The Phagocytosis and Toxicity of Amorphous Silica

BACKGROUND: Inhalation of crystalline silica is known to cause an inflammatory reaction and chronic exposure leads to lung fibrosis and can progress into the disease, silicosis. Cultured macrophages bind crystalline silica particles, phagocytose them, and rapidly undergo apoptotic and necrotic death. The mechanism by which particles are bound and internalized and the reason particles are toxic is unclear. Amorphous silica has been considered to be a less toxic form, but this view is controversial. We compared the uptake and toxicity of amorphous silica to crystalline silica. METHODOLOGY/PRINCIPAL FINDINGS: Amorphous silica particles are phagocytosed by macrophage cells and a single internalized particle is capable of killing a cell. Fluorescent dextran is released from endo-lysosomes within two hours after silica treatment and Caspase-3 activation occurs within 4 hours. Interestingly, toxicity is specific to macrophage cell lines. Other cell types are resistant to silica particle toxicity even though they internalize the particles. The large and uniform size of the spherical, amorphous silica particles allowed us to monitor them during the uptake process. In mCherry-actin transfected macrophages, actin rings began to form 1-3 minutes after silica binding and the actin coat disassembled rapidly following particle internalization. Pre-loading cells with fluorescent dextran allowed us to visualize the fusion of phagosomes with endosomes during internalization. These markers provided two new ways to visualize and quantify particle internalization. At 37 °C the rate of amorphous silica internalization was very rapid regardless of particle coating. However, at room temperature, opsonized silica is internalized much faster than non-opsonized silica. CONCLUSIONS/SIGNIFICANCE: Our results indicate that amorphous and crystalline silica are both phagocytosed and both toxic to mouse alveolar macrophage (MH-S) cells. The pathway leading to apoptosis appears to be similar in both cases. However, the result suggests a mechanistic difference between FcγRIIA receptor-mediated and non-opsonized silica particle phagocytosis

Maxillary fungus ball: zinc-oxide endodontic materials as a risk factor

open7openNICOLAI, Piero; MENSI, Magda; Marsili, F.; Piccioni, M.; SALGARELLO, Stefano Alessandro; GILBERTI, Maria Enrica; Apostoli, P.Nicolai, Piero; Mensi, Magda; Marsili, F.; Piccioni, M.; Salgarello, Stefano Alessandro; Gilberti, Maria Enrica; Apostoli, P

A yeast-based repurposing approach for the treatment of mitochondrial DNA depletion syndromes led to the identification of molecules able to modulate the dNTP pool

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients

Optical properties of highly n-doped germanium obtained by in situ doping and laser annealing

High n-type doping in germanium is essential for many electronic and optoelectronic applications especially for high performance Ohmic contacts, lasing and mid-infrared plasmonics. We report on the combination of in situ doping and excimer laser annealing to improve the activation of phosphorous in germanium. An activated n-doping concentration of 8.8  ×  1019 cm−3 has been achieved starting from an incorporated phosphorous concentration of 1.1  ×  1020 cm−3. Infrared reflectivity data fitted with a multi-layer Drude model indicate good uniformity over a 350 nm thick layer. Photoluminescence demonstrates clear bandgap narrowing and an increased ratio of direct to indirect bandgap emission confirming the high doping densities achieved

Association of the immature platelet fraction with sepsis diagnosis and severity

Management of Sepsis would greatly benefit from the incorporation of simple and informative new biomarkers in clinical practice. Ideally, a sepsis biomarker should segregate infected from non-infected patients, provide information about prognosis and organ-specific damage, and be accessible to most healthcare services. The immature platelet fraction (IPF) and immature reticulocyte fraction (IRF) are new analytical parameters of the complete blood count, that have been studied as biomarkers of several inflammatory conditions. Recently, a study performed in critically-ill patients suggested that IPF could be a more accurate sepsis biomarker than C-reactive protein (CRP) and procalcitonin. In this retrospective study we evaluated the performance of IPF and IRF as biomarkers of sepsis diagnosis and severity. 41 patients admitted to two intensive care units were evaluated, 12 of which with severe sepsis or septic shock, and 11 with non-complicated sepsis. Significantly higher IPF levels were observed in patients with severe sepsis/septic shock. IPF correlated with sepsis severity scores and presented the highest diagnostic accuracy for the presence of sepsis of all studied clinical and laboratory parameters. No significant differences were observed in IRF levels. Our results suggest that IPF levels could be used as a biomarker of sepsis diagnosis and severity

Innate immune activation by inhaled lipopolysaccharide, independent of oxidative stress, exacerbates silica-induced pulmonary fibrosis in mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress. © 2012 Brass et al

LKB1 Destabilizes Microtubules in Myoblasts and Contributes to Myoblast Differentiation

Background: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings: We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMPactivated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions: Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeleta