidermal growth factor receptor gene (EGFR) related to the clinical response to tyrosine kinase inhibitors, has transformed the management of non-small cell lung cancer (NSCLC). Several methods have been developed for determination of mutations in EGFR, with different sensitivity and potential ability to detect a different number of mutations. METHODS: We developed a screening method by high resolution melting (HRM) to detect EGFR mutations, and compared the results of 123 fixed in formalin and paraffin embedded (FFPE) tumor tissue samples with the detection of mutations by allele-specific PCR. In samples with discordant results, Sanger and massive parallel sequencing (MPS) were additionally performed. RESULTS: Eight samples showed discordant results between both methods. Three samples with negative results by allele specific PCR and positive by HRM were confirmed by Sanger sequencing (p.S768I+p.V769L, T751_I759del and p.E709K+p.G719A; patients 1, 3 and 4, respectively). One sample with a negative result by HRM, and positive by allele specific PCR (p.T790M; patient 2), was confirmed by Sanger sequencing. Additionally, two positive samples for a deletion in exon 19 by allele-specific PCR, were negative by Sanger sequencing and HRM (patients 2 and 5) and finally, two samples were negative by allele-specific PCR and positive by HRM and Sanger sequencing due to synonymous variants in exon 21. CONCLUSIONS: HRM is a good method for mutational screening in EGFR. It is able to detect any change in the sequence of exons 18-21, providing high cost/effectiveness, but samples with low tumor burden may produce false negatives results.

Targeting oxidant-dependent mechanisms for the treatment of COPD and its comorbidities.

Chronic obstructive pulmonary disease (COPD) is an incurable global health burden and is characterised by progressive airflow limitation and loss of lung function. In addition to the pulmonary impact of the disease, COPD patients often develop comorbid diseases such as cardiovascular disease, skeletal muscle wasting, lung cancer and osteoporosis. One key feature of COPD, yet often underappreciated, is the contribution of oxidative stress in the onset and development of the disease. Patients experience an increased burden of oxidative stress due to the combined effects of excess reactive oxygen species (ROS) and nitrogen species (RNS) generation, antioxidant depletion and reduced antioxidant enzyme activity. Currently, there is a lack of effective treatments for COPD, and an even greater lack of research regarding interventions that treat both COPD and its comorbidities. Due to the involvement of oxidative stress in the pathogenesis of COPD and many of its comorbidities, a unique therapeutic opportunity arises where the treatment of a multitude of diseases may be possible with only one therapeutic target. In this review, oxidative stress and the roles of ROS/RNS in the context of COPD and comorbid cardiovascular disease, skeletal muscle wasting, lung cancer, and osteoporosis are discussed and the potential for therapeutic benefit of anti-oxidative treatment in these conditions is outlined. Because of the unique interplay between oxidative stress and these diseases, oxidative stress represents a novel target for the treatment of COPD and its comorbidities.

Informe del Taller Ibérico de Histocompatibilidad 2013. Componente de análisis de situación de procedimiento de pruebas cruzadas en guardias de trasplante de órganos / Report of the 2013 Iberian Histocompatibility Workshop of 2013. Component analysis of the proceedings of cross-matching procedures in emergency organ transplantation

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Mucosal memory CD8⁺ T cells are selected in the periphery by an MHC class I molecule.

The presence of immune memory at pathogen-entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the nonclassical major histocompatibility complex (MHC) class I molecule thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αß(+) T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αß(+) memory T cells. The memory process driven by TL and CD8αα was essential for the generation of CD8αß(+) memory T cells in the intestine and the accumulation of highly antigen-sensitive CD8αß(+) memory T cells that form the first line of defense at the largest entry port for pathogens.

Phenotypic and functional evaluation of CD3+CD4-CD8- T cells in human CD8 immunodeficiency.

BACKGROUND: Human CD8 immunodeficiency is characterized by undetectable CD8(+) lymphocytes and an increased population of CD4(-)CD8(-) (double negative) T lymphocytes. DESIGN AND METHODS: We hypothesized that the double negative subset corresponds to the cellular population that should express CD8 and is committed to the cytotoxic T lymphocyte lineage. To assess this, we determined the phenotype and function of peripheral blood mononuclear cells and/or magnetically isolated double negative T lymphocytes from two CD8-deficient patients. To analyze the expression and co-localization with different organelles, 293T cells were transfected with plasmids bearing wild-type or mutated CD8α. RESULTS: CD8α mutated protein was retained in the cytoplasm of transfected cells. The percentages of double negative cells in patients were lower than the percentages of CD8(+) T cells in healthy controls. Double negative cells mostly had an effector or effector memory phenotype whereas naïve T cells were under-represented. A low concentration of T-cell receptor excision circles together with a skewed T-cell receptor-V repertoire were observed in the double negative population. These data suggest that, in the absence of CD8 co-receptor, the thymic positive selection functions suboptimally and a limited number of mature T-cell clones would emerge from the thymus. In vitro, the double negative cells showed a mild defect in cytotoxic function and decreased proliferative capacity. CONCLUSIONS: It is possible that the double negative cells are major histocompatibility complex class-I restricted T cells with cytolytic function. These results show for the first time in humans that the presence of the CD8 co-receptor is dispensable for cytotoxic ability, but that it affects the generation of thymic precursors committed to the cytotoxic T lymphocyte lineage and the proliferation of mature cytotoxic T cells.

Preformed antibodies detected by cytotoxic assay or multibead array decrease liver allograft survival: role of human leukocyte antigen compatibility.

The significance of human leukocyte antigen (HLA) compatibility and preformed antibodies in liver transplantation remains unclear. The objectives of this study were to evaluate, in a single-center cohort comprising 896 liver transplants, whether the degree of donor-recipient compatibility and preformed antibodies modified graft survival. Univariate Kaplan-Meier analysis demonstrated that donor-recipient HLA compatibility had a marginal impact on allograft survival. As for compatibility at individual antigen loci, 2 mismatches at HLA-A conferred a survival advantage in retransplanted allografts (P = 0.011). HLA-B and HLA-DR loci did not play a significant role in outcome in any pathology. The concordance of results on preformed antibodies detected by complement-dependent cytotoxicity (CDC) and a multiple bead assay (Luminex xMAP) showed a strong correlation between both techniques (P < 0.0001). Both CDC-detected and Luminex-detected antibodies were associated with shorter graft survival within the first year post-transplant (P = 0.01 and P = 0.016, respectively). Positive CDC T crossmatches and Luminex-detected HLA class II antibodies played a significant role in decreasing graft survival (P = 0.043 and P = 0.0019 at 1 year, respectively, and P = 0.005 and P = 0.038 at 5 years, respectively). A correlation was also observed between the presence of preformed Luminex-detected class II or Luminex I and II antibodies and allograft rejection (P = 0.001 and P = 0.042, respectively). In conclusion, although HLA typing is not a prerequisite for transplantation, screening of HLA antibodies with Luminex techniques and CDC crossmatch may be useful in the detection of at-risk patients that could benefit from increased surveillance and tailored therapy following transplantation.

Peripheral blood lymphocyte populations in end-stage liver diseases.

GOALS/BACKGROUND: The aim of this study was to decipher whether end-stage liver failure modifies peripheral blood lymphocytes (PBL) in a homogeneous manner, independently of the base pathology, or, if on the contrary, PBL subsets show a different profile in each hepatic disease. METHODS: We studied PBL subsets in 71 patients with end-stage liver disease, before liver transplant, and 74 healthy controls by flow cytometry. The results were statistically compared between patients and controls, and cohorts of patients classified according to their base pathology. RESULTS: We observed lower absolute numbers in all lymphocyte populations in patients compared with controls. We found an increment of CD3+ activated cells (P<10) and CD45RO+CD4+ (P<10) in chronic hepatitis C virus versus controls; hepatitis B virus showed high TCRgammadelta+ and CD8+ T cells with respect to controls (P=0.008 and P=0.029, respectively); alcoholic cirrhotic patients showed low CD8+, mainly CD45RA+CD8+ (P=0.007) and high CD45RO+CD4+ (P<10) compared with the normal population; autoimmune diseases showed lower CD3+ and TCRalphabeta+ (P=0.002 and P=0.0001) than controls. CONCLUSIONS: Regardless of the base pathology, patients with end-stage liver disease show a low absolute number of lymphocyte populations compared with controls. However, PBL profiles are different, characteristic, and specific of every disease causing chronic liver failure.

Rapid molecular prenatal diagnosis of ataxia-telangiectasia by direct mutational analysis.

Mutations of the ataxia-telangiectasia-mutated (ATM) gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). This study reports the first A-T prenatal diagnosis performed in Spain by direct molecular analysis. The pregnant woman had a previous child suffering from A-T due to a deletion in the ATM gene. The ATM coding region was sequenced in the A-T patient and her parents. Then, a specific polymerase chain reaction (PCR) to detect the deletion was performed for prenatal diagnosis. Additionally, polymorphic HLA loci were examined in order to exclude the possible contamination by maternal DNA. In this family of Gypsy origin, we carried out a rapid molecular diagnosis of A-T. Then, a prenatal diagnosis was carried out, identifying the deletion in the fetal DNA. Additionally, we performed a population study in unrelated Spanish Gypsies and in unrelated controls, showing that the deletion described could be a hotspot in the Spanish Gypsy population. The size of the coding region and the genomic structure, together with the absence of hotspots, make the mutation screening of the ATM gene difficult. The ability to identify ATM mutations provides a tool that can be applied in confirmatory diagnosis, genetic counselling, carrier prediction and prenatal diagnosis.