The screening of albumin as a key serum component to prevent neutrophil extracellular traps release by selectively inhibiting mitochondrial ROS generation

Neutrophil extracellular traps (NETs) are extracellular DNA webs released from neutrophils to mediate host anti-microbial defense. As NETs could also induce thrombosis and cause organ injury, their release should be strictly controlled. However, it is not well understood about the intrinsic mechanisms that prevent unfavorable NETs. Herein, an accidental finding of NETs release from human peripheral neutrophils was firstly described in serum free culture, and it was also determined as a conserved effect for serum to prevent NETs. In contrast to canonical NETs induced by phorbol-12-myristate-13-acetate (PMA), NETs formation by serum free culture was rapid and without prevalent NETosis. Next, albumin was screened out as a key serum component that mediated the suppression of NETs. Moreover, NETs induced upon serum or albumin deficiency were independent of the canonical pathway that involves NOX2 activation and cytosol ROS production. Instead, the generation of mitochondrial ROS (mtROS) was upregulated to promote NETs release. Albumin exhibited mtROS scavenging activity and thus inhibited NETs. Serum free culture also induces the release of NET-bound oxidized mtDNA which stimulated IFN-β production. Overall, our research provides new evidences that characterize the NETs production in serum free culture and determine the mechanisms of serum albumin to inhibit NETs.

Bovine fetal tissue extracts as an alternative to fetal serum for in vitro reproduction of viruses

The authors assessed the possibility of substitution of the serum component with tissue extracts (muscles, liver, kidneys) of bovine fetuses in the culture medium during the cultivation of transplanted LEK and Vero cell lines, as well as the reproduction of infectious rhinotracheitis IR, PI-3 viruses and reovirus on them. The greatest stimulating effect on LEK and Vero cells was obtained from bovine fetuses muscle extract. The effect of this extract on the proliferative activity of LEK and Vero cells is significant and amounts to 27 and 25%, respectively. The power of the effect of liver and kidney extracts is significantly lower and equal, respectively, 15 and 18% for LEK and 14 and 19% for Vero, although it is reliable. The reproductive activity of IR and PI-3 viruses when using tissue extracts was inferior to that when using blood serum. The stimulating effect of blood serum and muscle extract on the reproduction of reovirus was comparable. The effect of fetal muscle extract on the reproduction of IR, PI-3 viruses and reovirus is reliable and amounts to 29, 31 and 33%, respectively. In general, it is close to that of the blood serum of bovine fetuses - 30, 35 and 36%. The power of the influence of the liver and kidney extracts of the bovine fetuses is significantly lower and comparable to that of the blood serum of the cows themselves: 25, 23 and 20%, although it is reliable.

Serum cholesterol selectively regulates glucocorticoid sensitivity through activation of JNK

Glucocorticoids (Gc) are potent anti-inflammatory agents with wide clinical application. We have previously shown that increased serum concentration significantly attenuates regulation of a simple Gc-responsive reporter. We now find that glucocorticoid receptor (GR) regulation of some endogenous transactivated but not transrepressed genes is impaired, suggesting template specificity. Serum did not directly affect GR expression, activity or trafficking, implicating GR crosstalk with other signalling pathways. Indeed, a JNK inhibitor completely abolished the serum effect. We identified the Gc modulating serum component as cholesterol. Cholesterol loading mimicked the serum effect, which was readily reversed by JNK inhibition. Chelation of serum cholesterol with methyl-β-cyclodextrin or inhibition of cellular cholesterol synthesis with simvastatin potentiated the Gc response. To explore the effectin vivowe usedApoE−/−mice, a model of hypercholesterolaemia. Consistent with ourin vitrostudies, we find no impact of elevated cholesterol on the expression of GR, or on the hypothalamic–pituitary–adrenal axis, measured by dexamethasone suppression test. Instead we find selective Gc resistance on some hepatic target genes inApoE−/−mice. Therefore, we have discovered an unexpected role for cholesterol as a selective modulator of Gc actionin vivo. Taken together these findings reveal a new environmental constraint on Gc action with relevance to both inflammation and cancer.

Technetium-99m-Labeled Autologous Serum Albumin: A Personal-Exclusive Source of Serum Component

Technetium-99m human serum albumin (99mTc-HSA) is an important radiopharmaceutical required in nuclear medicine studies. However, the risk of transfusion-transmitted infection remains a major safety concern. Autopreparation of serum component acquired from patient provides a “personal-exclusive” source for radiolabeling. This paper is to evaluate the practicality of on-site elusion and subsequent radiolabeling efficacy for serum albumin. Results showed that the autologous elute contained more albumin fraction than serum without extraction procedure. Good radiochemical purity and stability were demonstrated after radiolabeling. Biodistribution study showed that labeled albumin accumulated immediately in the lung, liver, and kidney. It was cleared steadily and excreted in the urine. The biologic half-life was defined, and all samples passed the pyrogenicity and sterility tests. In conclusion, autoalbumin could be extracted and radiolabeled properly in a nuclear medicine setting. Moreover, the risk of transfusion-transmitted infection associated with nonautologous, multisource99mTc-HSA agents can be reduced.

Coordinating epidermal growth factor-induced motility promotes efficient wound closure

Wound healing is a response to injury that is initiated to reconstruct damaged tissue. In skin, reepithelialization involves both epithelial cells and fibroblasts and contributes to the reformation of a barrier between the external environment and internal milieu. Growth factors including epidermal growth factor (EGF) play important roles in promoting this process. In the present studies we employed CV-1 fibroblasts in a tissue culture model of reepithelialization to develop strategies for optimizing wound closure stimulated by EGF. We found that EGF enhanced cell motility within 6–8 h of EGF treatment in serum-free medium but wounds failed to close within 24 h. However, if medium on these cultures was exchanged for medium containing serum, cells pretreated with EGF closed new scrape wounds more rapidly than did cells that were not pretreated. These results indicate that serum factors work in concert with EGF to coordinate cell motility for efficient wound closure. Indeed, EGF enhanced the rate of wound closure in the presence of serum, and this effect also persisted for at least 24 h after EGF was removed. This coordination of EGF-induced cell motility was accompanied by an increase in the transient phosphorylation of ERK1 and ERK2. The persistent effects of EGF were blocked by transient exposure to reversible inhibitors of transcription and translation, indicating that the expression of new proteins mediated this response. We propose that EGF-stimulated CV-1 fibroblast motility is coordinated by a serum component that induces cell-cell adhesive properties consistent with an epithelial phenotype, thereby enhancing the reepithelialization process.