The Dual Role of Platelets in the Cardiovascular Risk of Chronic Inflammation

Patients with chronic inflammatory diseases often exhibit cardiovascular risk. This risk is associated with the systemic inflammation that persists in these patients, causing a sustained endothelial activation. Different mechanisms have been considered responsible for this systemic inflammation, among which activated platelets have been regarded as a major player. However, in recent years, the role of platelets has become controversial. Not only can this subcellular component release pro- and anti-inflammatory mediators, but it can also bind to different subsets of circulating lymphocytes, monocytes and neutrophils modulating their function in either direction. How platelets exert this dual role is not yet fully understood

Diferències fenotípiques i funcionals dels limfócits CD4+ de sang de cordó umbilical i sang perifèrica d'adult

Des de l'any 1989 en que es va realitzar el primer trasplantament de cèl·lules hematopoiètiques procedents de la sang de cordó umbilical (SCU) a un pacient amb anèmia de Falconi, ha anat creixent l'interès per les característiques immunològiques de la sang de cordó umbilical. Un dels principals avantatges que presenta la SCU és la baixa incidència en la malaltia de l'injert contra l'hoste (GVHD) que es desencadena després del trasplantament entre pacients no emparentats. Fins el moment tots els treballs han demostrat que els limfòcits T de SCU presenten un fenotip CD45RA+ que indicaria en un principi la manca d'exposició prèvia a antígens i aquesta característica ha comportat que es considerés als limfòcits naive de SCU com immunològicament immadurs i amb una resposta peculiar enfront a diversos estímuls. L'ús de diferents condicions experimentals i sistemes d'activació han donat lloc a resultats no comparables i a vegades fins i tot contradictoris. Per una banda, l'ús de tècniques de separació que comportin la pèrdua sel·lectiva de poblacions cel·lulars o produeixin pertorbacions funcionals, i per l'altra la utilització de diversos agents per activar les cèl·lules que intervenen a diferents nivells de la cascada de senyalització, han donat lloc a resultats difícils d'interpretar. L'objectiu general d'aquest treball ha estat de caracteritzar la població majoritària CD4+CD45RA+ de SCU utilitzant un anticòs monoclonal anti-CD3 humà (OKT3), que permet una activació de tots els limfòcits independentment de la seva especificitat. Per abordar aquest objectiu, es va obtenir la població majoritària CD4+CD45RA+ de la sang de cordó umbilical i de sang perifèrica d'adult, utilitzant una selecció negativa, que consistia en l'obtenció dels limfòcits CD3+ a partir de les cèl·lules mononuclears obtingudes per gradient de Ficoll, i una segona selecció negativa a partir dels limfòcits CD3+ per poder obtenir la població CD4+CD45RA+ purificada. El grau de purificació es va determinar per citometria de flux. L'estudi fenotípic i funcional dels limfòcits CD4+CD45RA+ de sang de cordó umbilical va demostrar que aquesta població limfocitària presentava característiques que la diferenciava de la població CD4+CD45RA+ de la sang perifèrica d'adult. Així s'observà, que els limfòcits naive de SCU presentaven una major tendència a entrar en apoptosi espontània comparat amb la SPA, i que aquesta mort podia ser en part revertida per la presència d'interleuquina 2 (IL2). Una activació via TCR/CD3 amb una sola senyal anti-CD3 portà als limfòcits naive de SCU a activar-se, dividir-se i finalment a morir per apoptosi. Aquesta activació anava acompanyada d'una fosforilació diferencial en Tyr de proteïnes implicades en els primers processos de la cascada de senyalització via TCR, transformació blàstica, expressió de marcadors d'activació en superfície i divisió cel·lular. La coestimulació via CD28 feia que els dos tipus cel·lulars assolissin uns graus d'activació similars, excepte en l'coexpressió de CD45RO que només s'observava amb la SCU. No sabem si la resposta de les cèl·lules naive observada -in vitro- pot ser sobrepassada in vivo per altres factors i tampoc coneixem el paper de les cèl·lules de SCU en l'expansió perifèrica després del trasplantament. Després del trasplantament amb cèl·lules de SCU, la primera onada de reconstitució de cèl·lules T s'origina per les cèl·lules T donants madures presents en l'injert. L'estimulació de les cèl·lules de SCU a través del receptor per l'antigen sense coestimulació podria induir un estímul toleritzant mitjançant l'activació subòptima i la posterior eliminació d'una gran part de les cèl·lules activades. En aquest cas, l'apoptosi és possiblement sel·lectiva per cèl·lules reactives donants, fenomen que es contempla com la base de la tolerància en el trasplantament i que podria explicar l'aparent reducció del GVHD en els individus trasplantats amb SCU

Selective Loss of Chemokine Receptor Expression on Leukocytes after Cell Isolation

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5±2.9%) whereas 32.8±6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4±7.5% and 57.1±5.5%; Ficoll: 29.5±2.2% and 5.4±4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5±0.4 and Ficoll: MFI 3.3±0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2±4.5% and Ficoll: 55±4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured

Salivary immunity and lower respiratory tract infections in non-elite marathon runners

Peer ReviewedPostprint (published version

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish

Immunological status of bladder cancer patients based on urine leukocyte composition at radical cystectomy

Altres ajuts: Roche donationBackground: Bladder cancer (BC) is the ninth most common malignancy worldwide, with high rates of recurrence. The use of urine leukocyte composition at the time of radical cystectomy (RC) as a marker for the study of patients' immunological status and to predict the recurrence of muscle-invasive bladder cancer (MIBC) has received little attention. Methods: Urine and matched peripheral blood samples were collected from 24 MIBC patients at the time of RC. Leukocyte composition and expression of PD-L1 and PD-1 in each subpopulation were determined by flow cytometry. Results: All MIBC patients had leukocytes in urine. There were different proportions of leukocyte subpopulations. The expression of PD-L1 and PD-1 on each subpopulation differed between patients. Neoadjuvant chemotherapy (NAC), smoking status, and the affectation of lymph nodes influenced urine composition. We observed a link between leukocytes in urine and blood circulation. Recurrent patients without NAC and with no affectation of lymph nodes had a higher proportion of lymphocytes, macrophages, and PD-L1+ neutrophils in urine than non-recurrent patients. Conclusions: Urine leukocyte composition may be a useful tool for analyzing the immunological status of MIBC patients. Urine cellular composition allowed us to identify a new subgroup of LN− patients with a higher risk of recurrence

Neuregulins mediate calcium-induced glucose transport during muscle contraction

Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction

Bacteriophages immunomodulate the response of monocytes

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic

Antimicrobial peptides, disease severity and exacerbations in bronchiectasis

Rationale: Recently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be proinflammatory in chronic lung disease. Objectives: To determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis. Methods: A total of 135 adults with bronchiectasis were prospectively enrolled at three European centres. Levels of cathelicidin LL-37, lactoferrin, lysozyme and secretory leucocyte protease inhibitor (SLPI) in serum and sputum were determined at baseline by ELISA. Patients were followed up for 12 months. We examined the ability of sputum AMP to predict future exacerbation risk. Measurements and main results: AMP levels were higher in sputum than in serum, suggesting local AMP release. Patients with more severe disease at baseline had dysregulation of airway AMP. Higher LL-37 and lower SLPI levels were associated with Bronchiectasis Severity Index, lower FEV1 (forced expiratory volume in 1 s) and Pseudomonas aeruginosa infection. Low SLPI levels were also associated with the exacerbation frequency at baseline. During follow-up, higher LL-37 and lower SLPI levels were associated with a shorter time to the next exacerbation, whereas LL-37 alone predicted exacerbation frequency over the next 12 months. Conclusions: Patients with bronchiectasis showed dysregulated sputum AMP levels, characterised by elevated LL-37 and reduced SLPI levels in the frequent exacerbator phenotype. Elevated LL-37 and reduced SLPI levels are associated with Pseudomonas aeruginosa infection and can predict future risk of exacerbations in bronchiectasis

CSR and related terms in SME owner-managers' mental models in six European countries: national context matters

As a contribution to the emerging field of corporate social responsibility (CSR) cognition, this article reports on the findings of an exploratory study that compares SME owner–managers’ mental models with regard to CSR and related concepts across six European countries (Belgium, Italy, Norway, France, UK, Spain). Utilising Repertory Grid Technique, we found that the SME owner–managers’ mental models show a few commonalities as well as a number of differences across the different country samples. We interpret those differences by linking individual cognition to macro-environmental variables, such as language, national traditions and dissemination mechanisms. The results of our exploratory study show that nationality matters but that classifications of countries as found in the comparative capitalism literature do not exactly mirror national differences in CSR cognition and that these classifications need further differentiation. The findings from our study raise questions on the universality of cognition of academic management concepts and warn that promotion of responsible business practice should not rely on the use of unmediated US American management terminology