Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study.

Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.

Serum bilirubin concentration in healthy adult North-Europeans is strictly controlled by the UGT1A1 TA-repeat variants.

The major enzyme responsible for the glucuronidation of bilirubin is the uridine 5'-diphosphoglucose glucuronosyltransferase A1 (UGT1A1) enzyme, and genetic variation in the UGT1A1 gene is reported to influence the bilirubin concentration in the blood. In this study, we have investigated which gene-/haplotype variants may be useful for genetic testing of Gilbert's syndrome. Two groups of samples based on serum bilirubin concentrations were obtained from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA): the 150 individuals with the highest bilirubin (>17.5 µmol/L) and the 150 individuals with normal bilirubin concentrations (<17.5 µmol/L). The individuals were examined for the TA6>TA7 variant in the UGT1A1 promoter and 7 tag-SNPs in an extended promoter region of UGT1A1 (haplotype analysis) and in selected SNPs in candidate genes (SLCO1B3, ABCC2 and NUP153). We found significant odds ratios for high bilirubin level for all the selected UGT1A1 variants. However, in stepwise multivariate logistic regression analysis of all genetic variants together with age, sex, country of origin and fasting time, the repeat variants of UGT1A1 TA6>TA7 and SLCO1B3 rs2117032 T>C were the only variants significantly associated with higher bilirubin concentrations. Most individuals with high bilirubin levels were homozygous for the TA7-repeat (74%) while only 3% were homozygous for the TA7-repeat in individuals with normal bilirubin levels. Among individuals heterozygous for the TA7-repeat, a low frequent UGT1A1-diplotype harboring the rs7564935 G-variant was associated with higher bilirubin levels. In conclusion, our results demonstrate that in testing for Gilbert's syndrome, analyzing for the homozygous TA7/TA7-genotype would be appropriate.

Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs).

Recognition of conserved bacterial structures called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), may lead to induction of a variety of "early immediate genes" such as chemokines. In the current study, we have in an ex vivo whole blood model studied the induction of the chemokines MIP-1alpha, MCP-1 and IL-8 by various PAMPs. The rate of appearance of Escherichia coli-Lipopolysaccharide (LPS) induced chemokines differed. The production of MIP-1alpha and IL-8 was after 1 h of stimulation significantly higher when compared to unstimulated whole blood, whereas MCP-1 was not significantly elevated until after 3 h. At peak levels the MIP-1alpha concentration induced by E. coli-LPS was 3-5-fold higher than MCP-1 and IL-8. By specific cell depletion, we demonstrated that all three chemokines were mainly produced by monocytes. However, the mRNA results showed that IL-8 was induced in both monocytes and granulocytes. The production of all three chemokines, induced by the E. coli-LPS and Neisseria meningitidis-LPS, was significantly inhibited by antibodies against CD14 and TLR4, implying these receptors to be of importance for the effects of LPS in whole blood. The chemokine production induced by lipoteichoic acid (LTA) and non-mannose-capped lipoarabinomannan (AraLAM) was, however, less efficiently blocked by antibodies against CD14 and TLR2. E. coli-LPS and LTA induced a dose-dependent increase of CD14, TLR2 and TLR4 expression on monocytes in whole blood. These data show that PAMPs may induce chemokine production in whole blood and that antibodies against PRRs inhibit the production to different extent.

Effects of bacterial cell wall components (PAMPs) on the expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and the chemokine receptor CCR2 by purified human blood monocytes.

Regulation of chemokine production and the expression of chemokine receptors play an important role during inflammation and infectious diseases. The present study was designed to study the effects of five different bacterial cell wall components (PAMPs) on the production of MCP-1 and MIP-1alpha and the expression of CCR2 by highly purified human blood monocytes. All five PAMPs induced high expression of mRNA and protein synthesis of both chemokines. Generally, MCP-1 mRNA and protein levels were higher than MIP-1alpha levels. Expression of MCP-1 and MIP-1alpha differed both at the mRNA and at the protein levels, MIP-1alpha always showing a more rapid initial increase, attaining lower protein levels than MCP-1. Antibodies against CD14 significantly inhibited the inducing effects of all the PAMPs used. Antibody against TLR2 inhibited the chemokine production induced by LTA and AraLAM by more than 36% (P < 0.05) while chemokine production induced by Escherichia coli-LPS, purified E. coli-LPS and Neisseria meningitidis-LPS was inhibited by more than 60% by antibody against TLR4 (P < 0.05). The inducing effects of all five PAMPs could be inhibited by rIL-4, rIL-10 and rIL-13. rIL-4 was the most effective. Generally, IC(50) of these anti-inflammatory cytokines were lower for the MIP-1alpha than for the MCP-1 production. The cell surface expression of CCR2 was significantly down-regulated by all five PAMPs in addition to a decrease in cytosolic free calcium and binding of rMCP-1. We conclude that MCP-1 and MIP-1alpha as well as the MCP-1 receptor CCR2 will be substantially regulated upon monocyte contact with various cell wall components (PAMPs) from Gram-negative and Gram-positive bacteria as well as from mycobacteria.