In Vitro Macrophage Phagocytosis Assay

The key roles of macrophages in atherosclerosis include the phagocytosis of apoptotic and necrotic cells and cell debris, whose accumulation in atherosclerotic lesions exacerbates inflammation and promotes plaque vulnerability. Evidence is accumulating that macrophage phagocytic functions peak at the early stages of atherosclerosis and that the reduced phagocytosis at the late stages of disease leads to the generation of necrotic cores and a defective resolution of inflammation, which in turn promotes plaque rupture, thrombus formation, and life-threatening acute ischemic events (myocardial infarction and stroke). The impaired resolution of inflammation in advanced lesions featuring loss of macrophage phagocytic activity may be in part due to an imbalance between M1 and M2 subsets of polarized macrophages. A better understanding of the mechanisms that regulate macrophage phagocytic activity in the context of atherosclerosis may therefore help identify novel therapeutic targets. This chapter presents a protocol for establishing primary mouse macrophage cultures, a method for polarizing macrophages to the M1 and M2 states, and a method for the in vitro study of macrophage phagocytosis of IgG-opsonized or IgM/complement component 3-opsonized erythrocytes.M.R.H. is supported by an FPI predoctoral fellowship from the Spanish Government (BES-2011-043938) and R.V-B. by a Juan de la Cierva postdoctoral contract from the Spanish Government (JCI-2011- 09663). Work in V.A.’s laboratory is supported by grants from the Spanish Ministry of Economy and Competitivity (MINECO) (SAF201346663-R), Fondo Europeo de Desarrollo Regional (FEDER), Instituto de Salud Carlos III (RD12/0042/0028), the Progeria Research Foundation (Innovator Award 2012, Established Investigator Award 2014), and the European Union (Liphos, Grant Agreement 317916). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the MINECO and the Pro-CNIC Foundation.S

Effectiveness of dry needling for headache: A systematic review.

Introducción El uso de tratamientos no farmacológicos en pacientes con cefalea, como la punción seca (PS), está asociado a una baja morbimortalidad y a un bajo coste sanitario. Algunos han demostrado utilidad en la práctica clínica. El objetivo de esta revisión fue analizar el grado de evidencia de la efectividad de la PS en la cefalea. Métodos Revisión sistemática de ensayos clínicos aleatorizados sobre cefalea y PS en las bases de datos biomédicas PubMed, Web of Science, Scopus y PEDro. Se evaluó la calidad de los estudios incluidos mediante la escala PEDro por 2 evaluadores de forma independiente. Resultados De un total de 136 estudios, se seleccionaron 8 ensayos clínicos publicados entre 1994 y 2019, incluyendo en total 577 pacientes. Dos estudios evaluaron pacientes con cefalea cervicogénica, otros 2, pacientes con cefalea tensional, y otro, pacientes con migraña. Los otros 3 estudios evaluaron pacientes con cefalea de características mixtas (tensional/migraña). La calidad de los estudios incluidos osciló entre «baja» (3/10) y «alta» (8/10). La eficacia de la PS sobre los episodios de cefalea fue similar a la de los tratamientos con los que se comparó. No obstante, obtuvo mejoras significativas respecto a variables funcionales y de sensibilidad. Conclusiones La punción seca es una técnica a considerar para el tratamiento de las cefaleas en la consulta, pudiendo utilizarse de forma rutinaria, bien de forma aislada, bien en combinación con terapias farmacológicas. Introduction Non-pharmacological treatment of patients with headache, such as dry needling (DN), is associated with less morbidity and mortality and lower costs than pharmacological treatment. Some of these techniques are useful in clinical practice. The aim of this study was to review the level of evidence for DN in patients with headache. Methods We performed a systematic review of randomised clinical trials on headache and DN on the PubMed, Web of Science, Scopus, and PEDro databases. Methodological quality was evaluated with the Spanish version of the PEDro scale by 2 independent reviewers. Results Of a total of 136 studies, we selected 8 randomised clinical trials published between 1994 and 2019, including a total of 577 patients. Two studies evaluated patients with cervicogenic headache, 2 evaluated patients with tension-type headache, one study assessed patients with migraine, and the remaining 3 evaluated patients with mixed-type headache (tension-type headache/migraine). Quality ratings ranged from low (3/10) to high (7/10). The effectiveness of DN was similar to that of the other interventions. DN was associated with significant improvements in functional and sensory outcomes. Conclusions Dry needling should be considered for the treatment of headache, and may be applied either alone or in combination with pharmacological treatments

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with CSC. We evaluated specific markers for contractile [i.e., actin, aortic smooth muscle (ACTA2), calponin-1 (CNN1), the Kruppel-like factor 4 (KLF4), and myocardin (MYOCD) genes] and inflammatory [i.e., IL-1 beta, and IL-6, IL-8, and galectin-3 (LGALS-3) genes] phenotypes. CSC increased the expression of inflammatory markers and reduced the contractile ones in both cell types, with KLF4 modulating the SMC phenotypic switch. Next, we performed a mass spectrometry-based differential proteomic approach on human SMCs and could show 11 proteins were significantly affected by exposition to CSC (FC >= 2.7, p <= 0.05). These proteins are active in signaling pathways related to expression of pro-inflammatory cytokines and IFN, inflammasome assembly and activation, cy-toskeleton regulation and SMC contraction, mitochondrial integrity and cellular response to oxidative stress, proteostasis control via ubiquitination, and cell proliferation and epithelial-to-mesenchymal transition. Through specific bioinformatics resources, we showed their tight functional correlation in a close interaction niche mainly orchestrated by the interferon-induced double-stranded RNA-activated protein kinase (alternative name: protein kinase RNA-activated; PKR) (EIF2AK2/PKR). Finally, by combining gene expression and protein abundance data we obtained a hybrid network showing reciprocal integration of the CSC-deregulated factors and indicating KLF4 and PKR as the most relevant factors

HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages

Macrophages secrete matrix metalloproteinases (MMPs) that may weaken the fibrous cap of atherosclerotic plaque, predisposing its fissuration. The 92- kDa gelatinase B (MMP-9) has been identified in abdominal aortic aneurysms and in atherosclerotic tissues. Fluvastatin, through the inhibition of the isoprenoid pathway, inhibits major processes of atherogenesis in experimental models (smooth muscle cell migration and proliferation and cholesterol accumulation in macrophages). We studied the effect of fluvastatin on the activity of MMP-9 in mouse and human macrophages in culture. Conditioned media of cells treated for 24 hours with fluvastatin were analyzed by gelatin zymography. In mouse macrophages, fluvastatin (5 to 100 \u3bcmol/L) significantly inhibited in a dose-dependent manner MMP-9 activity from 20% to 40% versus control. The drug, at a concentration as low as 5 \u3bcmol/L, inhibited MMP-9 activity ( 4330%) in human monocyte-derived macrophages as well. Phorbol esters (TPA, 50 ng/mL) stimulated MMP-9 activity by 50%, and fluvastatin inhibited this enhanced activity up to 50% in both mouse and human macrophages. The above results on the secretion of MMP-9 were confirmed by Western blotting and ELISA. The inhibitory effect of fluvastatin was overcome by the simultaneous addition of exogenous mevalonate (100 \u3bcmol/L), a precursor of isoprenoids. Fluvastatin's effect was fully reversible, and the drug did not cause any cellular toxicity. The statin did not block directly the in vitro activation of the secreted protease. Similar data were obtained with simvastatin. Altogether, our data indicate an inhibition of MMP-9 secretion by the drug. This effect is mediated by the inhibition of synthesis of mevalonate, a precursor of numerous derivatives essential for several cellular functions

NaAlH4 production from waste aluminum by reactive ball milling

Due to its thermodynamic properties and high reversibility, Ti doped sodium alanateis considered as a prototype hydrogen storage material. In this work we show how sodium alanate can be synthesized by reactive ball milling using aluminum particles obtained from recycled waste incineration slag. The synthesis was monitoredwith an in situ milling vial and characterized stepwise by PXD and DTA analyses. The sorption properties of the material were investigated using in situ synchrotron radiation PXD and volumetric analyses. A complete conversion of the starting reactants was obtained

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD

Sorption behavior of the MgH2-Mg2FeH6 hydride storage system synthesized by mechanical milling followed by sintering

The hydrogen sorption behavior of the Mg2FeH6eMgH2hydride system is investigated via in-situ synchrotron and laboratory powder X-ray diffraction (SR-PXD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), particle size distribution (PSD) and volumetric techniques. The Mg2FeH6eMgH2 hydride system is obtained by mechanical milling in argon atmosphere followed by sintering at high temperature and hydrogen pressure. In-situ SR-PXD results show that upon hydriding MgH2 is a precursor for Mg2FeH6 formation and remained as hydrided phase in the obtained material. Diffusion constraints preclude the further formation of Mg2FeH6. Upon dehydriding, our results suggest that MgH2 and Mg2FeH6 decompose independently in a narrow temperature range between 275 and 300 C. Moreover, the decomposition behavior of both hydrides in the Mg2FeH6eMgH2 hydride mixture is influenced by each other via dual synergetic-destabilizing effects. The final hydriding/dehydriding products and therefore the kinetic behavior of the Mg2FeH6eMgH2 hydride system exhibits a strong dependence on the temperature and pressure conditions.Fil: Puszkiel, Julián Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Gennari, Fabiana Cristina. Comision Nacional de Energia Atomica. Gerencia de Area de Aplicaciones de la Tecnologia Nuclear. Gerencia de Investigacion Aplicada; . Universidad Nacional de Cuyo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arneodo Larochette, Pierre Paul. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comision Nacional de Energia Atomica. Gerencia de Area de Aplicaciones de la Tecnologia Nuclear. Gerencia de Investigacion Aplicada; . Universidad Nacional de Cuyo; ArgentinaFil: Karimi, Fahim. Materials Technology. Institute of Materials Research; AlemaniaFil: Pistidda, Claudio. Materials Technology. Institute of Materials Research; AlemaniaFil: Gosalawit Utke, Rapee. Materials Technology. Institute of Materials Research; Alemania. Suranaree University of Technology. Institute of Science, School of Chemistry; TailandiaFil: Jepsen, Julian. Materials Technology. Institute of Materials Research; AlemaniaFil: Jensen, Torben R.. University of Aarhu. Center for Energy Materials, iNANO and Department of Chemistry; DinamarcaFil: Gundlach, Carsten. Lund University. MAX-lab; SuizaFil: Bellosta von Colbe, José. Materials Technology. Institute of Materials Research; AlemaniaFil: Klassen, Thomas. Materials Technology. Institute of Materials Research; AlemaniaFil: Dornheim, Martin. Materials Technology. Institute of Materials Research; Alemani

Complex hydrides for hydrogen storage - New perspectives

Since the 1970s, hydrogen has been considered as a possible energy carrier for the storage of renewable energy. The main focus has been on addressing the ultimate challenge: eveloping an environmentally friendly successor for gasoline. This very ambitious goal has not yet been fully reached, as discussed in this review, but a range of new lightweight hydrogen-ontaining materials has been discovered with fascinating properties. State-of-the-art and future perspectives for hydrogen-containing solids will be discussed, with a focus on metal borohydrides, which reveal significant structural flexibility and may have a range of new interesting properties combined with very high hydrogen densities

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

OBJECTIVE: To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549). METHODS: A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E(2 )(PGE(2)) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively. RESULTS: LPS increased the expression of COX-2 mRNA and production of PGE(2 )in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE(2). There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE(2 )(r = 0.947, P < 0.05). CONCLUSION: Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE(2 )in cultured A549 cells